Devices and methods for preparing macromolecule spreads

Abstract

Rapid, accurate, high throughput technologies for the preparation of cytogenetic spreads are disclosed herein. Through these approaches, tens or hundreds of chromosome spreads may be generated and analyzed in an automated fashion. These approaches allow for the rapid identification and quantification of cells with chromosomal aberrations or genomics diseases within a sample.

Description

DEVICES AND METHODS FOR PREPARING MACROMOLECULE SPREADS

[0001] This document claims priority to US Provisional Serial Number 63 / 580,087, filed September 1, 2023, the contents of which are hereby incorporated by reference in their entirety. FIELD OF THE INVENSION

[0002] Disclosed herein are devices and methods for preparing spreads of macromolecules originating from a package, on the surface of an open fluidic device. BACKGROUND

[0003] Genomic materials including chromosomes, extrachromosomal DNA, exogenous and transcribed RNAs are different in various species, and heterogenous among the individuals in the same species. These genomic materials can be distinct and heterogenous among the individual cells in tissues of the same individual, such as in the cases of mosaicism, cancer or neuron development. Furthermore, they can change dynamically within the same cell along the natural course of time, or from a pathological development such as an infectious event or mutation, or in divergent environment with external stimulus. Ideally, genomic and proteomic analysis technology should be able to detect and discern these differences and changes at individual cellular and subcellular level with structural, environmental, spatial, and chronological context, in real time. A chromosome is a deoxyribonucleic acid (DNA) molecule that contains all or part of the genetic material of an organism, its “genome”. Most eukaryotic chromosomes include packaging proteins which, aided by chaperone proteins, bind to and condense the DNA molecule to prevent it from becoming an unmanageable tangle [Hammond, 2017; Wilson, 2002]. For example, an average freely suspended human cell in solution with diameter of 20-100 um contains about 6.4 billion base pairs of DNA divided among 46 chromosomes (diploid). The length of each base pair is about 0.34 nm. Therefore, if the DNA molecule in a diploid cell were elongated, and laid out end to end, the total length of DNA would be approximately 2 meters, and yet remarkably this genomic material can fit in a cell nucleus of diameter 10 micrometers in an organized manner. This is accomplished by packaging the DNA in cells in highly ordered three-dimensional chromosomes. Furthermore, the genome structure of this packaging plays a significant role in gene transcriptional regulation [Bonev, 2016]. Chromosomes are physically visible in discrete forms under a light microscope when the cell is undergoing the metaphase of cell division (where all chromosomes are aligned in the center of the cell in their condensed form)[Alberts, 2014]. Before this happens, every chromosome is copied once (S phase), and the copy is joined to the original by a centromere, resulting either in an X-shaped structure if the centromere is located in the middle of the chromosome, or a two-arm of uneven length structure if the centromere is located near one of the ends. The original chromosome and the copy are now called sister chromatids. In cells, chromosomes go through progressively condensed stages from prophase, metaphase and reach their highest compaction level in anaphase during chromosome segregation [Antonin, 2016]. In human cells, the typical metaphase chromosome size has an approximate dimension of 1.4 micron in width to 10 microns in length. In these highly condensed forms, chromosomes are easiest to distinguish and study [Schleyden, 1847]. Chromosomal recombination during meiosis and subsequent sexual reproduction plays a significant role in genetic diversity. If these structures are manipulated incorrectly, through processes known as chromosomal instability, can result in changes ranging from simpler rearrangements such as inversion, translocation, to highly complex chromoanagenesis [Pihan, 2013] such as chromoplexy [Shen, 2013] and chromothripsis [Maher, 2012; Stephens, 2011]. Usually, this will make the cell initiate apoptosis leading to its own death, but sometimes mutations in the cell hamper this process and thus cause progression of cancer or developmental and congenital disorders.

[0004] Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosomes, either inside or outside the nucleus of a cell. Most DNA in an individual genome is found in chromosomes contained in the nucleus. Multiple forms of extrachromosomal DNA exist and serve important biological functions [Rush, 1985], for example they can play a role in disease, such as ecDNA in cancer [Verhaak, 2019]. In prokaryotes, nonviral extrachromosomal DNA are primarily found in plasmids whereas in eukaryotes extrachromosomal DNA are primarily found in organelles such as Mitochondria. Although found in normal eukaryotic cells, extrachromosomal DNA (ecDNA) are distinct entities that have been identified in the nuclei of cancer cells and have been shown to carry many copies of driver oncogenes [Nathanson, 2014; deCarvalho, 2018; Turner, 2017]. EcDNA molecules are considered to be a primary mechanism of gene amplification, resulting in many copies of driver oncogenes and very aggressive cancers. EcDNA in the cytoplasm have been found to be structurally different from nuclear DNA. Cytoplasmic DNA are less methylated than DNA found within the nucleus. It was also confirmed that the sequences of cytoplasmic DNA were different from nuclear DNA in the same organism, showing that cytoplasmic DNAs are not simply fragments of nuclear DNA [Koch, 1983]. In cancer cells, ecDNA have been shown to be primarily isolated to the nucleus. In addition to DNA foundoutside the nucleus in cells, infection of viral genomes also provides an example of extrachromosomal DNA. Clinical Samples are extremely complex, individualized and heterogeneous, at cellular and molecular levels. Large amounts of chromosomal lesions and rearrangements are well known large structural or numerical aberrations to affect biological functions and are associated with complex diseases such as developmental and mental disorders, rare & undiagnosed diseases, reproductive anomalies, blood and all cancers.

[0005] Cytogenetics is the study of chromosomes, which are long strands of DNA and associated proteins that contain most of the genomic information in a cell. Cytogenetics involves testing samples of tissue, blood, amniotic fluid or bone marrow in a laboratory to look for changes in chromosomes, including broken, missing, rearranged, or extra chromosomes, as potential signs of a genetic disease or condition or some types of cancer. Cytogenetics may be used to help diagnose a disease or condition, plan treatment, or find out how well treatment is progressing. Techniques used include karyotyping, analysis of G- banded chromosomes, other cytogenetic and optical banding techniques, as well as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). For example, the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer is just one of the databases, supported by National Cancer Institute (NCI), has catalogued a total number of published unique clinical cases of 69,551 (Oct, 2019), with a total number of unique gene fusions of 22,091 and a total number of genes involved of 12,044, since they started to collect information in 1983 (3844 cases) [Mitelman, 2019]. These documented chromosome aberrations were largely discovered by the gold standards of first-line clinical cytogenetics testing (karyotyping, FISH, Array, and CGH), with guidelines recommended by American College of Medical Genetics (ACMG), American Society for Clinical Pathology (ASCP), National Comprehensive Cancer Network (NCCN), American College of Medical Genetics (ACMG), American College of Obstetricians / Gynecologists (ACOG), and World Health Organization (WHO).

[0006] The advancement of next generation sequencing (NGS) technologies has rapidly expanded genome analysis. Using such technologies, a large data base of genomic changes mostly comprised with millions of point mutations and SNPs (Single Nucleotide Polymorphisms) in human or biological populations have been generated, but direct clinical utility of vast majority of these genomic changes remains unproven. There are over 8000 known genetic diseases, yet more than 2 / 3 remain without a clear understanding of the genetic cause. Despite the progress of NGS technology, the cost and technology limitations(for example limited native read length, library bias, and the bioinformatics complexity, to name a few) have limited NGS’s wider adoption into more clinical settings.

[0007] NGS provides a gain in genome nucleotide resolution, but at the expense of a loss in spatial and structural resolution of the chromosomes and genome analysis. In addition, with a large portion of the genome comprising “Dark Matter” such as instable repetitive regions that are still not easily accessible, NGS technologies have yet to provide true diploid / multiploidy medical grade genome data that is critical for clinical environment. In addition, complete extrachromosomal DNA (ecDNA) information and complex chromothripsis structures remains elusive, as NGS sample prep and algorithms cannot distinguish them a priori. NGS data for accurately identifying structural variants is largely limited to SNPs and short indels.

[0008] Clinical Samples are extremely complex, individualized and heterogeneous, at cellular and molecular levels. Large amounts of chromosomal lesions and rearrangements are well known large structural or numerical aberrations to affect biological functions and are associated with complex diseases such as developmental and mental disorders, rare & undiagnosed diseases, reproductive anomalies, blood and all cancers.

[0009] Genomic and proteomic analysis technology should be able to detect and discern these differences and changes at individual cellular and subcellular level with structural, environmental, spatial, and chronological context, in real time. If these structural genomic lesions generated by such as exposure of ionizing radiations, or infectious agents such as retrovirus (8% of human genome are of viral nature via integration process such as retrotransposition) causing chromosomal rearrangements and worse, genomic instability, can result in changes ranging from simpler rearrangements such as inversion, translocation, to highly complex chromoanagenesis, in somatic de novo fashion or permanently in germline. These would lead to cancer or genetic diseases, or failed gene therapy or editing or ineffective genetic engineering or molecular breeding, if not monitored correctly.

[0010] The well-established cytogenetics techniques of optically interrogating chromosome spreads represent an elegant “top down” true single-cell, single-molecule level test with full real time view of the entire genomic chromosomal sets. The techniques remain the gold standard of first-line tests used by clinicians in tens of thousands hospital and clinical labs with established protocols and guidelines, yielding results trusted by doctors and clinicians. However, despite the historical success of these techniques, significant technological challenges have limited their potential to achieve lower cost, consistent quality, reduced errors, and improved resolution of genomic changes. These limitations include: labor- intensive procedures requiring subjective manual involvement of highly trained professionals(low scalability with 100~500 tests / person / year) [NPAAC, 2013], low resolution of identifying genomic changes, limited to megabases or larger, long turn-around time from sample to answer (typically 3~28 days), and ambiguous or erroneous raw image datasets that hinder the adaption of machine learning that requires further manual curation. Technologies that overcome these limitations, while maintaining the clinical validity of traditional cytogenetic methods will allow for improved patient and doctor access to actionable genomic information of clinical value, unleash the power of medical AI analysis, and help fulfill the promise of personalized medicine.

[0011] Traditional cytogenetic methods for generating chromosome spreads on glass slides suffer from a multitude of limitations with respect to their clinical and research utility, even when prepared under automated or skilled hands. The fundamental source of these limitations is the random process of generating chromosome spreads on a glass slide surface. Among those familiar with the art of cytogenetics, the chromosome “spread” refers to the physical positioning of chromosomes from a single cell on the surface of a substrate, typically a glass slide, such that the chromosomes from a single cell can be identified as having originated from a single cell due to their physical proximity to each other, thus forming a chromosome cluster that can be identified by human inspection or image recognition algorithms.

[0012] Typically, spreads are prepared on the surface of the glass slide at random locations by dropping a solution of cells from a height above the slide, and rupturing the cells after contact with the slide, such that the chromosomes are then ejected from the ruptured cells to form a spread at the location of each ruptured cell [Grubb, 2015]. Within each spread is a collection of chromosomes co-located on the surface of the slide in a random fashion. These limitations: the random positioning of the ruptured cells on the slide, and the random physical positioning of the chromosomes within the cluster (spread) that originate from each ruptured cell results in significant ambiguities as to physical and genomic nature and / or count of the chromosomes, all of which may be essential for identifying or diagnosing a variety of diseases or genetic information in the originating cell or organism.

[0013] A chromosome spread on a glass slide generated by traditional cytogenetic methods of dropping a solution a few inches above the slide held at a 45-degree angle is shown in Figure 1, and demonstrates some of the common challenges of identifying and thus characterizing chromosomes with such techniques. Overlapping chromosomes can obfuscate the portion of the chromosome that is hidden, potentially hiding critically important banding information which may be necessary to identify the chromosome, or a structural variation, or a disease. Additionally, when two or more chromosomes are in contact or overlapping, theirresponsiveness to post-processing chemicals, such as trypsin, diminishes, resulting inconsistent chemical activity across the chromosomes in a spread. Furthermore, if two or more chromosomes are in physical contact, analysis done by human or image recognition software may incorrectly identify which portions of chromosomes belong to the same chromosome, thus erroneously reporting the incorrect number of chromosomes that originated from the cell, or erroneously reporting an incorrect structural variation, mutation, or modification.

[0014] Critical to the successful identification of spreads, and their ultimate utility for generating genetically relevant information, are the size, shape, distribution, nature, and density of the spreads. Spreads that contain chromosomes which can be unambiguously identified as having originated from a single cell, and with all individual chromosomes within the spread unambiguously identified from each other, are referred to as the “useful spreads”. The absolute number of useful spreads on a glass slide is the “yield”. A fundamental requirement within the field of cytogenetics is the ability to prepare glass slides with a high yield. Most clinical diagnostics require independent confirming observations of a genetic characteristic from multiple cells, thus a high yield is essential to provide such confirmation. If the spreads are too small in area, the probability of physical interaction or overlapping between the chromosomes within the spread increases, lowering the overall yield. If the spreads are too large in area, or the density of the spreads on the surface is too high, the probability of neighboring spreads overlapping each other increases, which reduces the confidence in the ability to identify the boundaries of the spreads, and thus again lowering the yield. If the density of the spreads is too low, the probability of a single cell generating genetically relevant information increases, however often at the cost of overall yield due to the lower number of spreads.

[0015] Another common source of error is determining an accurate identification of the complete set chromosomes that originate from a single cell. Mis-identification of the set of chromosomes, by incorrectly subtracting or adding chromosomes via human recognition or image processing algorithms of the chromosome clusters may inappropriately associate a loss or gain of chromosomes with the originating cell, and thus, erroneously associating the loss or gain with a disease. Figure 2 demonstrates an example of a typical collection of chromosome spreads where by the boundaries of many of the spreads is ambiguous. One important observation with respect to traditional cytogenetic methods for generation of spreads is the physical boundary that contains the spread has a significant amount of variation in its shape and size within a single slide preparation, due to the random and violent nature ofrupturing cells via dropping from a height. Chromosomes may be randomly ejected from the rupture point in any direction on the surface, often 100s of microns away from the rupture epicenter. Because of such large variation in spread geometry profiles, spreads generated from traditional cytogenetic techniques must ensure a sufficiently low density of ruptured cells on the surface of slide, and thus sufficiently large separation between ruptured cells, to ensure an output with a reasonable yield of useful spreads.

[0016] As an example of the challenges faced by traditional cytogenetic methods for preparing spreads, the spread in Figure 2 identified as 201 can reasonably be assumed to have originated from a single cell due to the condensed packing the chromosomes and substantial physical spacing from neighboring spreads. Dense packing of chromosomes facilitates spread identification with greater confidence, however at a cost of increased probability of chromosome overlapping, thus lowering the yield. When spreads from two cells are too close, as demonstrated by region 202 which presumably contains spreads from two different cells, the boundary conditions between the spreads become ambiguous. Furthermore, when spreads are not condensed, but eject their chromosomes in random directions and distances, as demonstrated by region 203, again the boundary condition for the spread is ambiguous as it is not entirely clear which genomic material should be included with the spread. Such ambiguities are highly problematic in a clinical application where an accurate single-cell chromosome count and chromosome health evaluation are essential. SUMMARY

[0017] In some aspects, provided herein is a device or method for generating spreads of size or shape in a consistent manner. In the preferred embodiment, the device or method allows for maximizing the spread density on a surface without significant cost of yield reduction. In another aspect of the device or method, spread density can be further increased by biasing the location of the spreads on the surface of the fluidic device, further increasing the yield. In another aspect of the device or method, the quality of an individual spread can be increased by reducing the probability of chromosome physical interaction within the spread, further increasing the yield. In another aspect of the device or method, at least a portion of at least one chromosome is removed from the surface of the fluidic device and undergoes additional processing.

[0018] In some aspects, provided herein is a device or method that relates to a fluidic device for the interrogation of macromolecules, more particularly for the preparation of spreads of single macromolecules originating from a single package for subsequent processing,including interrogation, in particular, interrogation for the purpose of extracting genomic information. In a further aspect, a method or device is provided for preparing spreads of single macromolecules originating from a single package for subsequent processing. The device or method is particularly suited for preparing, processing, and interrogating macromolecules, including chromosomes and ecDNA, originating from a single package.

[0019] According to a further aspect, the present device or method relates to consumables, systems, apparatus, kits, algorithms and methods for handling, preparing, analyzing and characterizing (in any order) biological samples. The device or method also describes uses of the device or method, particularly in relation to nucleic acid physical mapping techniques.

[0020] As provided herein, devices and methods to integrate clinical single-cell cytogenomic optical imaging techniques with microfluidic devices. Here we present devices, systems and methods to automate, standardize, and accelerate the digital cytopathological sample preparation and data generation. In some device and method embodiments, the sample input cell quantity may be substantially reduced over traditional cytogenetic methods, thus allowing for a reduction in time required to multiply the cells via growth. In some device and method embodiments, the chromosomes are presented for interrogation in an ordered and controlled manner, reducing the ambiguity when compared to traditional cytogenetic methods in determining the number and physical structure of the chromosomes, and allowing for greater automation and reduced manual curation. In some device and method embodiments, the chromosomes originating from a single package are presented as spreads with a predictable or definable cluster boundary, thus reducing the ambiguity of the originating package of any particular chromosome when compared to traditional cytogenetic methods. In some device and method embodiments, the chromosomes are presented in a state such that the chromosome arms are pulled apart from each other, allowing for reduced ambiguity in performing analysis of the individual chromosome arms when comparted to traditional cytogenetic methods. In some device and method embodiments, the chromosomes are presented in a substantially elongated format over tradition chromosome methods, such that with optically equivalent interrogation tools, improved resolution of identifying genomic features within the chromosomes are feasible. In some device and method embodiments, the genomic material may be physically manipulated, transported, stretched, compressed, or pinched in a fluidic device. In some device and method embodiments, single chromosomes, or portions of single chromosomes representing regions of interest may be isolated. In some device and method embodiments, these isolated regions of interest may then be further processed, including amplified, sequenced, or genotyped, on the device, or off the device.

[0021] The present disclosure provides devices and methods that facilitate the characterization of genomic variation in large nucleic acid molecules. The devices and methods disclosed herein may enable improved quality of physical maps, for example karyotyping or FISH spreads. In another example, the devices and methods herein may enable the manipulation of large macromolecules in a microfluidic environment to improve the quality and reliability of the genomic data obtained from the interrogation of said molecules. In another example, a specific package from which the macromolecule will be selected from in order to perform interrogation is selected from a collection of packages. In another example, the macromolecule is manipulated in a fluidic device such that the macromolecule’s physical conformation can be altered to obtain interrogation data of the desired region and resolution in the macromolecule.

[0022] In some embodiments, the package comprises a single cell, or single nucleus, contained within are macromolecules. In the preferred embodiment, the macromolecules are condensed such as metaphase or prophase chromosomes. In some embodiments, the cells or nucleus are in a hypotonic state.

[0023] In some embodiments, the package comprises a type of droplet containing macromolecules. The droplet type may comprise: oil-in-water, water-in-oil, or water-in-oil- in-water droplet. In some embodiments, the macromolecules are entire chromosomes that originate from a single cell. In some embodiments, the macromolecules are a selection of chromosomes that are selected on the basis of a certain criteria, for example chromosome length, number, or disease trait. In some embodiments, the macromolecules may not originate from a single cell.

[0024] In some embodiments, a package containing a package fluid and at least two macromolecules is ruptured on the surface of an open fluidic device, with said device comprising fluidic features, such that a fluid comprising the package fluid positions the at least two macromolecules within the fluidic features. In the preferred embodiment, the positioning of the at least two macromolecules within the fluidic features forms a spread.

[0025] In some embodiments, the probability of the at least two macromolecules being in physical contact when positioned within the fluidic features may be reduced when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods.

[0026] In some embodiments, the probability of the at least two macromolecules overlapping each other when positioned within the fluidic features may be reduced when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods.

[0027] In some embodiments, the probability of the at least two macromolecules physically separating from each other beyond a maximum distance when positioned within the fluidic features may be reduced when compared to a similar package being ruptured on a glass slide. In some embodiments, the orientation of the maximum distance is determined at least in part by the fluidic features.

[0028] In some embodiments, the point of rupture of at least one package may be determined at least in part by at least one fluidic feature.

[0029] In some embodiments, the center-of-mass of the spread may be determined at least in part by at least one fluidic feature.

[0030] In some embodiments, the probability of the at least two macromolecules maintaining a minimum distance from each other when positioned within the fluidic features may be increased when compared to a similar package being ruptured on a glass slide. In some embodiments, the minimum distance is determined at least in part by at least one fluidic feature.

[0031] In some embodiments, the probability of the at least two macromolecules having a substantially similar orientation with respect to each molecule’s major axis when positioned within the fluidic features may be increased when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods.

[0032] In some embodiments, the at least two macromolecules are positioned within the fluidic features in a substantially linear manner with respect to each molecule’s major axis.

[0033] In some embodiments, the at least two macromolecules’ respective major axis have a relative angle that is at least in part determined by at least one fluidic feature.

[0034] In some embodiments, the probability of the at least two macromolecules having a similar nucleic acid density along the length of the each molecule’s major axis when positioned within the fluidic features may be increased when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods.

[0035] In some embodiments whereby the at least two macromolecules are chromosomes, a pair of chromatids from at least one of the chromosomes’s arm are separated from each other by a minimum distance. In some embodiments, the minimum distance may be determined at least in part by at least one fluidic feature.

[0036] In some embodiments whereby the at least two macromolecules are chromosomes, a pair of chromatids from at least one of the chromosomes’s arm have a substantially similar orientation with respect to each chromatid’s major axis.

[0037] In some embodiments whereby the at least two macromolecules are chromosomes, a pair of chromatids from at least one of the chromosomes’ arm have a substantially similar nucleic acid density with respect to each chromatid’s major axis. In some embodiments, the arm is a q arm. In some embodiments, the arm is a p arm.

[0038] In some embodiments, at least two packages, each containing a package fluid and at least two macromolecules are ruptured on the surface of an open fluidic device, with said device comprising fluidic features, such that each package’s respective package fluid positions each package’s respective at least two macromolecules within the fluidic features.

[0039] For all embodiment devices and methods described herein, the interrogation of the macromolecule(s) may comprise the use of an interrogation system. In some embodiments, the interrogation may comprise optical interrogation techniques, including but not limited to: fluorescent, TIRF, epifluorescence, brightfield, darkfield, phase contrast. In some embodiments, the interrogation may comprise a contact probe, preferably an AFM.

[0040] For all embodiment devices and methods described herein, at least one portion of at least one macromolecule may be further processed after interrogation, with said processing comprising amplification, sequencing, or genotyping. In some embodiments, at least a portion of said processing may occur with at least a portion of said at least one macromolecule in contact with said fluidic device. In some embodiments, at least a portion of said processing may occur with at least a portion of said at least one macromolecule while not-in-contract with said fluidic device.

[0041] For all embodiment devices and methods described, the interrogation of the at least one portion of at least one macromolecules may be performed while the at least one portion of at least one macromolecule is at least partially contained within a gel, or semi-gel like material. The gelling material may be introduced as part of the input solution that contains the input sample, or may be introduced at a later time-point, or may be present before the introduction of the molecule. In one preferable embodiment, the gelling material is added to the device after at least a portion of the packages have been ruptured. INCORPORATION BY REFERENCE

[0042] All publications, patents, and patent applications mention in this specification are herein incorporated by reference to the same extent as if individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] For all drawings, the used of roman numbers i), ii), iii), iv), etc are to denote a passage of time. Unless specifically stated, the figures are not drawn to scale.

[0044] Figure 1 is a bright field image that demonstrates a typical spread of banded chromosomes from a single cell generated by traditional cytogenetic methods of dropping a solution of cells on a glass slide, demonstrating a common issue of overlapping and touching chromosomes which can challenge or completely hinder the ability to unambiguously identify individual chromosomes and their disease state.

[0045] Figure 2 is a bright field image that demonstrates the challenges associated with identifying spreads from single cells when traditional cytogenetic methods are employed. In some cases the spread boundaries can be reasonably identified with minimal ambiguity. In many other cases, the spread boundaries have a high degree of ambiguity.

[0046] Figure 3 is a schematic that demonstrates examples of different fluidic features used for containing fluid within a fluidic channel of a fluidic device.

[0047] Figure 4 is a schematic that demonstrates examples of different patterned surface modifications.

[0048] Figure 5 is a schematic that demonstrates the rupture of a package and release of package contents including the package solution and macromolecules into the fluidic features of the fluidic device, so as to position the macromolecules within the fluidic features to generate a spread.

[0049] Figure 6 is a time series of fluorescent images showing the rupturing of a cell immobilized on the surface of fluidic features of a fluidic device. (i) time point 2 seconds: the cell immediately prior to rupture, with the chromosomes stained with a fluorescent dye. (ii) time point 8 seconds: after rupture of the cell, and release of the cell fluid and chromosomes, the individual chromosomes start to become observable as they flow into the fluidic features of the fluidic device. (iii) time point 14 seconds: all of the cell’s chromosomes are now distributed within the channels of the fluidic device.

[0050] Figure 7 is a scanning electron microscope (SEM) image demonstrating individual chromosomes positioned within channels of a fluidic device.

[0051] Figure 8 is a bright field image demonstrating the oval chromosome spreads on the surface of fluidic features on the fluidic device, and the consistency of the spread sizes and chromosome distribution within the spreads.

[0052] Figure 9 is a fluorescent image demonstrating the impact of the size of the fluidic features on the shape of the chromosome spread. Here, a fluidic device with 3 micron widechannels and spacing produces spreads with a major oval axis diameter of 75 microns. The white scale bar is 100 microns.

[0053] Figure 10 is a fluorescent image demonstrating the impact of the size of the fluidic features on the shape of the chromosome spread. Here, a fluidic device with 1.3 micron wide channels and spacing procduces spreads with a major oval axis diameter of 120 microns. The white scale bar is 100 microns.

[0054] Figure 11 is a fluorescent image demonstrating chromosome spread originating from a single cell, whereby the chromosomes are positioned between the hydrophobic pillars.

[0055] Figure 12 is a fluorescent image demonstrating a chromosome & ecDNA spread generated from a single cell, whereby the chromosomes are positioned between the hydrophobic pillars (which in this image are outlined with a white box to enhance visibility).

[0056] Figure 13 is a schematic demonstrating the (i) rupture of a package and release of package contents including package solution and macromolecules of small and large size onto the fluidic device composed of hydrophilic channels with a porous roof, such that after rupture, (ii) the large macromolecules are captured on the top of the porous roof while the smaller macromolecules are able to pass through the porous roof and enter inside the channels.

[0057] Figure 14 is a schematic demonstrating various different and non-limiting embodiments of capture points.

[0058] Figure 15 is a time series of bright field images demonstrating the immobilizing cells on the surface of an open fluidic device at designed capture points. At time point (i), the cells are suspended on a thin film of delivery fluid on the surface of the fluidic device. At time point (ii), the cells have become immobilized at their respective capture points on the surface of the fluidic device, and the delivery fluid has almost completely been removed from the surface of the device. At time point (iii), the cells have been ruptured, and positioned their respective chromosomes into the fluidic features of device to generate spreads.

[0059] Figure 16 is a cross section scanning electron microscope (SEM) image demonstrating an open fluidic device composed of silicon with 2 micron wide channels that are 1 micron deep and separated from each other by 2 microns.

[0060] Figure 17 is a cross section scanning electron microscope (SEM) image demonstrating an open fluidic device composed of silicon pillars. In this particular embodiment, the substrate is silicon, and the features include pillars approximately 1 micron long, 250 nm wide, and 1 micron tall.

[0061] Figure 18 is a scanning electron microscope (SEM) image demonstrating an open fluidic device composed of glass with 0.8 micron wide channels that are 0.5 micron deep that are interspersed with larger 2.0 micron wide channels. In this particular embodiment, the wider channels are designed to accommodate a single human chromosome, while the narrower channels are designed to exclude chromosomes.

[0062] Figure 19 is a scanning electron microscope (SEM) image demonstrating an open fluidic device composed of silicon with 0.8 micron wide pits that are 1.0 micron deep.

[0063] Figure 20 is a scanning electron microscope (SEM) image demonstrating an open fluidic device composed of silicon with 0.3 micron wide pits that are 1.0 micron deep.

[0064] Figure 21 is a scanning electron microscope (SEM) image demonstrating an open fluidic device composed of silicon with 0.8 micron wide pillars that are 1.0 micron tall.

[0065] Figure 22 is a scanning electron microscope (SEM) image demonstrating an open fluidic device composed in borofloat glass with a central 20 micron diameter pit, with 1.5 micron wide channels radiating outwards approximately 25 microns in length.

[0066] Figure 23 is a fluorescent image demonstrating chromosomes physically confined within a 3 micron wide channel, and pushed up against a filter region whereby the channel is branched into two smaller channels, each with a width of 1 micron, preventing the chromosomes from progressing further (in the direction of the white arrows) into the branched structure by physical obstruction.

[0067] Figure 24 is a fluorescent image demonstrating a chromosome spread physically confined within a 100 micron wide region on the open fluidic device by filter elements integrated into the fluidic channels.

[0068] Figure 25 is a fluorescent image demonstrating chromosomes and ecDNA positioned in 3 micron wide channels of a fluidic device, whereby the chromosomes and ecDNA are physically separated from each other, and positioned differently due to their respective physical size difference. The chromosomes span the width of the channel, contacting both sides, while the ecDNAs are drawn into the corners of the channels.

[0069] Figure 26 is a fluorescent image demonstrating the physical separation of ecDNA from chromosomes in a spread generated on an open fluidic device.

[0070] Figure 27 is a schematic demonstrating an embodiment method of manufacturing a porous roof by selective growth of a material on the tips of pillars in a fluidic channel or chamber.

[0071] Figure 28 is a fluorescent image that demonstrates a high degree of major axial alignment of chromosomes originating from a single cell spread, wherein the axial alignment is in one predominant direction (horizontal).

[0072] Figure 29 is a fluorescent image demonstrating a high degree of major axial alignment of macromolecules (chromosomes) originating from a single cell spread, wherein the axial alignment is in two predominant directions (horizontal and vertical).

[0073] Figure 30 is a fluorescent image demonstrating chromosomes from a ruptured cell being positioned along radial channels.

[0074] Figure 31 is a fluorescent image demonstrating chromosomes from a ruptured cell being positioned along radial channels.

[0075] Figure 32 is a fluorescent image demonstrating sub-micron wide channels sufficiently narrow to physically confine the chromosomes and enable elongation of the chromosomes along the lengths of the sub-micron channels.

[0076] Figure 33 is a fluorescent image demonstrating chromosome chromatids being separated from each other such that the centromere of the chromosome is readily observable.

[0077] Figure 34 is a fluorescent image demonstrating chromosome chromatids being separated from each other such that the centromere of the chromosome is readily observable. In particular, an example of a chromosome with two centromeres is readily observable. The 2.5 micron wide channels are appropriately spaced to enable the chromatids to be physically separated for interrogation of the centromeres.

[0078] Figure 35 is a fluorescent image demonstrating a chromosome spread with a FISH label.

[0079] Figure 36 is a schematic demonstrating an embodiment for the application of a solution containing at least one reagent to a portion of a macromolecule.

[0080] Figure 37 is a schematic demonstrating an embodiment for the application of different solution compositions to different portions of a macromolecule.

[0081] Figure 38 is a schematic demonstration of an embodiment for the application of a solution containing at least one reagent to a macromolecule confined within a pit.

[0082] Figure 39 is a schematic demonstration of an embodiment for the selective removal of a portion of a macromolecule by a contract probe.

[0083] Figure 40 is a transmitted light micrograph of a karyotype ruptured onto a glass open fluidic device with fluidic features comprising an array of channels. The chromosomes have been banded with Giemsa Trypsin.DETAILED DESCRIPTION Definitions

[0084] All publications, patents, patent applications, and information available on the internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0085] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the devices and methods of the disclosure and how to make and use them. It will be appreciated that way. Consequently, alternative language and synonyms may the same thing can typically be described in more than one be used for any one or more of the terms discussed here. Synonyms for certain terms are provided. However, a recital of one or more synonyms does not exclude the use of other synonyms, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0086] The device or method is also described by means of particular examples. However, the use of such examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the device or method or of any exemplified term. Likewise, the device or method is not limited to any particular embodiments described herein. Indeed, many modifications and variations of the device or method will be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The device or method is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.

[0087] As used herein, “about” or “approximately” in the context of a number shall refer to a range spanning + / - 10% of the number, or in the context of a range shall refer to an extendedrange spanning from 10% below the lower limit of the listed range to 10% above the listed upper limit of the range.

[0088] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0089] The words “a” and “an,” when used in conjunction with the word “comprising” in the claims or specification, denotes one or more, unless specifically noted.

[0090] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise,” “comprising,” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to.” Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below,” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0091] The use of the term “combination” is used to mean a selection of items from a collection, such that the order of selection does not matter, and the selection of a null set (none), is also a valid selection when explicitly stated. For example, the unique combinations including the null of the set {A,B} that can be selected are: null, A, B, A and B.

[0092] Sample. The term “sample,” as used herein, generally refers to a biological sample of a subject which at least partially contains nucleic acid originating from said subject. The biological sample may comprise any number of macromolecules, for example, cellular long nucleic acid molecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample may be a CTC (circulating tumor cells) or CFC (circulating fetal cells) sample. The sample can include one or more cells. The sample may be one or more droplets containing a biological material. The sample can include one or more microbes. The biological sample may be a nucleic acid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be a cell-free or cell free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.

[0093] Nucleic Acid. The terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide” and “polynucleotide”, “nucleic acid polymer”, “nucleic acid fragment”, “polymer” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, including both naturally occurring and synthesized. The terms encompass, e.g., DNA, RNA and modified forms thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNAs (mRNA), transfer RNAs, ribosomal RNAs, lncRNAs (Long noncoding RNAs), lincRNAs (long intergenic noncoding RNAs), ribozymes, cDNA, ecDNAs ( extrachromosomal DNAs), Circular Polynucleotide, artificial minichromosomes, cfDNAs (circulating free DNAs), ctDNAs (circulating tumor DNAs), cffDNAs (cell free fetal DNAs), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence and configuration including circular RNA, nucleic acid probes, and primers.

[0094] Unless specifically stated otherwise, the nucleic acid molecule can be single stranded, double stranded, or a mixture there-of. For example, there may be hairpin turns or loops. Unless specifically stated otherwise, the nucleic acid molecule may contain nicks.

[0095] Long Nucleic Acid Molecule. Unless specifically stated otherwise, a “long nucleic acid fragment” or “long nucleic acid molecule” is double strand nucleic acid of at least 1 kbp or 1 kb in length, and is thus a kind of macromolecule, and can span to an entire chromosome. It can originate from any source, man-made or natural, including single cell, a population of cells, droplets, an amplification process, etc. It can include nucleic acids that have additional structure such as structural proteins histones, and thus includes chromatin. It can include nucleic acid that has additional bodies bound to it, for example labeling bodies, DNA binding proteins, RNA.

[0096] Circular Polynucleotide. Used herein, “Circular Polynucleotide” or “Circular DNA” or “Circular Nucleic Acid” refers to a polynucleic acid molecule that forms a complete loop, and for which, is at least partially double-stranded. In some embodiments, the entirety of the molecule may double-stranded. In some embodiments, the molecule may have at least one nick, or at least one higher order nucleic acid structure, for example a hair pin.

[0097] The molecule may be a plasmid, a circular bacterial artificial chromosome, a circular yeast artificial chromosome, a circular Mitochondrial DNA, a circular Extrachromosomal circular DNA, a circular Chloroplast DNA, a circular chromosome, a circular virus, a covalently closed circular DNA. The molecule may comprise synthetic nucleotides, or naturalnucleotides. The molecule may comprise synthetic sequence of nucleotides, wild sequence of nucleotides, cloned sequence of nucleotides, inserted sequence of nucleotides.

[0098] Macromolecule. Used herein, “macromolecule” refers to a very large molecule composed of a thousand or more covalently bonded atoms. In the preferred embodiment, macromolecules are involved in biological processes, and are naturally formed. Examples include nucleic acid polymers, chromosomes, portions of chromosomes, ecDNA, eccDNA, circular polynucleotide, plasmids, mitochondrial DNA, chromatin, long nucleic acid molecules, long nucleic acid molecules with a higher order structure, proteins, carbohydrates, or lipids. Non-biological macromolecules may comprise dendrimers, polymers, and carbon nanotubes, DNA origami.

[0099] Higher order nucleic acid structure. A “higher order nucleic acid structure”, or “structure”, or “higher order structure” refers to any 2nd, 3rd, or 4th order DNA structure, including any body bound to said nucleic acid molecule. The nucleic acid molecule may be linear or circular. Nucleic acids can have any of a variety of structural configurations, e.g., be single stranded, double stranded, triplex, replication loop or a combination of both, as well as having higher order intra- or inter-molecular secondary or tertiary or quaternary structures, e.g., chromosomal territories, chromosome boundaries, chromosome regions, compartments, Topologically Associating Domains (TAD), chromatin loop and local direct regulatory factors binding, condensing associated loops, cohesion, associated loops, guide nucleic acid, argonaut complexes, CRISPR Cas9 complexes, nucleoprotein complexes, insulator complexes, enhancer-promoter complexes, ribonucleic acid (RNA), small interfering RNA (siRNA), micro RNA (miRNA), guide RNA (gRNA), long non-coding RNA (lncRNA), repeat region binding proteins, telomere modification proteins, nucleic acid repair proteins, regulatory factor binding proteins, nucleic acid binding proteins, proteins, histone deacetylase (HDAC), chromatin remodeling protein, methyl-binding protein, transcription factor transcription complexes, bending with kinks of the genomic DNA polymers such as hairpins, replication loops, triple stranded regions, in cis or trans fashion etc. The nucleotides within the nucleic acid may have any combination of epigenomic state including but not limited to such as methylation or acetylation states. The nucleic acid can originate from any source, man-made or natural, including single cell, a population of cells, droplets, an amplification process, etc. In some embodiments, these structures include compounds and / or interactions of nucleic acids and proteins. In some embodiments, these structures include 2D and 3D configurations of the nucleic acid beyond the linear 1D polymer chain. These 2D and 3D configurations can be formed via interactions with proteins, other nucleic acid molecules, orexternal boundary conditions. Non limiting examples of boundary conditions include a micro or nanofluidic chamber, a well on or in substrate or defined within a fluidic device, a droplet, a nucleus. The nucleic acid can include nucleic acids that has additional structure such as structural proteins including but not limited to such as any regulatory binding sites complexes, enhancer or transcription factor complex and their interaction with a nucleic acid molecule, Cohesins complex SMC (structural maintenance of chromosomes), ATPase subunits (Smc1 and Smc3), non-SMC regulatory subunits (Rad21 / Scc1 / Mcd1 and SA1 / SA2 / Scc3), Sgo1, mitotic kinases (pololike kinase 1 (Plk1) and aurora B), protein phosphatase 2A (PP2A), chromosome passenger complex (CPC), topo II decatenation, condesins, CTCF proteins, PDS5 proteins, WAPL proteins, condensin, condensing nt II, CAP-G, histones and their derivative complexes, and thus includes chromatin. In some embodiments, higher order structure can include exogenous nuclei acid genome integration complex, in particular, an exogenous nuclei acid genome integration complex that comprises viral genome integration complexes or recombinant nucleus acid. In some embodiments, higher order structure can include extrachromosomal episomes physical docking complexes, in particular, where such complexes host chromosomes through binding sites. In some embodiments, the higher order nucleic acid structure comprises extrachromosomal nucleic acid deriving from a host chromosome. All of above, not limiting, could be target of labelling, physical or conformational biomarkers indicating the presence of certain state of genome organization or the shift between the states, that could be associated with pathogenomic consequences.

[0100] In particular, higher order nucleic acid structure can refer to the various levels of genome organization contained within a cell nucleus [Jerkovic, 2021; Kempfer, 2020] either individually, collectively, or a sub-set there-of. Such genomic organization starts with linear primary DNA winding around histones to form nucleosomes, which are organized into clutches, each containing ~1–2 kb of DNA. Nucleosome clutches form chromatin nanodomains (CNDs) ~100 kb in size, where most enhancer–promoter (E–P) contacts take place. At the scale of ~1 Mb, CNDs and CCCTC-binding factor (CTCF)–cohesin-dependent chromatin loops form topologically associating domains (TADs) and loop domains. On the higher scale up to 100s of megabases, chromatin segregates into gene-active and gene- inactive compartments (A and B, respectively) and into compartment-specific contact hubs, formation of sister chromatid axes. At the highest topological level, the nucleus is organized into chromosome territories.

[0101] Labelling Body. A “labelling body” or “label” used herein is a physical body that can bind to a target body directly or indirectly, which can be used to generate a signal that can be detected with interrogation, that differs from a detected signal (or lack there-of) that would be generated by said target body without said label. A target body may comprise a macromolecule, a nucleic acid molecule, a long nucleic acid molecule, a nucleic acid molecule with at least one higher order structure, a cell, a droplet, a package, a protein, an enzyme. A labelling body may comprise a fluorescent intercalating dye that when bound to nucleic acid, can be used in a fluorescent imaging system to identify the presence of said nucleic acid. In another example, a labelling body may comprise a compound that binds specifically to methylated nucleotides, and gives a current blockade signal when transported through a nanopore, thus reporting a signal as to said molecule’s methylation state. In another example, a labelling body may comprise a quantum dot. In another example, a labeling body may comprise a fluorescent probe specifically hybridized to a sequence of a nucleic acid, thus providing confirmation with a fluorescent imaging system that the sequence is present on said nucleic acid. In another example, a labeling body may comprise a fluorescent probe that specifically binds to a specific protein (e.g.: DNA binding protein), with said protein bound to a long nucleic acid molecule or a macromolecule. In some cases, the absence of the labelling body, may comprise the signal. In some cases, the signal associated with the labelling body may comprise an attenuation, blocking, displacement, quenching, or modification of a signal from another labelling body. Non-limiting examples include: binding of a dark labelling body to the nucleic acid molecule to displace an existing bound fluorescent body; binding of a dark labelling body to the nucleic acid to block a fluorescent labelling body from binding; quenching a near-by fluorescent labelling body bound to a nucleic acid; directly, or indirectly, reacting with a fluorescent labelling body bound to a nucleic acid to reduce its fluorescence. In some cases, the labelling body is not physically attached to the target body at the time of interrogating said target body and labelling body. For example, a labelling body may be attached to a target body via a cleavable linker. At the desired time, the linker is cleaved, releasing said labelling body which is then detected by interrogation.

[0102] Interrogation. “Interrogation” is a process of assessing the state of a target body with an interrogation system. A target body may comprise a macromolecule, a nucleic acid molecule, a long nucleic acid molecule, a nucleic acid molecule with at least one higher order structure, a cell, a droplet, a package, a protein, an enzyme. In some embodiments, the state of the target body is assessed by interrogating the state of at least one labelling body on thetarget body by measuring a signal generated directly, or indirectly from the labelling body. It may be a binary assessment, such as the labelling body is present, or not. It may be quantitative, such as how many labelling bodies are present on the target body. It may be a signal density or intensity along a line, an area, or volume. It may be a physical count, or distance between labelling bodies along the length the target body.

[0103] In some embodiments, interrogation is used to generate a digitized or an in-silico representation of a physical map.

[0104] In some embodiments, interrogation is used to assess the physical state of a higher order nucleic acid structure. The physical state of the structure being interrogated may comprise the topology of the molecule such as the presence of a loop structure, a set of hierarchical loop structures, the number of supercoils present in a loop or the degree to which one or more loops from the same or separate molecules are intertwined. The physical state of the structure being interrogated may comprise the accessibility of a region of the nucleic acid to a binding partner or a cis or trans acting factor. The physical state of the structure being interrogated may comprise the presence of partially replicated nucleic acid still in close proximity such as Okazaki fragments or a marker of newly synthesized nucleic acid such as results from a pulse of BrdU. The physical state of the structure being interrogated may comprise the level of cohesin left on metaphase chromosomes that has been manipulated experimentally or affected by genetic anomalies (e.g., by depleting either cohesin itself or Wapl), the resulting chromatids display substantially different lengths and shapes, becomes a quantitatively measurable biomarkers indicating of certain pathological states [Losada 2005; Gandhi 2006; Shintomi 2009]. The physical state of the structure being interrogated may comprise the amount, ratio, and distribution of condensins I and II in these chromatids. The physical state of structure being interrogated may comprise dynamic changes in genome organization, as in Cohesin release and sister chromatid resolution.

[0105] In some embodiments, the signal being interrogated may be fluorescent, photoluminescent, electro-magnetic, electrical, magnetic, physical, chemical, exhibit plasmon resonance or enhance raman signals by means of surface enhanced plasmon resonance.

[0106] The signal being interrogated may be analog or digital in nature. For example, the signal may be an analog density profile of the labelling body along the length of the nucleic acid in which the signal measured originates from multiple labelling bodies. In some embodiments, the state of the nucleic acid is directly interrogated without a labelling body, for example direct interrogation of macromolecule or long nucleic acid molecules in a cell via phase microscopy, or direct interrogation of nucleic acid via a current blockade nanopore.Non exhaustive examples of different interrogation methods that may be used an interrogation systems either separately, or in combination include fluorescent imaging, bright-field imaging, dark-field imaging, phase contrast imaging, epi-florescent imaging, total internal reflection fluorescence imaging, nearfield or evanescent field imaging, a wave guide, a zero mode waveguide, plasmonic signaling, confocal, scattering, light sheet, structured illumination, stimulated emission depletion, super resolution, stochastic activation super resolution, stochastic binding super resolution, multiphoton, nanopore sensing of a current, voltage, power, capacitive, inductive, or reactive signal (either column blockade through the pore, and tunneling across the pore), chemical sensing (for example: via a reaction), physical sensing (for example: interaction with a sensing probe), SEM, TEM, STM, SPM, AFM. In addition, combinations of different labelling bodies and interrogation methods are also possible. For example: fluorescent imaging of an intercalating dye on a nucleic acid, while translocating said nucleic acid through a nanopore and measuring the pore current.

[0107] The “interrogation region” is the region in the device in which interrogation occurs.

[0108] Interrogation System. Used herein, “Interrogation System” is an automated, semi- automated, or manual system for interrogating the sample. In some embodiments, whereby the sample is interrogated while within or on a fluidic device, the interrogation system interfaces with the fluidic device and controls the operation of the fluid device. In some embodiments, the interrogation system comprises a multitude of separate systems that together can be coordinated by a controller or user. For example, an instrument for loading sample into a fluidic device, an instrument for flowing said sample in said fluidic device, an instrument for imaging said sample in said fluidic device, a controller for operating software for analysis of said imaging data. In some embodiments, the interrogation system comprises an integration of all or a sub-set of systems.

[0109] In some embodiments whereby a sample is contained within, or on, a fluidic device, operation of the device by the interrogation system can comprise: manipulating the physical position and conformation of the package, macromolecule, or long nucleic acid molecule via the application of external forces on said bodies; exposing the package, macromolecule, or long nucleic acid molecule to an environmental condition or reagent for a time period; optically interrogating the static or dynamic configuration of the package, macromolecule, or long nucleic acid molecule to facilitate analysis of their composition or as part of a feedback system to control operation of the device; extracting desired packages, macromolecules, or long nucleic acid molecules from the device. The fluidic device and interrogation system can interface in a number of ways. A non-exhaustive list includes: fluidic ports (both open andsealed), electrical terminals, optical windows, mechanical pads, heat pipes or sinks, inductance coils, fluid dispensing, surface scanning probes. A non-exhaustive list of potential functions the interrogation system may perform on the device include: temperature monitoring, applying heat, removing heat, applying pressure or vacuum to ports, measuring vacuum, measuring pressure, applying a voltage, measuring a voltage, applying a current, measuring a current, applying electrical power, measuring electrical power, exposing the device to focused and / or unfocused electromagnetic waves, collecting the electromagnetic waves light generated or reflected from the device, in far or near-fiend setting, creating and measuring a temperature, electromagnetic force, surface energy or chemical concentration differential or gradient, dispensing liquid into a device well or port, or on the device surface, contacting the device surface or entity on the device surface with a contact probe (for example: an AFM tip).

[0110] In some embodiments, confirmation of the presence of a macromolecule, long nucleic acid molecule, or package in a certain region of a fluid device and control over its physical position within said device is controlled by the interrogation system using a feedback controller system. Detection of the macromolecule, long nucleic acid molecule, or package is via detection of at least one interrogated signal. In the preferred embodiment, the signal is an electromagnetic signal originating from a labelling body bound to said macromolecule, long nucleic acid molecule, or package. In one embodiment, the control instrument feedback control system at least in part utilizes as input information the identification of a physical map profile within the macromolecule or long nucleic acid molecule, or absence of a physical map profile within the molecule.

[0111] In some embodiments, the interrogation system comprises localized computational processing modules within the system, adjacent computational processing modules via a direct communication connection, external computational processing modules via a network connection, or combination there-of. Various examples of computational processing modules include: a PC, a micro-controller, an application specific integrated micro-chip (ASIC), a field-programmable gate array (FPGA), a CPU, a GPU, System on Chip, a network server, cloud computing service, or combinations there-of.

[0112] The interrogation system may include at least one fluidic dispensing tip that Is capable of dispensing fluid drops at the desired x,y,z coordinates on the surface of the device, and in some embodiments, extracting fluid drops at the desired x,y,z coordinates on the surface of the fluidic device. Fluid dispensing and extracting may be in volumes of microliters, nanoliters, picoliters, femtoliters, or attoliters.

[0113] The interrogation system may be able to illuminate multiple light sources simultaneously, or in series, and be able to image multiple colors simultaneously, or in series. If imaging multiple colors simultaneously, this may be done on different cameras, on a single camera but different regions of the sensor array, or on the same sensor of the same camera. In some embodiments, the wavelength of light illuminated by the control instrument is chosen so as to interact with the sample, the sample labelling body, or a functionalized surface in some way. Non limiting examples include: photo-cleaving of the nucleic acid, photo-cleaving photo-cleavable linkers, manipulating optical tweezers, activating photo-activated reactions, de-protecting photolabile protecting groups, IR thermal heating.

[0114] Sequence. The term “sequence” or “nucleic acid sequence” or “oligonucleotide sequence” refers to a contiguous string of nucleotide bases and in particular contexts also refers to the particular placement of nucleotide bases in relation to each other as they appear in an oligonucleotide.

[0115] Similarly, the term “polypeptide sequence” or “amino acid sequence” refers to a contiguous string of amino acids and in particular contexts also refers to the particular placement of amino acids in relation to each other as they appear in a polypeptide.

[0116] Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina, Pacific Biosciences, Oxford Nanopore, Life Technologies (Ion Torrent), BGI, GenapSys, Element Biosciences, Singular Genomics Systems, Ultima Genomics. Sequencing can be performed by various technologies currently available, such as, without limitation: sanger sequencing, nanopore sequencing, nanogap sequencing, sequencing by ligation, sequencing by combinatorial probe anchor synthesis, sequencing by synthesis, pyrosequencing, polony sequencing, DNA nanoball sequencing, tunnelling current sequencing, sequencing by hybridization, sequencing by mass spectrometry, array-based sequencing, RNA polymerase based sequencing.

[0117] Structural variation. As used herein, “structural variation”, “structural variant”, or “SV” is the variation in structure of an organism’s chromosome with respect to a genomic reference. These variations include a wide variety of different variant events, including insertions, deletions, duplications, retrotransposition, translocations, inversions short and long tandem repeats, rearrangements, and the like. These structural variations are of significant scientific interest, as they are believed to be associated with a range of diverse genetic diseases. In general, the operational range of structural variants includes events > 50bp, while the “large structural variations” typically denotes events > 1,000 bp or more. The definition of structural variation does not imply anything about frequency or phenotypical effects.

[0118] Genomic information. The term “genomic information” or “genomic data” here includes any information content obtained directly or indirectly from the interrogation of a nucleic acid molecule that relates directly or indirectly to the underlying conventional genomic and epigenomic content of said molecule. Such information may include at least a portion of sequence information, the orientation (5-prime, 3-prime) of the molecule with respect to said molecule’s physical environment or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule. The physical position of a base, or sequence with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule. The physical position of a structural variant with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule. The physical position of a higher order nucleic acid structure with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule. The physical position of epigenetic data with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule. The physical position of epigenetic data with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule. The physical position of a labelling body bound to said molecule with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one additional labeling body bound to said molecule. The physical position of a body bound to said molecule with respect to said molecule’s physical environment, or the molecule itself, where the positional reference within the molecule may be physical length, sequence, a physical map along the molecule, or at least one labeling body bound to said molecule.

[0119] Examples can include the relative position of a gene within a molecule as identified by an analysis of a physical map alignment to a reference along the length of said molecule measured in base-pairs, or the relative position of a cohesin loop along the length of saidmolecule measured in physical length distance, or the relative position of a methylation pattern with respect to the underlying sequence of the molecule.

[0120] In some embodiments, the genomic information may be the relative position of at least two independent portions of genomic information with respect to each other within the molecule, or some other physical reference location or fiducial. For example, the relative position of a TAD with respect to a labelling body within the molecule, or some other physical reference location or fiducial.

[0121] Elongated Nucleic Acid. The majority of linear physical mapping methods that use fluorescent imaging or electronic signals to extract a signal related to the underlying genomic, structural, or epigenomic content employ some form of method to at least locally ‘elongate’ the long nucleic acid molecule such that the resolution of the physical mapping in the region of elongation can be improved, and disambiguates reduced. A long nucleic acid molecule in its natural state in a solution will form a random coil. Thus, a variety of methods have been developed to ‘uncoil’ and elongate the molecule.

[0122] By binding a portion of long nucleic acid molecules on a functionalized solid surface, the molecule is elongated by flowing a solution and ultimately pulled taut, coming into full contact with the substrate surface [US Patent 7,368,234” Physical mapping method using molecular combing technique allowing positioning of a great number of clones within a genome”], a technique typically called ‘combing’ DNA. Alternatively, there are other long polymer elongation methods such as fluid flow induced elongation with ends anchoring on surface [Gibb, 2012], aqueous solution hydrodynamic focusing by laminar flows [US Patent 6,696,022 ”Methods and apparatuses for stretching polymers”], linearization by confining nanochannels [Wang, 2005], long nucleic acid molecules in microfluidic device pulled by two angled opposing externally applied forces in a presence of physical obstacle features [Volkmuth, 1992], molecules hydrodynamically trapped in a fluidic device by simultaneously exposed to two opposing externally applied forces [Tanyeri, 2011].

[0123] Most of the time, the elongation state of at least a portion of the long nucleic acid molecule has to be sustained by an external force before otherwise returning to its natural random coiled state, unless at least a portion of the nucleic acid is retained in the elongated state by physical confinement without a sustaining external force [Dai, 2016].

[0124] Unless specifically stated otherwise, an ‘elongated’ or ‘partially elongated’ nucleic acid is a long nucleic acid fragment for which at least one segment of the major axis of the molecule comprising at least 1kb can be projected against a 2D plane, and does not overlap with itself. For clarity, for embodiments where-by long nucleic acid includes additionalstructure, for example as when the nucleic acid is contained in chromatin, compacted with histones, the major axis refers to the larger chromatin molecule, not the nucleic acid strand contained within. In another example, for embodiments where-by the long nucleic acid molecule is a chromosome or portion of a chromosome, the major axis refers to the larger body of the chromosome, such as along a chromosome arm, not the nucleic acid strand contained within. Therefore statements in this disclosure such as “along the length of the molecule” when referring to long nucleic acid molecules, refers to along the length of the major axis.

[0125] Binding. “Binding”, “bound”, “bind” as used herein generally refers to a covalent or non-covalent interaction between two entities (referred to herein as “binding partners”, e.g., a substrate and an enzyme or an antibody and an epitope). Any chemical binding between two or more bodies is a bond, including but not limited to: covalent bonding, sigma bonding, pi ponding, ionic bonding, dipolar bonding, metallic bonding, intermolecular bonding, hydrogen bonding, Van der Waals bonding. As “binding” is a general term, the following are all examples of types of binding: “hybridization”, hydrogen-binding, minor-groove-binding, major-groove-binding, click-binding, affinity-binding, specific and non-specific binding. Other examples include: Transcription-factor binding to nucleic acid, protein binding to nucleic acid.

[0126] Substrate. As used herein, the term “substrate” is intended to mean a solid or semi- solid support that can serve as the foundation for the definition of features. Non limiting examples of features include wells, immobilized molecules, pillars, channels, pits. The features can randomly positioned on the substrate, or patterned. A substrate as provided herein can be modified to accommodate attachment of biopolymers by a variety of methods well known to those skilled in the art. Exemplary types of substrate materials include glass, modified glass, functionalized glass, inorganic glasses, silicon, silicon di-oxide, silicon nitride, quartz, fused silica, microspheres, including inert and / or magnetic particles, polysaccharides, nitrocellulose, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene polycarbonate, or combinations thereof.

[0127] Those skilled in the art will know or understand that the composition and geometry of a substrate as provided herein can vary depending on the intended use and preferences of the user. Therefore, although planar substrates such as slides, chips or wafers are often exemplified herein, given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of other substrates exemplified herein or well known in the art also can be used in the methods and / or compositions herein.

[0128] The substrate may comprise of multiple substrates that are physically connected, for example using any combination of bonding mechanism, an adhesive, a film, a vacuum. The substrate may include various combinations of coatings. The substrate may have a patterned surface. The patterning may be additive or subtractive in nature, or combination of both. The substrate may comprise a component of a microfluidic device or a flow-cell. A substrate may be a film, which itself, may be in contact with another substrate.

[0129] A substrate can be of any desired shape. For example, a substrate can be typically a thin, flat shape (e.g., a square or a rectangle or oval). In some embodiments, a substrate structure has rounded corners (e.g., for increased safety or robustness). In some embodiments, a substrate structure has one or more cut-off corners (e.g., for use with a slide clamp or cross-table). In some embodiments, where a substrate structure is flat, the substrate structure can be any appropriate type of support having a flat surface (e.g., a chip or a slide such as a microscope slide).

[0130] In some embodiments where the substrate is modified to contain one or more features, including but not limited to, wells, projections, ridges, features, or markings, the features can include physically altered sites. For example, a substrate modified with various features can include physical properties, including, but not limited to, physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, surface energy altered sites, hydrophobic or hydrophilic altered sites, and / or electrostatically altered sites.

[0131] In some embodiments, a substrate includes one or more markings on the surface of the substrate, for example to provide guidance for correlating spatial information with the characterization of interest. For example, to allow the size of objects seen under magnification to be easily estimated and / or to provide reference areas for counting objects. In some embodiments, fiducial markers can be included on a substrate. Such markings can be made using techniques including, but not limited to, printing, etching, sand-blasting, and depositing on the surface.

[0132] In some embodiments, a fiducial marker can be present on a substrate to provide orientation of the sample with features on the substrate, or the substrate itself.

[0133] Functionalized Surface. A “functionalized” surface is a surface of a substrate that has been modified or engineered such as by certain chemicals, or macromolecules to elicit certain desired properties. For example: to bind specifically or non-specifically to a macromolecule, or to provide a reagent.

[0134] Immobilized. As used herein, the term “immobilized” when used in reference to a molecule, macromolecule, or body in direct or indirect attachment to a substrate via covalent or non-covalent bond(s) or stationery state by physical confinement, local energy minimization, local entropy minimization, or held stationery by an external force. Indirect attached to the substrate may be via at least one additional intermediary molecule or body. In certain embodiments, covalent attachment can be used, but all that is required is that the molecules, macromolecules, or bodies remain co-localized to the substrate under conditions in which it is intended to use. Non limiting examples include the entire molecule, macromolecule, or body may be held stationary with respect to the substrate, or a portion of the molecule, macromolecule, or body held stationary with respect to the substrate, while the remainder of the molecule, macromolecule, or body has limited freedom of movement, or the molecule, macromolecule, or body is indirectly attached to the substrate via an intermediary, and the entire molecule, macromolecule, or body has some limited freedom of movement. For example, immobilization of an oligonucleotide to a substrate can occur via hybridization of said oligonucleotide to a secondary oligonucleotide, said secondary oligonucleotide at least partially containing a complementary sequence to the first, and itself immobilized to the substrate.

[0135] In certain embodiments, the molecule, macromolecule, or body may be immobilized on a surface via a process that comprises physisorption, hydrogen bonding, van der waals forces, or stiction.

[0136] In certain embodiments, the molecule, or macromolecule can comprise biomolecules, nucleic acid molecules, proteins, peptides, nucleotides, or any combination thereof. In certain embodiments, the body can comprise cells, droplets, dendrimers, packages, nucleosomes, beads, nanoballs, nanoparticles, nanodots, quantum dots.

[0137] Certain embodiments may make use of a substrate which has been functionalized, for example by application of a layer or coating of an intermediate material comprising reactive groups which permit covalent attachment to biomolecules, such as polynucleotides. Examples of such substrates include, but are not limited to, polyacrylamide hydrogels supported on an inert secondary substrate such as glass, particularly polyacrylamide hydrogels as described in [WTO Patent Publication WO 2005 / 065814 “Modified Molecular Arrays”] and [US PatentPublication 2008 / 0280773 “Method of Nucleotide Detection”], the contents of which are incorporated herein in their entirety by reference. In such embodiments, the biomolecules (e.g. polynucleotides) may be directly covalently attached to the intermediate substrate (e.g. the hydrogel) but the intermediate substrate may itself be non-covalently attached to the secondary substrate.

[0138] Exemplary bonding examples include click chemistry techniques, non-specific interactions (e.g. hydrogen bonding, ionic bonding, van der Waals interactions etc.) or specific interactions (e.g. affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). Exemplary bonding mechanism are set forth in: [US Patent 6,737,236 “Bioconjugation of macromolecules”], [US Patent 7,259,258 “Methods of attaching biological compounds to solid supports using triazine”], [US Patent 7,375,234 “Copper- catalysed ligation of azides and acetylenes”], [US Patent 7,427,678 “Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method”], and [US Patent Publication 2011 / 0059865 “Modified Molecular Arrays”], each of which is incorporated herein by reference.

[0139] Surface Energy. Surface tension of a fluid is the energy parallel to the surface that opposes extending the surface. Surface tension and surface energy are often used interchangeably. “Surface energy” is defined here as the energy required to wet a surface. To achieve optimum wicking, wetting and spreading, the surface tension of a fluid is decreased and is less than the surface energy, of the surface to be wetted. The wicking movement of a fluid through the channels of a fluid device occurs via capillary flow. Capillary flow depends on cohesion forces between liquid molecules and forces of adhesion between liquid and walls of channel. The Young / Laplace Equation states that fluids will rise in a channel or column until the pressure differential between the weight of the fluid and the forces pushing it through channel are equal. [Moore, 1962]

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[0145] ZKLFK^GHVFULEHV^WKH^UHODWLRQVKLS^EHWZHHQ^WKH^FRQWDFW^DQJOH^^^DQG^VXUIDFH^WHQVLRQ^RI^ liquid-YDSRU^LQWHUIDFH^Ȗ / 9^^WKH^VXUIDFH^WHQVLRQ^RI^WKH^VROLG-YDSRU^LQWHUIDFH^Ȗ69^^DQG^VXUIDFH^ tension of the liquid-YDSRU^LQWHUIDFH^Ȗ6 / ^^:KHQ^WKH^FRQWDFW^DQJOH^^^EHWZHHQ^OLquid and solid is zero or so close to 0, the liquid will spread over the solid. A contact angle measurement test is used as an objective and simple method to measure the comparative surface tensions of solids. In general, a material is considered to be hydrophilic when the contact angle in this test is below 90°. If the contact angle is above 90°, the material is considered to be hydrophobic.

[0146] Fluidic Device. The term “microfluidic device” or “fluidic device” or “chip” as used herein generally refers to a device configured for fluid transport and / or transport of bodies through a fluid, and having a fluidic channel in which fluid can flow with at least one minimum dimension of no greater than about 100 microns. The minimum dimension can be any of length, width, height, radius, or cross-sectional axis. A microfluidic device can also include a plurality of fluidic channels. The dimension(s) of a given fluidic channel of a microfluidic device may vary depending, for example, on the particular configuration of the channel and / or channels and other features also included in the device.

[0147] A fluidic device may comprise any device that can contain milliliters volume (or less) of fluid. Physical boundaries that contain the fluid may be not necessarily be all solid, and can also include gas and / or secondary liquid boundaries. In some embodiments, the microfluidic device can be exposed to a large volume of fluid during at least some portion of the operation, whereby the total volume exceeds milliliter scale. Non limiting examples include submerging the device in a reservoir, or submerging the device in a bath of liquid, or flowing large volumes of liquid through the device, or flowing large volumes of liquid over the device.

[0148] It should be understood that some of the principles and design features described herein can be scaled to larger devices and systems including devices and systems employing channels and features reaching the millimeter or even centimeter scale channel cross-sections. Thus, when describing some devices and systems as “microfluidic,” it is intended that the description apply equally, in certain embodiments, to some larger scale devices. In addition,it should be understood that some of the principles and design features described herein can be scaled to smaller devices and systems including devices and systems employing channels and features that are 100s of nanometers, or even 10s of nanometers, or even single nanometers in scale channel cross-sections. Thus, when describing some devices and systems as “microfluidic,” it is intended that the description apply equally, in certain embodiments, to some smaller scale devices. As an example, a device may have input wells to accommodate liquid loading from a pipette that are millimeters in diameter, which are in fluidic connection with channels that are centimeters in length, 100s of microns wide, and 100s of nm deep, which are then in fluidic connection with nanopore constriction devices that are 0.1-10 nm in diameter.

[0149] Microfluidic devices described herein can also include any additional components that can, for example, aid in regulating fluid flow, such as a fluid flow regulator (e.g., a pump, a source of pressure, etc.), features that aid in preventing clogging of fluidic channels (e.g., funnel features in channels; reservoirs positioned between channels, reservoirs that provide fluids to fluidic channels, etc.) and / or removing debris from fluid streams, such as, for example, filters. Moreover, microfluidic devices may be configured as a fluidic chip that includes one or more reservoirs that supply fluids to an arrangement of microfluidic channels and also includes one or more reservoirs that receive fluids that have passed through the microfluidic device. In addition, microfluidic devices may be constructed of any suitable material(s), including polymer species and glass, or channels and cavities formed by multi- phase immiscible medium encapsulation. Microfluidic devices can contain a number of microchannels, valves, pumps, reactors, mixers and other components for producing and manipulating (merging, sorting, splitting, etc.) the droplets. Microfluidic devices may contain active and / or passive sensors, electronic and / or magnetic devices, integrated optics, or functionalized surfaces. The physical substrates that define the microfluidic device channels can be solid or flexible, permeable or impermeable, or combinations there-of that can change with location and / or time. Microfluidic devices may be composed of materials that are at least partially transparent to at least one wavelength of light, and / or at least partially opaque to at least one wavelength of light.

[0150] A microfluidic device is typically designed and operated to manipulate a sample contained in a solution in order to achieve some desired outcome. The sample may be loaded into the device manually, for example using a pipette, or in an automated fashion, for example via an automated liquid handling system. The sample may further be introduced by exposing the microfluidic device to a fluidic stream. In addition, various other solutionsincluding buffers and reagents may be added simultaneously with the sample input, or separately, at different input ports and / or different time points. In some cases, the microfluidic device is manufactured with liquids and reagents contained within.

[0151] A microfluidic device can be fully independent with all the necessary functionality to operate on the desired sample contained within. The operation may be completely passive, such as with the use of capillary pressure to manipulate fluid flows [Juncker, 2002], or may contain an internally power supply such as a battery. Alternatively, the fluidic device may operate with the assistance of an external device that can provide any combination of power, voltage, electrical current, electrical waveform, magnetic field, pressure, vacuum, light, heat, cooling, sensing, imaging, digital communications, encapsulation, acceleration forces, movement of one part of the microfluidic device relative to another, movement of a fluidic meniscus across at least a portion of the microfluidic device, sonic or ultrasonic energy, environmental conditions, etc. The fluidic device may also operate by chemical energy supplied by one or more fluids, solids or gasses applied to the device, such as energy from decomposition, combustion, chemical reaction of one more fluids solids or gasses, energy of mixing, hydration, solvation or desolvation of solids, fluids, and or gasses, wetting or meniscus forces, energy of phase transition from one state to another such as energy of vaporization or fusion. The external device maybe a mobile device such as a smart phone, a microcontroller based device, embedded computing platform, a larger instrument, with or without direct human control.

[0152] In some embodiments, the fluidic device includes an “electrowetting device” or “droplet microactuator”, which is a type of microfluidic device capable of controlled droplet operations within the fluidic device via specific application of local electric fields. Non limiting examples of such devices include a liquid droplet surrounded by air on an open surface, and a liquid droplet surrounded by oil sandwiched between two surfaces. A detailed review of the various configurations of use, and physics of droplet control are provided by [Mugele, 2005] and [Zhao, 2013], both of which are provided here for reference.

[0153] Unless specifically stated otherwise, a “fluidic feature” is a feature within or on the fluidic device with a property that comprises the ability by itself, or conjunction with other fluidic features, to at least partially contain, directly or indirectly, a liquid or body within said fluidic feature(s), or a property that comprises the ability to interact with a body. Examples of bodies include macromolecules, cells, nucleus, nucleosomes, microconidia, DNA, chromosomes, ecDNA, circular polynucleotides, proteins, enzymes, nucleotides,carbohydrates, RNA, beads, dendrimers, nanoparticles, lipids, nanoballs, quantum dots, Pickering emulsion, and droplets.

[0154] In some embodiments, the fluidic feature’s interaction with the with a biological body may comprise: shear-force, or bonding, or repulsion, or attraction, puncture, compression, extrusion, expansion, electrochemical reaction, or charge exchange. In some embodiments, the fluidic feature comprises a physical obstacle.

[0155] In some embodiments the fluidic feature may comprise a channel, a wall, a corner, an edge, a barrier, a physical obstacle, a pillar, a pit, a cone, a rod, a sphere, a partial sphere, an oval, a circle, a polygon, a cylinder, a mound, a hill, a trough, a cube, a parallelogram, an ellipse, a triangle, a trapezoid, a trapezium, a crescent, a cuboid, an ellipsoid, a triangular prism, a pyramid, a tetrahedron, an octahedron, a line, a dash, a dot, a torus, a prism, a polyhedron. A fluidic feature may comprise a combination or permutation of at least two fluidic features.

[0156] In some embodiments, the fluidic feature may comprise a region with a desired surface energy property, or desired surface hydrophobicity property, or desired surface hydrophilicity property, or desired anti-fouling property, or desired surface-roughness property, or desired surface-smoothness property.

[0157] In some embodiments, an antifouling property or behavior includes preventing the adhesion of an intact package, semi-intact package, macromolecule, or molecule to at least a portion of the surface with said property. In some embodiments, the antifouling property is further influenced by the chemical composition of the delivery fluid or at least one environmental condition. The optimum antifouling surface treatments for the various classes of intact packages, semi-intact packages, macromolecules, and molecules are not necessarily the same, and the effects of a particular antifouling coating can be influenced by prior treatment of the package, macromolecule, or molecule.

[0158] In some embodiments, a surface roughness property is correlated with the preventing or promoting of the adhesion of an intact package, a semi-intact package, a macromolecule, or a molecule to at least a portion of the surface with said property. In some embodiments the roughness is more than 0.1 nm rms, or more than 0.2 nm rms, or more than 1 nm rms, or more than 2 nm rms, or more than 5 nm rms, or more than 10 nm rms, or more than 20 nm rms, or more than 50 nm rms, or more than 100 rms, or more than 200 nm rms, or more than 500 rms, or more than 1000 nm rms. In some embodiments the surface roughness is selected to achieve a certain hydrophobic property. In some embodiments, the surface roughness is selected to achieve a certain hydrophilic property. In some embodiments, the surfaceroughness is selected to achieve a certain anti-fouling property. In some embodiments, the surface roughness is tuned to achieve a certain adhesion property.

[0159] The containment or flow of a fluid or body “within the fluidic feature(s)” of the fluidic device can be by any means in which at least a portion of the fluid or body can be maintained within, among, along, or on, at least a portion of at least one fluidic feature defined within or on the fluidic device for a period of time.

[0160] In some embodiments, at least a portion of the fluid is contained by the solid or semi- solid physical boundaries of the channel walls. Figure 3 shows embodiments where-by channel walls with cross-sections such as rectangles (302), triangles (303), ovals (304), and mixed geometry (305) are all defined within a fluidic device (301). In other embodiments, fluidic containment within the fluidic device may be at least partially contained via solid physical features in combination with surface energy features [Casavant, 2013], or an immiscible fluid [Li, 2020]. Examples of a fluid being at least partially confined within physical boundaries include various channels physically defined on the surface of a fluidic device (306) such as grooves (307, 308) and rectangles (309, 310), all of which can contain at least a portion of a fluid via surface tension. In other embodiments, the channel (311) could be a defined by a groove in a corner (312) of a fluidic device, or the channel (314) could be defined by two physically separated boundaries (313 and 315) of a fluidic device, or the channel (321) could be defined by a corner (320) of a fluidic device. In other embodiments, the channel (317) is defined by a hydrophilic section (318) on the surface of a fluidic device (316) where-by the hydrophilic section is bounded by hydrophobic sections (319) on the surface of the fluidic device. In all cases, these embodiments are non-limiting examples and additional embodiments include combinations thereof. Used herein, an “open fluidic device” is a fluidic device that comprises at least one fluidic feature (for example: a channel) in which the solution in said fluidic feature is at least partially exposed to a gas-phase interface. Examples include air, water vapor, solvent vapor, oxygen, nitrogen, or mixtures thereof. In particular, with regards to the operation of an open fluidic device, the selection of the gas composition, pressure, and other environmental conditions may be controlled, and may be critical to the desired operation of the open fluidic device. For example, for a particular period of time, a particular temperature, or humidity, or dew point, or solvent vapor partial pressure, or wavelength exposure may be desired.

[0161] A multitude of fluidic features with desired surface property regions are possible. Non-limiting examples of surface properties include a degree of hydrophobicity, degree of hydrophilicity, or degree of anti-fouling, or degree of roughness, or degree of smoothness, orsurface charge density, or surface functionalization, or surface energy. In some embodiments, a fluidic feature may comprise one region with a desired surface property. In some embodiments, a fluidic feature may comprise at least one region with a desired surface property. In some embodiments, a fluidic feature may comprise a multitude of regions, each with a specific desired surface property.

[0162] Figure 4 demonstrates some embodiments of fluidic features with selective patterning of different desired surface properties of type “A” (402) and type “B” (403) on the surface of a fluidic device (401). The fluidic features defined on the fluidic device may take on any shape. In addition, any surface region on any said shape, including the entirety or portion of a surface plane, may comprise a desired surface property. For example, fluidic features comprising of open fluidic rectangular cross-section channels (404, 405, 406) may have different regions with a surface property “B” inside the channel, and surface property “A” outside the channel. In another example, fluidic features comprising of open fluidic triangular cross-section channels (407, 408, 409) may have different regions with a surface property “B” inside the channel, and surface property “A” outside the channel.

[0163] There are numerous different methods for modifying the surface energy properties or anti-fouling properties of a surface. For example, by contact transfer stamping of a hydrophobic or anti-fouling molecule to the glass fluidic device surface, leaving the naturally hydrophilic open and depressed channels on the fluidic device surface un-modified. Other methods may comprise spin coating a film, dip coating a film, growing a film, depositing a film, printing a film, dispensing a film, spraying a film, self-assembling a monolayer. Modification may comprise the removal of a certain surface property or material. For example, the removal of a film, a monolayer, a polymer, a material. Modification may comprise a change in the physical or chemical structure of a certain region. For example, by selective exposure to a plasma, a UV light, an ozone gas. Modification of the surface may be selective to certain regions, wherein the modified regions are selected by the means of modification, or their physical position relative to other regions, or use of a pattering technology such as a mask or direct write tool, or use of a temporary sacrificial material. Modification of the surface may be selective to certain regions, wherein the modified regions are selected by the means of chemistry, such that only materials of certain surface chemistry properties will participate in the modification process. Modification of the surface may be selective to certain regions that comprise a seed, or additional material that comprises a surface property that allows for selective modification.

[0164] Transfer contact printing (or micro-contact printing) is a widely used method of transferring a molecule from a donor substrate to an acceptor substrate. The most common donor substrates are typically flexible elastomers that provide some degree of compliance with the acceptor substrate. For a review of different micro-contact methods and materials refer to [Smith, 2004], [Lamping, 2019], and [Qiu, 2021]. In some embodiments the transferred molecules form a monoloyer on the acceptor substrate, or a bilayer, or a multi- layer, or a concatemer, or a film. In some embodiments, multiple contact printing steps may be employed.

[0165] In some embodiments the surface modifications modulate the hydrophobicity of a portion of the fluidic device, and work in concert with topological features to direct fluidic flow, specifically the flow of an interface that separates phases, most preferably the meniscus between a fluid and a gas phase. Surface modifications with a high degree of hydrophobicity are not limited to the class of molecules with one or more pendant halogenated chains such as poly(heptadecafluorodecyl acrylate), Octadecyltrichlorosilane or 1H,1H,2H,2H- Perfluorodecylamine. Surface modifications with a high degree of hydrophobicity are not limited to the class of molecules with aliphatic groups such as lauryl methacrylate, Octadecyltrimethoxysilane, or Dodecylamine.

[0166] In some embodiments, the hydrophobicity of the surface modifications applied to a portion of the device can be altered by mild reaction or environmental conditions. The adaptable hydrophobicity surface modifications are not limited to polymers, present as homopolymers, copolymers or block-co-polymers, made from labile or cleavable groups that include (Trimethylsilyl)methacrylate, t-BOC protected amines such as N-(3-BOC- aminopropyl)methacrylamide, 3-[(2-Aminoethyl)dithio]propionic acid, enzymatically cleavable phenylalanine rich peptides conjugated to polyacrylic acid, and polymers decorated with terminal nitrobenzyl groups that are further conjugated to fluorinated alkanes. The adaptable hydrophobicity surface modifications are not limited to the group of thermoresponsive polymers that includes cellulose nanocrystals, Poly(N- isopropylacrylamide), Pluronic F127, poly-(organophosphazene), and copolymers of the above including poly(N-isopropylacrylamide) copolymerized with poly(ethylene glycol).

[0167] In some embodiments, the surface modifications add a chemical moiety that interacts with a portion of the package or with a portion of the macromolecules. Further, surface modifications can be combined with one or more of the above strategies for dynamic modification of the surface modifications. The surface modifications are not limited to polymers and molecules containing moieties that are multivalent, including linear andbranched polyamines and including spermidine and spermine moieties, peptides rich in charged amino acids including lysine, glutamic acid, arginine, histidine and aspartic acid, oligonucleotides, homopolymers of charged amino acids, copolymers, graft polymers, and co-block polymers of charged amino acids, polyacrylic acid, poly(methacryol-l-lysine), poly(ethyleneimine), poly(2-Aminoethyl methacrylate), poly(ethylene glycol), poly(N-[3- (N,N-Dimethylamino)propyl] acrylamide), poly(2-(tert-Butylamino)ethyl methacrylate), poly(2-Diisopropylaminoethyl methacrylate).

[0168] The surface modifications include and are not limited to molecules, polymers and macromolecules that react to form covalent bonds with packages and macromolecules, as homopolymers, copolymers or co-block polymers, either self-reactive or reactive in combination with chemical crosslinkers such as formaldehyde, paraformaldehyde and EDC / NHS, the group including Poly(ethylene glycol) diamine, Cinnamyl methacrylate, poly(glycidyl methacrylate), poly(propargyl methacrylate), poly(2-Aminoethyl methacrylate).

[0169] The surface modifications include and are not limited to molecules, polymers and macromolecules that interact specifically with the membranes, lipid bilayers or other such components that form the envelope of packages or interact with the meniscus separating multiple fluidic or gaseous phases. These surface modifications, whether as homopolymers, copolymers, graft polymers, or co-block polymers, are not limited to pendant phospholipids and aromatic groups added to the end of a polymer including 1-decyne added to a NaN3 substituted ATRP polymer end group via a Cu catalyzed cycloaddition.

[0170] In some embodiments, the modification is a “grafting to” reaction that adds a small molecule, polymer or macromolecule to at least a portion of the fluidic device or a fluidic feature. Some embodiments utilize the reaction of silanes to glass, silicon or oxide surfaces, the silanes including but not limited to aminopropyl (3-Aminopropyl)triethoxysilane, mPEG5K-Silane and 3-(Trimethoxysilyl)propyl acrylate. In some embodiments, non- covalent interactions are formed with surface modifying chemicals including poly-l-lysine, most preferably with co-block polymers including the family of Pluoronic detergents, Tween 20 and Tween 80 and others described in this document.

[0171] A variety of materials and methods, according to certain aspects of the device or method, can be used to form articles or components such as those described herein, e.g., channels such as microfluidic channels, chambers, etc. For example, various articles or components can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, bonding techniques, depositiontechniques, lamination techniques, molding techniques, imprinting techniques, 3D printing techniques, Fused deposition modeling 3D printing, stereolithography 3D printing, selective laser sintering 3D printing, etching methods including wet chemical or plasma processes, multi-phase immiscible medium encapsulation and the like. For patterning, a variety of methods may be employed, including but not limited to: photolithography, electron-beam lithography, nanoimprint lithography, AFM lithography, STM lithography, laser writer, focused ion-beam lithography, stamping, embossing, molding, and dip pen lithography. For bonding, a variety of methods may be employed, including but not limited to: thermal bonding, adhesive bonding, pressure bonding, vacuum bonding, surface activated bonding, fusion bonding, anodic bonding, plasma activated bonding, laser bonding, and ultra sonic bonding.

[0172] In one set of embodiments, various fluidic features, structures, or components of the articles described herein can comprise of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE” or Teflon®), or the like. For instance, according to one embodiment, a microfluidic channel may be implemented by fabricating the fluidic system separately using PDMS or other soft lithography techniques [Xia, 1998; Whitesides, 2001].

[0173] Other examples of potentially suitable polymers include, but are not limited to, polyethylene terephthalate (PET), polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyethylene, polypropylene, polyvinylchloride, cyclic olefin copolymer (COC), cyclo-olefin polymer (COP), polytetrafluoroethylene, a fluorinated polymer, a silicone such as polydimethylsiloxane, polyvinylidene chloride, bis-benzocyclobutene (“BCB”), a polyimide, a fluorinated derivative of a polyimide, nylon, or the like. Combinations, copolymers, or blends involving polymers including those described above are also envisioned.

[0174] In some embodiments, various structures or components of the article are fabricated from polymeric and / or flexible and / or elastomeric materials, and can be conveniently formed of a phase changing fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The phase changeable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and / or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the phase changeable fluid comprises a polymeric liquid or a liquid polymeric precursor (for example: a “prepolymer”). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, waxes, metals, or mixtures orcomposites thereof heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use according to the device or method include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, dodecyltrichlorosilanes, etc.

[0175] Silicone polymers are used in certain embodiments, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of various structures of the device or method. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65° C. to about 75° C. for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the device or method. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

[0176] One advantage of forming structures such as microfluidic structures or channels from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials. Thus, structures can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). Oxidation and sealing methods useful in the context of the present device or method, as well as overall molding techniques, are described in the art, for example: [Duffy, 1998].

[0177] The device may comprise glass, silicon, silicon nitride, silicon oxide, quartz, metal, fused silica, mica, ceramics, aluminum, BK7 glass, linbo3, borofloat 33 glass, SD2 glass, 7740 pyrex glass, flexinity® glass, D263 glass, AF32 glass, mempax glass, B270 glass, AS87 ECO glass, foturan II glass, flexible glass, Schott UTG glass, flat planel display glass, KG2 glass, KG3 glass, NG11 glass, BG63 glass, EAGLE XG® glass, Astra™ glass, Lotus™ NXT glass, gallium arsenide, germanium, graphene, ITO, litao3, nitride, polysilicon, sapphire, silicon carbide, silicon on insulator, soda lime glass, InP, Polymers, Plastic, Polycarbonate (PC), PMMA, Acrylic, Polyethylene Terephthalate, Amorphous Copolyester (PETG), Polyvinyl Chloride (PVC), Liquid Silicone Rubber (LSR), Cyclic Olefin Copolymers (COC), Polyethylene (PE), Ionomer Resin, Polypropylene (PP), Fluorinated Ethylene Propylene (FEP), Styrene Methyl Methacrylate (SMMA), Styrene Acrylonitrile Resin (SAN), Polystyrene, Methyl Methacrylate Acrylonitrile Butadiene Styrene (MABS), acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), polyethylene terephthalate glycol (PETG), Nylon, thermoplastic polyurethane (TPU), polyvinyl alcohol (PVA), high impact polystyrene (HIPS), Resins, Polyurethan Resins, Elastic Resins, ESD Resins, Ceramic Resins, Nylon 12, Nylon 11, TPU, Titanium, Stainless steel, Nickel alloys, gel, hydrogel, xerogel, colloidal gels, organolgels, thermoplastics, thermosetting plastics.

[0178] The device may comprise filter papers, parafilm, permanent maker inks, or wax.

[0179] The device may comprise a patternable material, in particular, a material comprising a patternable polymer, or a patternable organic polymer, or a patternable inorganic polymer, ora patternable gel. In some embodiments, the patternable material may comprise a photo- patternable material, or an electron-patternable material, or material capable of being embossed, or a material capable of being imprinted, or a material capable of being 3D printed, or a material capable of being dispensed from a dispensing system, or a material capable of being patterned with ion beam lithography, or a material capable of being patterned with soft-lithography, or a material capable of being patterned with colloidal lithography, or a material capable of being patterned by self-assembly methods, or a material capable of being patterned by area-selective deposition (ASD), or a material capable of being patterned by micro-contact printing, or a material capable of being patterned by casting, or a material capable of being patterned by lamination, or a material capable of being patterned by roll-to-roll processing, or a material capable of being patterned by microthermoforming, or a material capable of being patterned by micro-molding in capillaries (MIMIC), or a material capable of being patterned by capillary force lithography (CFL), or a material capable of being patterned by scanning probe lithography (SPL), or a material capable of being patterned by micro-cutting, or a material capable of being pattered by ultrasonic machine, or a material capable of being patterned by laser ablation, or a material capable of being patterned by plasma etching, or a material being capable of bio-templated assembly, or a material capable of being patterned by electro-discharge machining (EDM), or a material capable of being patterned by micro-electrochemical machining (ECM), or a material capable of being patterned by X-ray LIGA. For a non-limiting review of several non- photolithography methods of patterning materials, in particular polymers, see [Qiu, 2023], [Acikgoz, 2011] and [Scott 2021], in particular 3D methods see [Yazdi, 2016].

[0180] In some embodiments whereby the patternable material is a patternable polymer, the patternable polymer may comprise any of the previously listed polymer materials.

[0181] In some embodiments whereby the patternable material is a photo-patternable material, the photo-patternable material may comprise a photoresist. In some embodiments, the photoresist is a negative photoresist, or a positive photoresist, or a light-curable resist, or a UV-curable resist. Examples of different photo-patternable photoresists include DNQ (diazonaphthoquinone) and novolac resin based resists, epoxy-based resists, Off- stoichiometry thiol-enes (OSTE) polymer resists, and hydrogen silsesquioxane (HSQ) based resists. In some embodiements the photo-patternable polymer is based on FOX-16 or its derivatives, or based on poly(methyl methacrylate) (PMMA) or its derivatives, or based on poly(methyl glutarimide) (PMGI) or its derivatives, or OSTE polymers or its derivatives, or based on Ma-N photoresists, or Shipley photoresists, or SPR photoresists. In someembodiments the photoresist comprises sensitivity for G-line, or I-line, or KrF, or ArF, or EUV, or electron beam. In some embodiments the photo-patternable polymer may comprise any Dupont SPR series of photoresists, or any Dupont MCPR series of photoresists, or any Dupont UV series of photoresists, or any Dupot UVN series of photoresist, or any Dupont LR series of photoresists, or any Dupont EPIC series of photoresists, or any Microchem AZ series of photoresist, or any Microchem TI series of photoresists, or any Microchem SU8 series of photoresists, or any Microchem Ordyl series of photoresists, of any microresist ma- N series of resists, or any microresist mr-DWL series of resists, or any microresist EpoCore series of resists, or any microresist EpoClad series of resists, or any microresist ma-P series of resists, or any microresist mr-P series of resists, or any microresist InkEpo series of resists, or any microchem InkOrmo series of resists, or any microchem SUEX series of resists, or any microresists ADEX series of resists, or any microresist UV series of resists, or any microresist microposit series of resists, or any microresist Ultra-I series of resists, or any microresist microposit LOL series of resists, or any microresist cyclotene series of resists, or any microresist intervia series of resists, or any microresist MX series of resist, or any microresist WBR series of resists, or any microresist SU-8 series of resists, or any microresist KMPR series of resists, or any microresist perminex series of resists, or any microresist KMSF series of resists, or any microresist LOR series of resits, or any Futurrex NR series of photoresists, or any Futurrex PR series of photoresists, or any Sumitomo PFI series of photoresist, or any Sumitomo PFM series of photoresists, or any Sumitomo PXi series of photoresists, or any Sumitomo NX series of photoresists, or any Sumitomo PEK series of photoresists, or any Sumitomo PAR series of photoresists, or any Tok TSMR series of photoresists, or any Tok THMR-iP series of resists, or any ToK TDMR-AR series of photoresists, or any ToK OFPR series of photoresists, or any Tok OEBR-CAP series of photoresists, or any ToK TFR-Di series of photoresists, or any Fujifilm OiR series of photoresists, or any Fujifilm GiR series of photoresists, or any Fujifilm HiPR series of photoresists, or any Fujifilm OiR series of photoresists, or any Fujifilm SC series of photoresists, or any Fujifilm IC series of photoresists, or any Fujifilm HNR series of photoresists, or any Fujifilm HR series of photoresists, or any Fujifilm GKR series of photoresists, or any Fujifilm GAR series of photoresists, or any Fujifilm FAiRS series of photoresists. In some embodiments, the photo-patternable polymer may comprise polymer resins, photoactive compounds, photoactivator compounds, photoinitiator compounds, detection additives, resist solvent, crosslinking agents, additives that assist the photoresistobtain the highest resolution, surfactants, quenchers, stabilizers, plasticizers, colorants, adhesive additives, surface leveling agents, or combinations thereof.

[0182] In some embodiments whereby the patternable material is an electron-patternable material, the electron-patternable material may comprise an electron beam resist. Examples of electron-patternable resists include any ZEP series of resists, or any HSQ and variants thereof, or any PMMA and variants thereof, or any polycarbonate and variants thereof, or any resists with increased sensitivity with the addition of halogenated acid generators, or any CSAR series of resists and variants thereof, or any AR series of resists, or any SML series of resists, or any microresist mr-EBL series of resists, or any microresist XR series of resists.

[0183] In some embodiments whereby the patternable material is an imprint capable material, the imprint capable material may comprise an imprint (or nano-imprint) resist. In some embodiments, the imprint resist comprises a UV curable polymer. In some embodiments, the imprint resist comprises a thermoset polymer. In some embodiments, the imprint resist comprises a thermoplastic polymer. In some embodiments, the imprint process may comprise nanoimprinting, electrochemical imprinting, laser assisted direct imprint, UV imprinting, ultrafast nanoimprint, roller imprint. Examples of imprint resists include any microresist mr- NIL series of resists, or any microresist mr-I series of resists, or any microresist SIPOL series of resists, or any microresist OrmoComp series of resists, or any microresist OrmoStamp series of resists, or any microresist OrmoClear series of resists, or any microresist OrmoCore series of resists, or any microresist OrmoClad series of resists, or any microresist mr-XNIL series or resists, or any microresist KER series of resists, or any microresist mr-UVCur series of resists, or any nanonex NRX series of resists.

[0184] In some embodiments the patternable materials may be applied to the fluidic device by any method comprising spin coating, or lamination, or roller coating, or spray coating, or dip-coating, or dry-film attaching, or air-knife coating, or curtain coating, or wire-rod coating, or wire-bar coating, or gravure coating, or extraction coating, doctor blade coating, or dispensing from a dispensing system, or ink-jet printing, or jetting, or tape dispensing, or combinations there-of.

[0185] The device may comprise organic films that are chemically bonded or grown on a surface of the device. In some embodiments, the organic film comprises polymers. In some embodiments, the exact thickness of the organic film is controlled by the bonding or growth conditions of the film. In some embodiments, the organic film’s growth is self-limiting. In some embodiments, the organic film’s growth can be controlled by tuning the reaction time during growth, or by turning the number of growth cycles. In some embodiments, the organicfilm may comprise a hydrophobic surface property, or a hydrophilic surface property, or a positive surface charge, or a negative surface charge, or an anti-fouling surface property. In some embodiments the location(s) of the organic film in the fluidic device may be controlled by bonding or growing the organic film on a seed material, such that the organic film will only bond or grow on said seed material. In some embodiments, the seed material may comprise silicon oxide, or silicon nitride, or silicon, or glass, or quartz, or gold, or nickel, or platinum, or ITO, or silver, or a polymer, or a photo-patternable polymer, or an imprint- patternable polymer, or an embossing patternable polymer, or a dispensable polymer. In some embodiments, the seed material first has its surface chemically modified before bonding or growing the organic film. In some embodiments, the location(s) of an absence of organic film in the fluidic device is controlled at least in part by the location of a blocking material.

[0186] In some embodiments, the surface modification of a fluidic device comprises the modification of a polymerizable patternable material that has undergone partial or complete polymerization, with the degree of polymerization effected by factors including photoinitiation dose, lithographic masking, dose of a maskless lithography apparatus, wavelength dependent absorption of the light used to activate the photoinitiator, local environment created by fine features and neighbor effects, pre-bake thermal profile, post- bake thermal profile, development, aging, etching, plasma etching and quenching. In preferred embodiments, the modification of the patternable material employs chemical reactions that specifically modify the patternable material and do not modify the underlying substrate. In some embodiments, the above effects create a patternable material of non- uniform reactivity for the context of further chemical modification.

[0187] In some embodiments the surface modifications react in a substantially uniform and specific manner to modify substantially all of the underlying substrate or substantially of the patternable material applied to the substrate. In some embodiments, the surface modifications rely of further photoactivation of a developed or partially developed patternable material to enhance the reactivity of the remaining patternable material in local areas to add an additional level of spatial control of the deposition of subsequent surface modification. In some embodiments, selective application of plasma modifies the reactivity of the substrate and or the patternable material. In some embodiments the surface modifications react non-uniformly with a partially polymerized or partially degraded patternable material as prepared using the effects described above. In some embodiments, the degree of surface modification is further influenced by the diffusion of oxygen within the three dimensional structures during the process of surface modification.

[0188] In some embodiments, surface modifications are performed on a partially polymerized patternable material. In some embodiments, modifications utilize residual acid from superacid photoactivators and / or free radicals left over from radical polymerization. In some embodiments, radical polymerization is performed using a mixture of one or more acrylates and methacrylates, not limited to the following: poly(ethylene glycol), methyl ether acrylate, poly(2-hydroxyethyl methacrylate), isobornyl methacrylate, Lauryl methacrylate, 2- (Trimethylsilyloxy)ethyl methacrylate, N-,VRSURS\ODFU\ODPLGH^^1^1ƍ- Methylenebis(acrylamide), 2-Hydroxyethyl methacrylate, Propargyl acrylate, acrylamide, N,N-Dimethylacrylamide, 2-N-Morpholinoethyl methacrylate, Pentafluorophenyl methacrylate, dextran methacrylate, methacrylated gelatin, methacrylated hyaluronic acid, methacrylated collagen, methacrylated alginate, methyl methacrylate, 2,2,2-trifluoroethyl acrylate, butyl acrylate, propyl acrylate, N-hydroxyethyl acrylamide, 2-hydroxy-3-phenoxy- propyl acrylate, tert-butyl methacrylate, tert-butyl acrylate, sodium methacrylate, benzyl acrylate. In some embodiments, radical polymerization, including radical polymerization with the above monomers is carried out in a controlled living polymerization using one of the Reversible-deactivation radical polymerization methods including but not limited to Atom transfer radical polymerization (ATRP), Nitroxide-mediated polymerization (NMP), and Reversible addition-fragmentation chain transfer (RAFT).

[0189] In some embodiments, unreacted epoxy moieties from a patternable material can be opened and used for further reactions, including acylation reactions, including succinic DQK\GULGH^DQG^Į-Bromoisobutyryl bromide. Furthermore, unreacted epoxy moieties react with primary amines under acid and base catalyzed conditions, and direct surface modifications of epoxy containing patternable materials are not limited to the addition of alkylamines including dodecylamine, octylamine, tert-Octylamine propylamine, cyclic aliphatic amines including cyclohexamine, fluorinated amines including 2,2,3,3,4,4,4- Heptafluorobutylamine, zwitterionic or charged amines including glutamatic acid, polar amines such as 2-(2-Aminoethoxy)ethanol, and secondary amines such as Dioctylamine.

[0190] In some embodiments, at least a portion of the surface of the fluidic device, or at least a portion of a fluidic feature is modified by a combination of two different reactions, applied one at a time or simultaneously, whereby the first reaction is the substrate for the second reaction. In such reactions, chemical amplification can be achieved under certain reaction conditions. In some embodiments, the first reaction is the surface initiated radical polymerization of the ATRP initiator (2-(2-Bromoisobutyryloxy)ethyl methacrylate), alone or as a mixture of different acrylates or methacrylates or bi or multifunctional crosslinkingacrylates or methacrylates, and the second step is surface initiated Atom Transfer Radical Polymerization (siATRP) using an acrylate or methacrylate as described above, and employing one of the established methods of Copper catalyzed ATRP including activators generated by electron transfer (AGET), activators regener-ated by electron transfer (ARGET), initiators for continuous activator regeneration (ICAR) and regeneration of Cu(I) from oxidation of Cu(0) in the presence of ligands including PMDETA, Me6TREN, TPMA, TMPA-NME2. In other embodiments, the first reaction is the thiol epoxy condensation of thiol containing ATRP reagents such as Bis[2-^^ƍ-bromoisobutyryloxy)ethyl]disulfide and Bis[2-(2-bromoisobutyryloxy)undecyl] disulfide, followed by siATRP using acrylates or methacrylates as described above. In other embodiments, the surface modification proceeds by radical polymerization of one or more monomer species containing carboxyls, including acrylic acid and methacrylic acid, and the second reaction utilizes N-Ethyl-N0-(3- (dimethylamino)-propyl)carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) to graft biomolecules onto the polymer, including commonly prepared proteins known for blocking ability or the ability to create a hydrogel or matrix including whole or partial Bovine Serum Albumen, Collagen, Gelatins, telocollagen, and tropoelastin. In other embodiments, the first reaction is radical polymerization of glycidyl methacrylate and the second reaction is the addition of primary amines present on proteins as described above with glycidyl ethers, or the addition of hyaluronic acid, dextran, or Carboxymethyl cellulose to the glycidyl ethers.

[0191] The device may comprise a combination of different materials that are mixed, bonded, laminated, layered, grown, adhered, patterned, joined, coated, deposited, evaporated, sputtered, melted, merged, or combination there-of..

[0192] Physical Obstacle. Unless specifically stated otherwise, a “physical obstacle” is a fluidic feature within a fluidic device in which a body in the presence of an applied force, physically interacts with, such that the body’s physical conformation or location is different than had said physical obstacle not been present. A body may comprise a macromolecule, a nucleic acid molecule, a long nucleic acid molecule, a nucleic acid molecule with at least one higher order structure, a cell, a droplet, a package, a protein, an enzyme. Non-limiting examples of obstacles include: pillars, corners, pits, traps, barriers, walls, bumps, constrictions, expansions, functionalized surfaces, modified surfaces. The physical obstacles need not be physically continuous with the fluidic device, but may also be additive to the device, with non-limiting examples including: beads, gels, particles inside a fluidic channel.

[0193] Droplet. The terms “droplet,” and “microdroplet” are used interchangeably herein, to refer to small, rounded structures (generally spherical in the unrestricted state), containing at least a first fluid phase, (for example: water), bounded by a second fluid phase (for example: oil) which is immiscible with the first fluid phase, or bounded by surface tension formed by the interface of the first fluid phase, a surface, and air.

[0194] In some embodiments, the second fluid phase will be an immiscible phase carrier fluid. Thus droplets according to the present disclosure may be provided as aqueous-in-oil emulsions or oil-in-aqueous emulsions. Droplets according to the present disclosure may be formed as multiple emulsions, such as double or higher level emulsions, for example resulting in aqueous-in-oil-in-aqueous droplets. In some embodiments, the subject droplets KDYH^D^GLPHQVLRQ^^IRU^H[DPSOH^^D^GLDPHWHU^^RI^RU^DERXW^^^^^^P^WR^^^^^^^P^^Lnclusive. Furthermore, in some embodiments, discrete entities as described herein have a volume ranging from about 1 aL to 1 uL, inclusive. Droplets according to the present disclosure may be used to encapsulate cells, nucleic acids (e.g., DNA), enzymes, reagents, and a variety of other entities. The droplet may contain a single entity (for example: a single cell, or a single long nucleic acid fragment), or multiple entities. The droplet may contain a mixture of different types of entities. The term droplet may be used to refer to a droplet produced in, on, or by a microfluidic device and / or flowed from or applied by a microfluidic device. The droplet may be externally generated, and applied to the microfluidic device. Alternatively, the droplet may be generated within a microfluidic device, and then removed from said device.

[0195] Depending on the desired operation, droplets can be partitioned into 2 or more droplets, or merged with at least one other droplet. The droplet to be merged may have identical, or dissimilar contents.

[0196] The composition and nature of the droplets may vary. For instance, in certain aspects, a surfactant may be used to stabilize the droplets. Accordingly, a droplet may involve a surfactant stabilized emulsion. Any convenient surfactant that allows for the desired reactions to be performed in the droplets may be used. In other aspects, a droplet is not stabilized by surfactants or particles.

[0197] In some embodiments droplets may be formed by the interface of liquid, surface, and air, and thus include droplets defined by an electrowetting device. Examples of such droplets are reviewed by [Zhao, 2013] and [Mugele, 2005].

[0198] Package. A “package” is any body capable of holding fluid contents within the defined boundary of the body. In some embodiments, the boundary is defined by a physical barrier such as a lipid bilayer, or a layer of surfactant molecules, or a layer of colloidalparticles, or a membrane. In some embodiments there is no barrier, such as a droplet formed by mixing two immiscible fluids. A non-exhaustive list of packages include: live cells, dead cells, nucleus, vesicles, exosomes, mitochondria, organelles, bacteria, swollen cells, physical- assisted fixed cells (heat, freezing, microwave-assisted fixation), chemical-assisted fixed cells (alcohol-based fixing, acid-based fixing, cross-linking-based fixing), bubble, artificial membrane package, water-in-oil droplets, oil-in-water droplets, water-oil-water droplets, oil- water-oil droplets, Pickering emulsions, a lipid particle, a lipid nanoparticle. In all cases, the package can be lysed (or ruptured) by various means to release the contents. The “package fluid” or “package solution” refers to the fluid content of the package. In some embodiments, the package boundary is non-permeable. In some embodiments, the package boundary is permeable, allowing for exchange of fluid or material into, or out of, the package. In some embodiments, the package boundary is selectively permeable, only allowing certain molecules or ions to pass through it. In some embodiments, the package comprises at least one labelling body. In some embodiments, the package contents comprises at least one other package. For example: a droplet that contains a cell.

[0199] In some embodiments, the package boundary is at least partially functionalized, in some embodiments functionalized with bioactive molecules. In some embodiments, the package boundary is at least partially ionized. In some embodiments, the package boundary is at least partially functionalized to promote adhesion to a surface. In some embodiments, the package boundary is at least partially functionalized to prevent adhesion. In some embodiments, the package boundary is at least partially functionalized to respond to the local environment in a certain area of a fluidic device. In some embodiments, the package boundary is chemically modified. In some embodiments, the package boundary is bound with at least one labelling body.

[0200] In some embodiments, the package can be lysed (or ruptured) by various means to release the contents.

[0201] Rupturing. “Lysing” (or “Rupturing”) is the process of releasing at least a portion of the contents of a package by disrupting or damaging the package boundary. In some embodiments, the rupturing process comprises weakening the package boundary such that the contents can more easily be released from the package. In some embodiments, the package boundary is non-recoverably destroyed with the contents released. In some embodiments, the package boundary is disrupted, and whose integrity may be recovered. In some embodiments, the package boundary is permanently or temporarily made more permeable. Examples of different lysing methods include chemical, enzymatic, electro-chemical, physical,mechanical, acoustic, electroporation and focused photons. Suitable lysing agents include, for example, enzymes (e.g., protease, glycosidases, nucleases). Exemplary enzymes include lysostaphin, pepsin, glucosidase, galactosidase, lysozyme, achromopeptidase, endopeptidases, N-acetylmuramyl-L-alanine amidase, endo-beta-N-acethylglucosaminidase, ALE-1, Dnase, and Rnase. Other lysing agents include salts (e.g., chaotrophic salts), solubilizing agents (e.g., detergents), reducing agents (e.g., beta-mercaptoethanol (BME), dithiothreitol (DTT), dithioerythritol (DTE), tris(2-carboxyethyl) phosphine hydrochloride (TCEP; Pierce Chemical Company, Rockford, Ill.), cysteine, n-acetyl cysteine), acids (e.g., HCl), and bases (e.g., NaOH). Various combinations of lysing agents and / or methods can be used if desired.

[0202] Focused photos to lyse packages, or “optical lysis”, has been demonstrated previously [US Patent 6,815,209 “Laser-induced cell lysis system”] by using a laser light of wavelength from 500 nm to 3500 nm to lyse cells in solution, or by short pulses [US Patent 6,156,576 “Fast controllable laser lysis of cells for analysis”]. A review of various methods for focused light lysing of cells was reported by [Islam, 2017] in which optically induced delectrophoresis (ODEP) is a process by-which a focused laser pulse at the cell-solution interface creates a cavitation bubble that lyses the cell. Focused laser lysing was further demonstrated [US Patent 10,221,443 “System and method for laser lysis”] as an effective means of lysing specific individual cells within a cluster of cells, typically a tissue sample.

[0203] Porous Material. A “porous material” is any composition of solid, or semi-solid matter that is porous in nature such that there exists a fluid or gas substance that can pass through said porous material. In some embodiments the porous material may comprise a gel, formed by cross-linking a gelling agent. In some embodiments the porous material may comprise an artificial gel, manufactured with either random, or controlled pore sizes. In some embodiments the porous material may comprise a solid state material with holes etched in random or deterministically. In some embodiments, the porous material may comprise a polymer. The porous material may be fluidic device channel in which there are patterned physical obstacles that between them have openings, for example: a collection of pillars. The pillars may be of consistent, random, or distribution of sizes. The pillars may be arranged in a regular, planned, or random manner. The porous material may be a collection of packed beads or packed isolated objects, such that the space between the beads or objects provides for the porous nature. The beads or isolated objects may be of consistent, random, or distribution of sizes. The packing can be regular or random. In some embodiments, the porous material may be a material that is grown, etched, or deposited [Plawsky , 2009]. Thematerial may be organic, inorganic, or a combination there-of. In some embodiments, the porous material may comprise only a single pore. In some embodiments, the porous material may comprise at least one pore. In some embodiments, the porous material may comprise a plurality of pores. In some embodiments at least two pores are connected to each other. In some embodiments, at least two pores are independent of each other. In some embodiments the porous material comprises at least one pore with a minimum cross-sectional dimension in the range of 50 microns to 5 nm. In some embodiments, the porous materials comprises at least one pore with a cross-sectional profile that is approximately circular, or oval, or square, or rectangle, or triangular, or polygon, or star shaped, or non-connected line, or a connected line. The line may vary in width. For clarity, examples of non-connected lines include “I” shape, or “S” shape, or “H” shape, while examples of connected lines include “8” shape, or “P” shape, or “Q” shape.

[0204] Environmental Condition. An “environmental condition” may comprise any property of physics, matter, chemistry that surrounds a body that may impact said body’s physical state, thermo-dynamic state, chemical state, or reactivity to other reagents. In some embodiments, the body may comprise a cell, a package, a droplet, a macromolecule, a long nucleic acid molecule, a nucleic acid molecule, a labeling body, a protein, an enzyme, a virus, a bacteria. The impact on the body may be due to the presence of the environmental condition, or a change in the environmental condition. An environmental condition may comprise a temperature, a pressure, a humidity level, a pH, an ionic concentration, a flow rate or direction. An environmental condition may comprise flux, polarization, intensity of a wavelength of light. An environmental condition may comprise a solution composition, for example a concentration of a certain reagent within a solution, or a ratio of certain reagents within a solution, or a salt composition used for a particular buffer. An environmental condition may comprise an external force acting on the body, for example a solution or air flow rate. An environmental condition may comprise thermal conductivity property, an electrical conductivity property, an optical opacity or transparency property. An environmental condition may comprise an electric or magnetic field. An environmental condition may comprise sound of a certain frequency or intensity. An environmental condition may comprise an ultrasonic wave of a certain frequency or intensity. An environmental condition may comprise a gravitation force, or an effective gravitational force. An environmental condition may comprise a vapor pressure. An environmental condition may comprise the composition of gases in an atmosphere. An environmental condition maycomprise the composition of vapors in an atmosphere. An environmental condition may comprise the relative volatility of an atmosphere.

[0205] Reagent. Used herein, a “reagent” is any substance or compound added to a system to cause, enhance, attenuate, supply, or stop a chemical reaction, including an enzymatic reaction. A reagent may comprise a nucleotide, a nucleotide of a certain type (for example: A, T, C, G, U), a terminating nucleotide, a reversibly terminating nucleotide, an enzyme, a polymerase, a protein, a restriction enzyme, a nicking enzyme, a polynucleotide, an at least partially double-stranded polynucleotide, an at least partially single-stranded polynucleotide, an RNA, a guide RNA, a CRISPR-associated protein (CAS).

[0206] External Force. An “external force” or an “external applied force” or “applied force” is any applied force on a body such that the force can perturb said body from a state of rest, or can accelerate (or deaccelerate) said body. Additionally, an external force is any applied force on a body wherein the removal of said force on said body can perturb said body from a state of rest, or can accelerate (or deaccelerate) said body. In some embodiments, the body may comprise a cell, a package, a droplet, a macromolecule, a long nucleic acid molecule, a nucleic acid molecule, a labeling body, a protein, an enzyme, a virus, a bacteria. Non-limiting examples include hydrodynamic drag exerted by a fluid flow [Larson, 1999] (which can be imitated by a pressure differential, gravity, capillary action, thermal-capillary convection, electro-osmotic, convection currents, surface tension, micro-convection, surface tension gradients, and the Maragoni effect), an electric field, electric-kinetic force, electrophoretic force, pulsed electrophoretic force, magnetic force, dielectric-force, centrifugal acceleration, gravitation force (real or effective), or combinations there-of. In addition, the external force may be applied indirectly, for example if bead is bound to the body, and then the bead is subjected to an external force such a magnetic field, or optical teasers.

[0207] Dispensing System. Used herein, a “dispensing system” or “dispenser” is an instrument, or a component of an instrument that is capable of dispensing a volume of liquid from a dispensing tip, nozzle, or orifice (herein, collectively referred to as “tip”) at a desired location in (x,y,z) space. In some embodiments the liquid is dispensed as a continuous stream. In some embodiments, the liquid is dispensed as a series of drops. The drop size may be 100 micro liters or less, 10 micro liters or less, 1 micro liters or less, 100 pico liters or less, 10 pico liters or less, 1 pico liters or less, 100 femto liters or less, 10 femto liters or less, 1 femto liter or less, 100 atto liters or less, 10 atto liters or less. In some embodiments, the tip is composed of a consumable pipette tip. In some embodiments, the dispenser tip is also capable of extracting solution from a target solution in (x,y,z) space, and so the dispenser is also an“extractor”. In some embodiments, the dispensing and extraction tips are different tips. In some embodiments, they are the same. In some embodiments, the tip is a micro-syringe, or the end of a capillary tube, or a nozzle. In some embodiments, the dispensing of liquid is controlled by air-displacement via a pressured air-line, or a syringe-pump moved via an electrical-mechanical system, such as a stepper motor.

[0208] In some embodiments, inkjet dispensers may be used. Inkjet printing includes continuous jet (CJ) and drop-on-demand jet (DODJ). The CJ based on the transducer, charging electrode and electric field can produce the droplet continuously, and the droplet location on a substrate can be determined by its charging density. There are several kinds of actuators for the DODJ device, including piezoelectric, thermal, solenoid, pneumatic, magnetostrictive and acoustic actuators. There are two actuation modes for the piezoelectric micro-jet devices in particular, including single actuation mode and hybrid actuation mode. The single actuation mode includes shear mode, squeeze mode, bend mode, push mode and needle-collision mode, while the hybrid actuation mode refers to electrohydrodynamic (EHD) assistant actuation. A detailed review of different inkjet technologies is provided by [Li, 2019], and included here for reference in its entirety.

[0209] In some embodiments the dispenser consists of a contact probe capable of transporting and depositing a drop of solution by contact wetting. In some embodiments, extraction of drop from a surface is done by a contact probe making contact with said drop, and wetting the contact probe.

[0210] Contact Probe System. Used herein, a “contact probe” system is an instrument, or a component within an instrument that is capable of positioning the point or tip of a contact probe within the desired location in (x,y,z) space with respect to a surface, preferably with nanometer position accuracy or better, and measuring a signal as a function of the xy, or xyz position. In the preferred embodiments, the contact probe is capable of measuring a signal based on its interaction with a physical object. In the preferred embodiment, the contact probe comprises part of a contact probe interrogation system, which itself is a type of interrogation system. In the preferred embodiments, the contact probe is a surface scanning probe, capable of generating a signal while the probe is physically moved in xyz space with respect to the surface by the instrument. Different types of contact probes include SPM (Scanning Probe Microscopy), AFM (Atomic Force Microscopy), HS-AFM (High Speed Atomic Force Microscopy), STM (Scanning Tunneling Microscopy), SPE (Scanning Probe Electrochemistry), CFM (Chemical Force Microscopy), LFM (lateral Force Microscopy), magnetic force microscopy (MFM), high frequency MFM, magneto-resistive sensitivitymapping (MSM), electric force microscopy (EFM), scanning capacitance microscopy (SCM), Scanning spreading resistance microscopy (SSRM), tunneling AFM and conductive AFM, contact AFM, non-contact AFM, dynamic contact AFM, tapping AFM, kelvin probe force microscopy (KPFM), piezo-response force microscopy (PFM), photothermal microscpectroscopy, scanning gate microscopy (SGM), scanning quantum dot microscopy (SQDM), scanning voltage microscopy (SVM), force modulation microscopy (FMM), ballistic electron emission microscopy (BEEM), electrochemical scanning tunneling microscopy (ECSTM), scanning Hall probe microscopy (SHPM), spin polarized scanning tunneling microscopy (SPSM), photon scanning tunneling microscopy (PSTM ), scanning tunneling potentiometry (STP), synchrotron x-ray scanning tunneling microscopy (SXSTM), Scanning Probe Electrochemistry (SPE), scanning electrochemical microscopy (SECM), scanning ion-conductance microscopy (SICM), scanning vibrating electrode technique (SVET), scanning Kelvin probe (SKP), fluidic force microscopy (FluidFM), feature-oriented scanning probe microscopy (FOSPM), magnetic resonance force microscopy (MRFM), near- field scanning optical microscopy NSOM, scanning near-field optical microscopy (SNOM), scanning SQUID microscopy (SSM), scanning spreading resistance microscopy (SSRM), scanning thermal microscopy (SThM), scanning single-electron transistor microscopy (SSET), scanning ontact-ionic microscopy (STIM), charge gradient microscopy (CGM), and scanning resistive probe microscopy (SRPM). For a review of different Scanning Probe Microscopy systems, refer to [Takahashi, 2017]. For clarity, a contact probe need not necessarily make intimate physical contact with the sample, or any object, to measure a signal from said sample.

[0211] In some embodiments where the contact probe comprises an AFM system, the system can operate in a variety of different modes, and thus measure a variety of different signals, depending on the selection of the probe type, its mode of operation, and the probe’s tip sharpness. Non limiting examples of different AFM modes include non-contact mode, contact mode, tapping mode, dry mode, wet mode, high-frequency mode, ultra-high frequency mode, force-modulation mode, conductive mode, magnetic mode, super-sharp tip mode, diamond tip mode, high-aspect ratio mode, electron beam deposited tip mode, and carbon-nano-tube tip mode. In some embodiments, the contact probe can operate in a dry environment, or a humid environment, or a liquid environment. In some embodiments, the point of the contact probe can be functionalized with chemical moieties, biological bodies, or affinity groups to enable biochemical interaction with the physical object being probed. For a review of various functionalization that have been demonstrated on contact probes, refer to[Ebner, 2019]. In some embodiments, the point of the contact probe may include a carbon nanotube, a nanorod, or a nanospike. In some embodiments, the tip of the contact probe may include a pore, or nanopore, that allows for a fluidic connection to a fluidic channel or fluidic chamber within the contact probe.

[0212] In AFM microscopy, the probe is attached to a spring-loaded or flexible cantilever that is in contact with the surface to be analyzed. Contact is made within the molecular force range (for example: within the range of interaction of Van der Waal forces). Within AFM, different modes of operation are possible, including contact mode, non-contact mode and TappingMode™. Fluidic Device for Cytogenetic Analysis.

[0213] In this set of embodiments, we disclose devices and methods for an open fluidic device that enables the positioning of macromolecules originating from packages to generate a spread within the fluidic features of the device. In the preferred embodiment, the spread is interrogated in a manner that improves the data quality via the controlled physical arrangement of the molecules within the fluidic features of the device when compared to spreads generated with traditional cytogetic methods on a glass slide using similar packages. In the preferred embodiment, no internal or external power source is required to control the movement and positioning of the packages and macromolecules after the input sample is dispensed into or onto the device.

[0214] In some device and method embodiments described herein, the generation of spreads originating from a single cell are co-located and substantially non-overlapping or non- touching with each other on the surface of the open fluidic device, providing higher yields and improved data quality when compared with traditional cytogenetic sample preparation methods of dropping a solution of similar cells on a microscope slide resulting in chromosomes that are positioned randomly on the surface of the slide.

[0215] In this set of embodiments, at least two packages, whereby each package contains a package fluid and at least two macromolecules, are ruptured on the surface of an open fluidic device that comprises fluidic features, such that each package’s respective package fluid allows for the placement of the each package’s respective macromolecules within the fluidic features of said device, such that the number of useful spreads per unit area when compared to traditional cytogenetic methods of dropping a solution of similar packages on a glass slide is higher. Package Rupture

[0216] In some embodiments, a package containing fluid, herein called a “package fluid” or a “package solution”, and at least two macromolecules is ruptured on the surface of an open fluidic device, with said device comprising fluidic features, such that a fluid comprising the package fluid flows within the fluidic features, and positions the at least two macromolecules within the fluidic features.

[0217] In some embodiments, the package boundary comprises a cell membrane. In some embodiments, the package membrane comprises a lipid bilayer. In some embodiments, the package boundary is weakened, or disturbed, or partially ruptured prior to rupturing. In some embodiments, the package membrane is weakened, or disturbed, or partially ruptured at least in part by the addition of a salt, a reagent, an enzyme, an acid, a base, a lysate, a virus, an electric field, an electric current, a gravitational force, a centripetal force, or a pH modifier. In some embodiments, the package boundary is weakened, or disturbed, or partially ruptured at least in part by the modification of at least one environmental condition.

[0218] In some embodiments, at least one package is treated prior to deposition and package rupture. In some embodiments, treatments may comprise methods to weaken the package boundary, including membrane permeabilization methods that dissolve lipids from cell membranes using organic solvents including methanol and acetone and detergents that remove cholesterol such as saponin and detergents that also remove lipids, including Triton X-100 and Tween 20. The number of detergents is large and the present application is in no way limited to the use of just the detergents listed herein. In some embodiments, a treatment of the package may comprise selectively removing a portion of the package prior to deposition, including the above solvents and detergents and related detergents in addition to DMSO, Ethanol, Methanol, Acetone, strong acid or base extractions including NaOH Acetic acid and HCl and deep eutectic solvents including Choline chloride and glycerol. In some embodiments, a treatment of the package may comprise altering the properties of at least one macromolecule inside the package, including the modification of chromatin through enzymatic means including acetylates, deacetylases, methyltransferases, phosphatases and kinases, partial cleavage of DNA, partial cleavage of RNA, partial proteolysis, addition of multivalent ions including spermine and spermidine, addition of co-block polymers comprised of various combinations of one more of the following list: Polyoxyethylene, polyoxypropylene, polyethylenimine, polyacrylic acid, polymethacrylic acid. In some embodiments, a treatment to the package may comprise the sever or partial sever of tethers or interactions between individual macromolecules inside the package, including poisoning of cellular organelles and inhibition of cell cycle by compounds not limited to Mebendazole,Colchicine, Griseofulvin, Vinca Alkaloids, Taxol, Epothilones, Ixabepilone, Vinorelbine, Flavopiridol, Nocodazole, vincristine, Centrinone, colchicine, Eribulin, Actinomycin D, Monasterol, Podophyllotoxin, Okadaic acid, Griseofulvin. In some embodiments, a treatment to the package may comprise the altering of the constituents of the package including genetic methods such as RNA interference (RNAi) against genes and gene products BAF, PP1, p53, Rb, Cyclin D, PP2A, B-Myb, CDK, USP28, PLK1, HEC1, Securin, p21, Aurora B, Separase.

[0219] In some embodiments, the package fluid comprises an aqueous solution. In some embodiments, the package fluid comprises cytoplasm, nucleoplasm, cytosol, ground plasm, or intracellular fluid. In some embodiments, the package fluid comprises a droplet fluid, or an oil. In some embodiments, the package fluid may have a pH of about 7, or about 8 or about 6, or about 9, or about 5, or about 10, or about 4, or about 11, or about 3. In some embodiments, the package fluid may have a net positive electric charge, or net negative electric charge, or a net neutral electric charge.

[0220] In some embodiments, the package comprises a hypotonic state. In some embodiments, the package comprises a hypertonic state. In some embodiments, the package comprises an isotonic state. In some embodiments, the package is under a positive osmotic pressure. In some embodiments, the package is under a negative osmotic pressure. In some embodiments the package is under a positive pressure. In some embodiments, the package is under a negative pressure.

[0221] In some embodiments, the macromolecules in the package are entire chromosomes that originate from a single cell. In some embodiments, the macromolecules in the package are a selection of chromosomes that are selected on the basis of a certain criteria, for example chromosome length, number, or disease trait. In some embodiments, the macromolecules in the package may not originate from a single cell. In the preferred embodiment, the macromolecules in the package are condensed such as metaphase or prophase chromosomes.

[0222] In some embodiments the macromolecules in the package have already undergone some degree of processing, for example digestion of proteins, binding of labeling bodies, binding of PCR primers, binding of MDA primers, binding of barcodes, digestion of nucleic acid with frequent or rare cutters to achieve the desired size distribution, or combination- there-of. In some embodiments, there may be at least one nucleic acid barcode incorporated into the package, which may be separately suspended in solution inside the package with the macromolecules, or bound to the molecules. In some embodiments, the barcode contains information unique to the package, or originating cell, or some other content.

[0223] In some embodiments, at least one package is delivered to the surface of the open fluidic device by first generating a solution (herein called the “delivery fluid” or “delivery solution”) containing the at least one package, and wetting said open fluidic device with said delivery fluid, allowing the at least one package to interact physically with the surface of the open fluidic device, and then removing at least a portion of said delivery fluid from at least a portion of the surface of the fluidic device comprising fluidic features. In some embodiments, the physical interaction results in at least a temporary immobilization of the interacting portion of the package.

[0224] In some embodiments, the physical interaction of the package with the open fluidic device comprises adhesion. In some embodiments, the physical interaction of the package with the open fluidic device comprises immobilization. In some embodiments, the physical interaction of the package with the open fluidic device comprises physically stretching of the package boundary. In some embodiments whereby the package is a cell, the physical interaction of the package with the open fluidic device comprises cell division, cell growth, progressing through at least a portion of a cell cycle stage, cell elongation, cell morphology changes. In some embodiments, surface of at least one fluidic feature can be modified to improve cell adhesion; In some embodiments, modification may comprise changing the surface charge, changing the surface hydrophobicity, changing the surface roughness, changing the surface anti-fouling property, conjugating a molecule to surface, covalently or ionically binding a molecule to the fluidic surfaces. In some embodiments, the surface of at least one fluidic feature can be modified to resist cell adhesion.

[0225] In some embodiments the delivery fluid comprises an aqueous solution. In some embodiments, the delivery fluid comprises a methanol solution. In some embodiments, the delivery fluid comprises chloroform. In some embodiments, the delivery fluid comprises acetic acid. In some embodiments, the delivery fluid comprises a Carnoy solution. In some embodiments, the delivery fluid comprises a fixative. In some embodiments, delivery fluid comprises organic acids such as citric acid, lactic acid, formic acid, malic acid, tartaric acid, ascorbic acid, acetylsalicylic acid, folic acid, benzoic acid. In some embodiments, delivery fluid comprises mineral acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, carbonic acid, hydrofluoric acid, chromic acid, boric acid, perchloric acid, sulfurous acid. In some embodiments, delivery fluid comprises of ethyl alcohol, acetone, toluene, ethyl acetate, hexane, dichloromethane, dimethyl sulfoxide, diethyl ether, acetonitrile, benzene, cyclohexane, isopropyl alcohol, butyl alcohol, cyclohexanol, benzyl alcohol, octanol, phenethyl alcohol, hexanol, glycerin, propylene glycol, ethylene glycol. In someembodiments, the delivery fluid comprises one or more volatile liquids, including but not limited to methanol, acetic acid, triethylamine, trifluoroacetic acid. In some embodiments, the volatile liquids are placed upon an open surface of the fluidic device and exposed to a gas. In some embodiments, the gas may comprise nitrogen, oxygen, water, ethanol, propanol, methanol, acetic acid, triethylamine, or trifluoroacetic acid. Preferably the gas is delivered as a stream of constant chemical composition that is blown over the device. In some embodiments, the delivery fluid exchanges with the gas and a net migration of delivery fluid occurs into the gas.

[0226] In some embodiments, the delivery fluid may comprise chelating agents such as EDTA (Ethylenediaminetetraacetic Acid), EGTA (Ethylene Glycol-%LV^ȕ-aminoethyl Ether)- N,N,N’,N’-Tetraacetic Acid), TPEN (N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine), and DTPA (Diethylenetriaminepentaacetic Acid).

[0227] In some embodiments, the delivery fluid may comprise reagents that can weaken, dissolve, or denature protein, spindle fibers, actin, cell membrane, cytoskeleton, extracellular matrix, intermediate filaments, nuclear envelope, cytoplasm, condensing I, condensing II, lipid structure, glycoprotein structure, or chromatin. In some embodiments, the delivery fluid may comprise a reagent, an enzyme, pepsin, Cytoskeletal drugs including but not limited to latrunculin A, latrunculin B, Colchicine, Cytochalasins, Demecolcine, Jasplakinolide, Nocodazole, Paclitaxel, Phalloidin, Swinholide, Vinblastine, Rotenone;Nocodazole, Paclitaxel, Phalloidin, Swinholide, Vinblastine, Rotenone; RNAse, DNAse, KCl; detergents including but not limited to Triton X-100, Tween 20, Tween 80, Pluronic® F-127, SDS (Sodium Dodecyl Sulfate), NP-40, Digitonin, CHAPS, Octylglucoside, Sarkosyl, Brij 35, Sodium Cholate;, lysozyme, proteinase K, guanidine hydrochloride, urea, phenol-chloroform, or HCl.

[0228] In some embodiments, immobilized packages on fluidic surfaces can be altered with addition of salt buffer (hypotonic buffer) solutions to change osmolarity of the packages. In some embodiments, immobilized packages on fluidic surfaces can be fixed, and the fixing solution can compromise alcohol and acid components.

[0229] In some embodiments, the at least one package ruptures on the surface of the fluidic device during or after the removing of said delivery fluid from at least a portion of the surface of the fluidic device comprising fluidic features, and said at least one package at least partially adhering to said at least a portion of the surface of the fluidic device comprising fluidic features.

[0230] In some embodiments, the wetting process comprises dispensing of the delivery fluid onto the surface of the fluidic device. In some embodiments, the dispensing is performed manually or by an automated or semi-automated dispensing system. In some embodiments, at least one environmental condition is controlled prior, during, or after the dispensing. In some embodiments, the dispensing comprises continuous dispensing of the delivery fluid over a period of time to achieve a certain total dispensed volume. In some embodiments, this dispensing comprises the dispensing of multiple drops of delivery fluid. In some embodiments, at least two drops merge together on the surface of the open fluidic device. In some embodiments, at least a single drop maintains an isolated liquid volume of delivery fluid from at least a second single drop of delivery fluid on the surface of the open fluidic device. In some embodiments, the isolation of the two drops is maintained at least in part by at least one fluidic feature on the surface of the open fluidic device. In some embodiments, dispensing comprises dispensing directly on the surface of the fluidic device in close proximity to at least one point of rupture, or dispensing from some distance from at least one point of rupture on the fluidic device. In some embodiments, as few a one package is contained in each drop of delivery fluid, or as few as 2 packages, or as few as 5 packages, or as few as 10 packages, or as few as 20 packages, or as few as 50 packages, or as few as 100 packages, or as few as 200 packages, or as few as 500 packages, or as few as 1,000 packages, or as few as 2,000 packages, or as few as 5,000 packages. In some embodiments the total volume of delivery fluid dispensed on the surface of the fluidic device is around 1-100 picolitres, or around 10-1000 picolitres, or around 0.1-10 nanoliters, or around 1-100 nanoliters, or around 10-1000 nanoliters, or around 0.1-10 microliters, or around 10-1000 microliters, or around 0.1-10 milliliters. In some embodiments, the volume of an individual dispensed drop of delivery fluid on the surface of the fluidic device is around 1-10 femtoliter, or around 10-100 femtoliter, or around 0.1-1 picoliter, or around 1-10 picoliter, or around 10- 100 picoliter, or around 0.1-1 nanoliter, or around 1-10 nanoliter, or around 10-100 nanoliter, or around 0.1-1 microliter, or around 10-100 microliter, or around 0.1-1 microliter, or around 1-10 microliter, or around 10-100 microliter, or around 0.1-1 milliliter, or around 1-10 milliliter. In some embodiments, the total quantity of packages dispensed on the surface of the fluidic device is 1 package or less, or 2 packages or less, or 5 packages or less, or 10 packages or less, or 20 packages or less, or 100 packages or less, or 200 packages or less, or 500 packages or less, or 1,000 packages or less, or 2,000 packages or less, or 5,000 packages or less, or 10,000 packages or less, or 20,000 packages or less, or 100,000 packages or less, or 200,000 packages or less, or 1,000,000 packages or less. In some embodiments, at leastone package delivered to the surface of the fluidic device was not incubated or cultured. In some embodiments, at least one package delivered to the surface of the fluidic device was grown from a incubation or culturing process of no more than 1 day, or no more than 2 days, or no more than 3 days, or no more than 4 days, or no more than 5 days, or no more than 6 days, or no more than 7 days, or no more than 8 days, or no more than 10 days, or no more than 12 days, or no more than 15 days.

[0231] In some embodiments, the wetting comprises dipping the open fluidic device into a pool or reservoir that comprises the delivery fluid. In some embodiments, the speed of the entry or exit of the open fluidic device into the pool or reservoir is controlled. In some embodiments, at least one environmental condition is controlled prior, during, or after the dipping. In some embodiments, the angle of entry or exit of the open fluidic device from the pool or reservoir is constant or variable. In some embodiments, the speed of entry or exit is constant, or variable. In some embodiments the speed of entry or exit for at least a period of time is approximately 1 micron / sec or less, or 10 microns / sec or less, or 100 microns / sec or less, or 1000 microns / sec or less, or 10 mm / sec or less, or 100 mm / sec or sell. In some embodiments, more than 1 dipping event is performed. In some embodiments, the more than 1 dipping event is performed in different reservoirs. In some embodiments, the different reservoirs contain different samples, or different reagents, or a different solution composition.

[0232] In some embodiments, the wetting comprises blade coating the open fluidic device with the delivery fluid. In some embodiments, at least one environmental condition is controlled prior, during, or after the blade coating. In some embodiments, the blade coating comprises coating the delivery fluid on the surface of the open fluidic device with a blade. In some embodiments, the speed of blade coating is constant, or variable. In some embodiments the speed of blade coating for at least a period of time is approximately 1 micron / sec or less, or 10 microns / sec or less, or 100 microns / sec or less, or 1000 microns / sec or less, or 10 mm / sec or less, or 100 mm / sec or sell. In some embodiments, more than 1 blade coating is performed. In some embodiments, the more than 1 blade coating are performed with different solutions or samples.

[0233] In some embodiments, the wetting comprises flowing a delivery fluid contained within at least one fluidic channel or chamber, wherein said at least one fluidic channel or chamber comprises the fluidic features of the fluidic device on at least one side, and a removable roof on at least one other side. In some embodiments, the wetting comprises flowing the delivery fluid into said at least one chamber or channel. In some embodiments, the wetting comprises applying at least a portion of the delivery fluid to the fluidic features ofthe open fluidic device and then positioning the removable roof to form the at least one channel or chamber. In some embodiments, the wetting comprises applying a least a portion of the delivery fluid to the removable roof, and then positioning the removable roof to form the at least one channel or chamber. In some embodiments, the removable roof is a coverslip, or a slide, or a transparent substrate, or a non-transparent substrate. In some embodiments, at least a portion of the removable roof has a modified surface. In some embodiments, the surface modification comprises a hydrophilicity, or hydrophobicity, or anti-fouling, or functionalization, or surface roughness. In some embodiments, the removeable roof comprises fluidic features. In some embodiments, the removal of the delivery fluid comprises a process of at least partially removing the removable roof.

[0234] In some embodiments, the wetting comprises flowing a delivery fluid contained a channel of the open fluidic device. In some embodiments the mechanism for flow comprises a capillary flow.

[0235] In some embodiments, at least one environmental condition is controlled prior, during, or after the wetting of the device with the delivery fluid. In some embodiments, at least one environmental condition is controlled prior, during, or after the removal of said delivery fluid from the at least a portion of the surface of the fluidic device comprising fluidic features. In some embodiments, at least one environmental condition is controlled prior, during, or after the rupture of the at least one package. In some embodiments, the environmental condition comprises temperature. In some embodiments, the temperature is less than -100 Celsius, or between -100 and 0 Celsus, or between 0 and 5 Celsius, or between 5 and 10 Celsius, or between 10 and 15 Celsius, or between 15 and 20 Celsius, or between 20 and 25 Celsius, or between 25 and 30 Celsius, or between 30 and 35 Celsius, or between 35 and 40 Celsius, or between 40 and 45 Celsius, or between 45 and 50 Celsius, or between 50 and 55 Celsius, or between 55 and 60 Celsius, or between 60 and 65 Celsius, or between 65 and 70 Celsius, or between 70 and 80 Celsius, or between 80 and 90 Celsius, or between 90 and 100 Celsius, or between 100 and 125 Celsius, or between 125 and 200 Celsius, or more than 200 Celsius. In some embodiments, the environmental condition comprises relative humidity In some embodiments, the relative humidity is between 0 and 5 percent, or between 5 and 10 percent, or between 10 and 15 percent, or between 15 and 20 percent, or between 20 and 25 percent, or between 25 and 30 percent, or between 30 and 35 percent, or between 35 and 40 percent, or between 40 and 45 percent, or between 45 and 50 percent, or between 50 and 55 percent, or between 55 and 60 percent, or between 60 and 65 percent, or between 65 and 70 percent, or between 70 and 75 percent, or between 75 and 80 percent, or between 80and 85 percent, or between 85 and 90 percent, or between 90 and 95 percent, or between 95 and 100 percent. In some embodiments, the environmental condition comprises pressure. In some embodiments, the pressure is less than 0.1 atmosphere, or between 0.1 and 0.5 atmosphere, or between 0.5 and 1 atmosphere, or between 1 and 1.5 atmosphere, or between 1.5 and 5 atmosphere, or between 5 and 10 atmosphere, or between 10 and 100 atmospheres, or between 100 and 1000 atmospheres, or more than 1000 atmosphere. In some embodiments, the environmental condition comprises an effective gravitational force. In some embodiments, the effective gravitational force is between 0 and 1. In some embodiments, the effective gravitational force is between 1 and 10. In some embodiments, the effective gravitational force is between 10 and 100. In some embodiments, the effective gravitational force is between 100 and 1000. In some embodiments, the effective gravitational force is between 1000 and 10,000. In some embodiments, the effective gravitational force is greater than 10,000. In some embodiments, controlling an environmental condition comprises maintaining said environmental condition at an approximate value for at least a period of time. In some embodiments, controlling an environmental condition comprises changing said environmental condition from one approximate value to a second approximate value over a period of time.

[0236] In some embodiments, the process of removal of said delivery fluid from the at least a portion of the surface of the fluidic device comprising fluidic features comprises evaporation of said delivery fluid.

[0237] In some embodiments, the process of removal of said delivery fluid from the at least a portion of the surface of the fluidic device comprising fluidic features comprises transport of said delivery fluid away from the portion of the surface. In some embodiments, transport of the said delivery fluid comprises capillary flow, evaporative flow, micro-convection flow, osmotic flow pressure driven flow, gravity driven flow, thermal-capillary convection flow, thermal flow, convection currents flow, surface-tension flow, Maragoni-effect flow, fluid flow from relatively more hydrophobic surface to a relatively more hydrophilic surface, wetting flow, de-wetting flow, flow to reduce the overall surface tension energy, electrokinetic flow, electroosmotic flow, or electrophoresis flow.

[0238] In some embodiments, the rupturing of the at least one package comprises a shear force between the package boundary and the fluidic features. In some embodiments, the rupturing of the at least one package comprises a tension, strain, or stress in boundary material of the package due to the package boundary reconfiguring to adhere or immobilize to at least some portions of the fluidic features. In some embodiments, the rupturing of the atleast one package comprises a downward pressure on the package against the fluidic device by the removal of at least a portion of the delivery fluid. In some embodiments, the removal of the delivery fluid is at least partially controlled by the interaction of the delivery fluid with the fluidic features, in particular an interaction that enables a capillary flow, or a wetting flow. In some embodiments, the rupturing of the at least one package comprises a rapid re- hydration of the package with the removal of the delivery fluid from the environmental atmosphere. In some embodiments, the rupturing of the at least one package comprises a controlled re-hydration of the package with the removal of the delivery fluid from the environmental atmosphere. In some embodiments, the rupturing of the at least one package comprises an osmotic pressure mechanism. In some embodiments, the rupturing of the at least one package comprises a virus. In some embodiments, the rupturing of the at least one package comprises an enzyme. In some embodiments, the rupturing of the at least one package comprises a lysate. In some embodiments, the rupturing of the at least one package comprises an electric field. In some embodiments, the rupturing of the at least one package comprises an electro-magnetic field. In some embodiments, the rupturing of the at least one package comprises temperature change or temperature gradient. In some embodiments, the rupturing of at the at least one package comprises triggering an exothermic reaction before, during or after removal of delivery fluid to the fluidic device. In some embodiments, the rupturing of at the at least one package comprises triggering an endothermic reaction before, during or after removal of delivery fluid to the fluidic device. In some embodiments, surface of the fluidic features might be pre-treated with cell lysing agents prior to package to fluidic feature interactions. In some embodiments, surface of the fluidic devices might be functionalized with package rupturing molecules prior to package to fluidic device interactions. In some embodiments, the rupturing of at least one package comprises sonication. In some embodiments, the rupturing of at least one package comprises laser dissection.

[0239] In some embodiments, the ruptured package on the surface of the open fluidic device releases said package’s package fluid and at least two macromolecules, the released package fluid and at least two macromolecules then flow within the fluidic features of said fluidic device, positioning the at least two macromolecules within the fluidic features. In some embodiments, the flow of the said package fluid within said fluidic features comprises a capillary flow, evaporative flow, micro-convection flow, osmotic flow, pressure driven flow, gravity driven flow, thermal-capillary convection flow, thermal flow, convection currents flow, surface-tension flow, Maragoni-effect flow, fluid flow from relatively morehydrophobic surface to a relatively more hydrophilic surface, wetting flow, de-wetting flow, flow to reduce the overall surface tension energy, electrokinetic flow, electroosmotic flow, or electrophoresis flow.

[0240] In some embodiments, the package fluid flow direction(s), speed(s), or location(s) within the fluidic features is at least in part, for at least a period of time, determined by the physical topology, geometry, layout, dimension, edges, corners, shapes, depth, length, width, surface roughness, surface properties, or surface energy of the fluidic features. In some embodiments, the package fluid flow direction(s), speed(s), or location(s) within the fluidic features is at least in part, for at least a period of time, determined by the relative prominence, or relative depression, of said fluidic features with respect to neighboring fluidic features. In some embodiments, the package fluid flow direction(s), speed(s), or location(s) within the fluidic features is at least in part, for at least a period of time, determined by at least one environmental condition. In some embodiments, the package fluid flow direction(s), speed(s), or location(s) within the fluidic features is at least in part, for at least a period of time, determined by at least one external force.

[0241] In some embodiments, the package fluid’s air-fluid interface topology, boundary, contours, shape, or movement within the fluidic features is at least in part, for at least a period of time, determined by the physical topology, geometry, layout, dimension, edges, corners, shapes, depth, width, length, surface roughness, surface properties, or surface energy of the fluidic features. In some embodiments, package fluid’s air-fluid interface topology, boundary, contours, shape, or movement within the fluidic features is at least in part, for at least a period of time, determined by the relative prominence, or relative depression, of said fluidic features with respect to neighboring fluidic features. In some embodiments, package fluid’s air-fluid interface topology, boundary, contours, shape, or movement within the fluidic features is at least in part, for at least a period of time, determined by at least one environmental condition. In some embodiments, package fluid’s air-fluid interface topology, boundary, contours, shape, or movement within the fluidic features is at least in part, for at least a period of time, determined by at least one external force.

[0242] Figure 5 is a schematic that demonstrates an embodiment whereby a package containing a package fluid and macromolecules is ruptured on the surface of the open fluidic device to generate spread of macromolecules. At time point (i), a package (503) is brought into contact with the surface of the open fluidic device (501) that comprises fluidic features (502) by means of a delivery fluid (not shown). The package contains macromolecules (505) which are suspended in a package fluid and contained within a package boundary (504). Inthis example, the fluidic features are channels, sized appropriately for the dimensions of the macromolecules contained within the package. At time point (ii), the delivery fluid has been completely or mostly removed from the surface of the fluidic device, and the package has started the process of rupturing. In this particular example, the package boundaries (512) become poorly defined as the package fluid (511) flows preferentially into the channels (515), directing the package fluid flow from the center of the package, outwards along the channels in both directions, as indicated by the arrows (516). The package fluid flow within the channels exerts a force on the macromolecules (514), and positions said macromolecules within the channels (515). The package fluid flow is preferentially maintained in the channels, as the channel surfaces (518) have a property that is largely hydrophilic, while the surface regions between the channels (519) have a property that is largely hydrophobic. This is drawn schematically with a cross-sectional view of the open fluidic device (517). At time point (iii), the package is completely ruptured, the package fluid and delivery fluid has been completely removed, and the result is the positioning of the macromolecules (532) within the open fluidic device channels (531) to form a spread.

[0243] Figure 6 is a time series of fluorescent images that demonstrate an embodiment of a cell rupturing on the surface of a fluidic device with fluidic features. In this particular example, the rupture of the cell distributes the chromosomes with a high degree of symmetry to generate an oval shaped spread. Such symmetry is due to the package fluid flowing in the left and right directions along the x-axis and within the channels from the center cell rupture position. In this particular example, the channels are parallel to each other and orientated horizontally in the image, so that the package fluid is constrained to flow horizontally along the x-axis within the channels, and inhibited from flow in the vertical y-axis direction across the hydrophobic barriers between adjacent channels. An oval spread profile is a predicable outcome with a parallel, uniform channel array, demonstrating the ability to tailor or design spread profiles by fluidic feature design.

[0244] At time point (i) in Figure 6, the delivery fluid has been mostly removed from the surface of the fluidic device via capillary flow and evaporation, and the cell has begun the process of rupturing on the surface of the fluidic device. The chromosomes were previously stained with a fluorescent dye prior to introduction to the device, and are collectively visible as a fluorescent blob (601). The cell has deformed, partially occupying the fluidic features underneath the cell, allowing the fluidic features of the device to be visible as the fluorescent chromosomes enter them. Six second later at time point (ii), the rupture of the cell continues, resulting in the cell fluid flowing in the left and right directions within the hydrophilicchannels as described in Figure 5, with the cell’s chromosomes following the cell fluid flow from the rupture center position to the left and right directions within the channels, while being inhibited from flowing up and down by the hydrophobic barriers between the channels. As the chromosomes disperse into the channels, individual chromosomes within the channels become visible (611 and 612). Another six second later at time point (iii), the rupture process has completed, and the chromosomes are now almost completely distributed and immobilized entirely within the channels to form a spread, with individual chromosomes within the channels clearly visible (621 and 622). The position of the channels is now clear from the fluorescent signature of the chromosomes, and are indicated by the set of arrows (623).

[0245] Figure 7 is a scanning election microscope (SEM) image demonstrating the positioning of chromosomes (702) within the fluidic features (701) of the fluidic device. In this particular example, the fluidic features are channels.

[0246] In some embodiments the spread generated from a package comprising a package fluid and at least two macromolecules rupturing and positioning the at least two macromolecules within the fluidic features of an open fluidic device has a parameter that is at least in part controlled by the said fluidic features. In the preferred embodiment, the parameter comprises the area of the spread. In some embodiments, the parameter comprises the major diameter of the spread. In some embodiments, the parameter comprises the minor diameter of the spread. In some embodiments, the parameter comprises the shape of the spread boundary. In some embodiments, the parameter comprises the relative orientation of the major axis of the spread. In some embodiments, the parameter comprises the position of the spread on the fluidic device.

[0247] In the preferred embodiment, the shape and size of the spread is controlled at least in part by the design of the fluidic features. In some embodiments whereby the fluidic features comprise hydrophilic channels, the direction, density, depth, width, cross-section, or pitch of the channels can be modified to generate oval-like spreads with a defined major axis or a defined minor axis. In some embodiments, the minor axis is defined by the cell diameter on the surface of the device. In some embodiments, the major axis is defined by the speed of the flow of the package fluid in the fluidic features of the device. An example of one preferred embodiment is shown in Figure 8 where-by a bright-field image of chromosome spreads on the surface of the fluidic device demonstrates the consistency of the spread’s oval shape. Within each rectangular box annotated in Figure 8 there is a single oval spread of chromosomes originating from a single cell. One such rectangular box annotation is identified as 801. Unlike traditional cytogenetic methods whereby the shape and orientationof the spreads are completely random, here the spread shape and orientation can be defined with a high level of control by the selection of the fluidic feature design.

[0248] In some embodiments, the spread shape can be selected based on the underlying patterned fluidic features. In some embodiments, the shape is oval, circular, square, polygon, triangular, or rectangular. In some embodiments, the patterned fluidic features are selected to generate a symmetry along at least one axis, or at least two axes in the spread. In some embodiments, the patterned fluidic features are selected to generate a rotational symmetry in the spread. For example, the patterned fluidic features may comprise a 2D array of pillars in a square or hexagonal lattice. In some embodiments, the patterned features are selected such that at least one axis of the spread has a maximum size limit. In some embodiments, the patterned features are selected such that at least one axis of the spread has a minimum size limit. In some embodiments, the patterned features are selected such that at least one axis of the spread has a variable size.

[0249] Figure 9 and Figure 10 are both fluorescent images of chromosome spreads on open fluidic devices that demonstrate the ability to tune a spread shape, and chromosome distribution within the spread, with the design and selection of the fluidic features. In Figure 9, the channel widths and separation spacing is 3 microns, resulting in oval spreads with a major axis diameter of 75 microns. In Figure 10, the channel widths and separation spacing is 1.3 microns, resulting in oval spreads with a major axis diameter of 120 microns.

[0250] Figure 11 and Figure 12 are both fluorescent images of chromosome spreads on open fluidic devices that demonstrate the ability to disperse and separate the chromosomes when fluid features are pillars that have hydrophobic roofs, and are separated from each other with hydrophilic trenches. In Figure 11, 2 micron wide pillars with 2 micron spacing are populated with fluorescently stained chromosomes positioned within the trenches. Unlike the previous examples of channels, the pillars offer a greater degree of freedom for the chromosomes to position themselves, and overall have no orientation bias between the x and y axis. Figure 12 is similar, except that the spacing between the pillars is larger (4 microns), allowing for an even greater degrees of freedom for the chromosomes to orientate themselves within the fluidic features. In this image, the hydrophobic roofs of the pillars are annotated with white boxes (1212), allowing for improved visibility that the chromosomes (1213) avoid the pillar roofs, and prefer the hydrophilic trenches separating the pillars. In addition, for this particular example, the cell contained both chromosomes and ecDNA (1214, 1215). The effective manner in which the fluidic device allows for the physical separation of the chromosomesand ecDNA demonstrates an embodiment application of using this device or method to facilitate the identification, count, and classification of a cell’s ecDNA.

[0251] In some embodiments, the shape of the spread may be controlled at least in part by the characteristics of the capillary flow of the package fluid within the fluidic features. In some embodiments, the shape of the spread may be controlled at least in part by the characteristics of the wetting flow of the package fluid within the fluidic features. In some embodiments, the shape of the spread may be controlled at least in part by the characteristics of the de-wetting flow of the package fluid within the fluidic features. In some embodiments, the shape of the spread may be controlled at least in part by the interaction of the package fluid and the surface energy characteristics of the fluidic features. In some embodiments, the shape of the spread may be controlled at least in part by the interaction between the macromolecules and the fluidic features.

[0252] In the preferred embodiment, for a particular package type and sample preparation method, the distribution of sizes for the spread shape is known, and the variation of in this distribution is less than the spreads generated by dropping a similarly prepared solution of packages on a glass slide using traditional cytogenetic methods.

[0253] In the preferred embodiment, for a particular package type and sample preparation method, the distribution of sizes for the spread shape is known, and can be used to calculate the package concentration in the delivery solution that provides the optimal spread density on the surface of the open fluidic device. In the preferred embodiment, the optimal spread density is that which provides the highest yield per unit area of the open fluidic device. In some embodiments, the calculated spread density as a function of starting package concentration comprises a random distribution placement of ruptured packages on the surface of the fluidic device, in particular, a Poisson distribution, or a Poisson point process. In some embodiments, the calculated spread density as a function of starting package concentration comprises a high likelihood of packages rupturing at certain specific locations on the surface of the fluid device.

[0254] In some embodiments, the fluidic features comprise a hydrophilic channel or hydrophilic chamber with a porous roof in which the package fluid may flow through. In some embodiments, the porous roof will allow a package macromolecule comprising a certain property to pass through. In some embodiments, the porous roof will forbid a package macromolecule comprising a certain property to pass through. In some embodiments, the property may comprise a size, a shape, a length, a width, a height, a diameter, a mass, a charge density, a quantity of charge, a surface charge density, a surface chemistry, a surfacehydrophobicity, a presence of a bound label, a presence of a bound body. In some embodiments whereby a molecule of a certain property is able to pass through the porous roof and enter the chamber or channel under said roof, said macromolecule can then be further isolated, collected, or processed. In some embodiments, the macromolecule is collected by flowing a solution through the chamber or channel to suspend said macromolecule in the solution. In some embodiments whereby a macromolecule of a certain property is prevented from passing through the porous roof, and remains on the surface of the porous roof, said molecule can then be further isolated, collected, or processed. In some embodiments, the macromolecule is further isolated, collected or processed with dispersion of a solution from a dispensing system, or by contact with a contact probe system.

[0255] Figure 13 is a schematic that demonstrates an embodiment whereby a package containing a package fluid and macromolecules is ruptured on the surface of the open fluidic device with a porous roof. At time point (i), immediately prior to rupture of the package (1304), its package boundary (1305) is in contact with the surface of a fluidic device (1301). After rupture, the package will release its package fluid, large macromolecules (1306), and small macromolecules (1307), allowing the fluid and small macromolecules to flow (1308) through the porous roof (1303) and enter the hydrophilic fluidic channels (1302) below, while the porous roof prevents the larger macromolecules from passing through. At time point (ii) after the rupture, the larger macromolecules (1314) remain positioned along the porous roofs (1313), while the smaller macromolecules (1316) are contained within the channels (1312) of the fluidic device (1311).

[0256] In some embodiments, after the package first physically interacts with the surface of the open fluidic device, but prior to the package at least partially adhering to the surface of the open fluidic device, the package is in a mobile state in which the package can transit along the surface of the open fluidic device.

[0257] In some embodiments, an applied external force transits, or aids in the transit, the package along the surface of the open fluidic device. In some embodiments, an applied external force prevents, or retards, the transit of the package along the surface of the open fluidic device. In some embodiments, the external force comprises a fluid flow. In some embodiments, the fluid flow comprises a capillary flow, an evaporative flow, a wetting flow, or a convection flow. In some embodiments the external force comprises a mechanical blade moving along the surface of the open fluidic device. In some embodiments the mechanical blade pushes the package. In some embodiments the mechanical blade pulls the package in the receding meniscus of the delivery fluid. In some embodiments, the external forcecomprises an electric field. In some embodiments the external force comprises a vibration of the fluidic device or the application of a sonication force.

[0258] In some embodiments, the direction(s) of transit on the surface of the device may be limited to a finite set of paths as determined by the interaction of the package and the surface of the device. In some embodiments, the package may be limited to travelling along a pathway, or a network of pathways. In some embodiments, the pathways are connected to, or incorporate, at least one capture point where-by once the package interacts with such capture point, the magnitude of the force or energy required to exit the package from the capture point is higher than the magnitude of the force or energy required to transit such package along the pathway prior to interacting with said capture point. In some embodiments, the force or energy necessary to transit the package along the surface of the open fluidic device is provided at least in part by the flow of the delivery fluid as the delivery fluid is at least partially removed from the surface of the open fluidic device. In the preferred embodiment, only one package can occupy a capture point at one time.

[0259] In some embodiments, the pathway may comprise fluidic features. In some embodiments, the pathway may comprise fluidic features into which the package’s macromolecules are to be positioned within after said package is ruptured. In some embodiments, the pathway may comprise at least one open fluidic channel. In some embodiments, the width of the at least one channel is on the order of the package itself, preferably around 1 / 100 of the package diameter, or around 1 / 75 of the package diameter, or around 1 / 50 of the package diameter, or around 1 / 40 of the package diameter, or around 1 / 30 of the package diameter, or around 1 / 25 of the package diameter, or around 1 / 20 of the package diameter, or around 1 / 15 of the package diameter, or around 1 / 10 of the package diameter, or around 1 / 8 of the package diameter, or around 1 / 6 of the package diameter, or around 1 / 5 of the package diameter, or around ¼ of the package diameter, or around 1 / 3 of the package diameter, or around ½ of the package diameter. In some embodiments, the depth of the at least one channel is on the order of the package itself, preferably around 1 / 100 of the package diameter, or around 1 / 75 of the package diameter, or around 1 / 50 of the package diameter, or around 1 / 40 of the package diameter, or around 1 / 30 of the package diameter, or around 1 / 25 of the package diameter, or around 1 / 20 of the package diameter, or around 1 / 15 of the package diameter, or around 1 / 10 of the package diameter, or around 1 / 8 of the package diameter, or around 1 / 6 of the package diameter, or around 1 / 5 of the package diameter, or around ¼ of the package diameter, or around 1 / 3 of the package diameter, or around ½ of the package diameter.

[0260] In some embodiments the capture point is an abrupt modification to the pathway. In some embodiments, the modification may be a fork, a connection, a branching, a pit, a wall, a barrier, or a corner in the pathway. In some embodiments, the modification may be a rapid reduction or increase in a channel width, depth, or density on the surface, in which the pathway comprises said channel. For example, a 1.5 fold reduction or increase, or a 2 fold reduction or increase, or a 3 fold reduction or increase, or a 5 fold reduction or increase, or a 10 fold reduction or increase. In some embodiments, the modification may be a transition from a pathway to a region that comprises at least one pit, or at least one hole, or at least one pillar, or at least one boundary, or at least one barrier, or at least one physical obstacle, or at least one fluidic feature. In some embodiments the modification comprises a change in surface energy, or surface roughness, or surface chemistry, or anti-fouling property.

[0261] In some embodiments, the positioning or density of capture points on the surface of the open fluidic device is selected to deterministically position the location of the ruptured packages on the surface of the open fluidic device. In the preferred embodiments, the positions of the capture points are selected such that regions of macromolecule spreads from neighboring packages do not overlap. In the preferred embodiments, the positions of the capture points are selected such that the density of ruptured packages on the surface of the open fluidic device is maximized. In some embodiments the separation distance of the capture points are around ½ the diameter of the package, or around ¾ the diameter of the package, or around the diameter of the package, or around 1.25 fold the diameter of the package, or around 1.5 fold the diameter of the package, or around 2 fold the diameter of the package, or around 3 fold the diameter of the package, or around 5 fold the diameter of the package, or around 10 fold the diameter of the package. In some embodiments, the separation distance of the capture points are around ½ the long axis diameter of the ruptured package spread, or around ¾ the long axis diameter of the ruptured package spread, or around the long axis diameter of the ruptured package spread, or around 1.25 fold the long axis diameter of the ruptured package spread, or around 1.5 fold the long axis diameter of the ruptured package spread, or around 2 fold the long axis diameter of the ruptured package spread, or around 3 fold the long axis diameter of the ruptured package spread, or around 5 fold the long axis diameter of the ruptured package spread, or around 10 fold the long axis diameter of the ruptured package spread. In some embodiments, the separation distance of the capture points are around 10 microns, or around 20 microns, or around 30 microns, or around 50 microns, or around 75 microns, or around 100 microns, or around 125 microns, or around 150 microns, oraround 200 microns, or around 250 microns, or around 300 microns, or around 400 microns, or around 500 microns.

[0262] Figure 14 is a schematic that demonstrates various embodiments of fluidic features on an open fluidic device, that can serve as capture points. In this example, a collection of hydrophilic channels (1402) that are orientated along the x-axis are separated from each other by hydrophobic walls (1401). A package in contact with such a fluidic device that is still mobile by the presence of delivery solution will have its freedom of movement largely limited to the x-axis, along the channels in the left or right direction which serve to be the pathway. However, when such a package interacts with a capture point, a fluidic feature that presents a barrier along the x-axis to a mobile package interacting with the surface of the device, the package is more likely to come to rest at this location. Examples of such capture point include a vertical channel connecting two adjacent channels (1403), or a vertical channel connection three channels (1404), or a multitude of vertical channels connecting adjacent channels (1405).

[0263] Figure 15 demonstrates an example of immobilizing cells at capture points. At time point (i) there is a thin film of delivery fluid on the surface of the open fluidic device (1502), wherein the device consists of an array of hydrophilic 3 micron wide horizontal channels separated by a 3 micron wide hydrophobic horizontal walls. Pairs of horizontal channels are connected with a vertical channel every 100 microns as shown by the arrows (1504), forming a capture point (1503) as described in the embodiment drawing of Figure 14 (1403). On the surface of the fluidic device are cells suspended in a thin film of delivery fluid. In particular a cell (1501) is shown that is mobile and moves with the flow of the thin film of delivery fluid as said fluid is removed from the surface of the device by evaporation and capillary flow, resulting in the movement of the cell on and along the surface of the device in the horizontal x-axis direction following the pathway of horizontal channels until it encounters the capture point (1503). At time point (ii) the cell (1511) is immobilized at the capture point, and the remaining delivery fluid on the surface of the device continues to be removed from the surface of the device. At time point (iii) the delivery fluid has largely been removed from the surface of the device, and the cell (1521) has ruptured, releasing its cell fluid and chromosomes into the fluidic features to generate an oval chromosome spread who’s center of mass is co-located at the cell’s respective capture point. Fluidic Device

[0264] In this set of embodiments, the positioning of macromolecules from a package on the surface of the fluidic device to generate a spread is at least in part facilitated via theinteraction of the package contents comprising the package fluid and the at least two macromolecules, with the patterned surface fluidic features of the open fluidic device.

[0265] In some embodiments, the patterned fluidic features may comprise patterned topologies or patterned surface energy regions that are patterned with at least one confining dimension suitably sized to accommodate at least a single macromolecule. In some embodiments, the patterned fluidic features may comprise patterned topologies or patterned surface energy regions that are patterned with at least one confining dimension suitably sized to accommodate only one macromolecule. In some embodiments, the at least one confining dimension is a length dimension, a width dimension, a depth dimension, a height dimension, cross-sectional dimension, a radius dimension, a diameter dimension, an average distance between two planes dimension.

[0266] In some embodiments, the patterned fluidic features may comprise at least one confining or non-confining dimension that is constant over a length scale, or changes randomly over a length scale, or changes due to some design intent over a length scale on surface of the fluidic device. In some embodiments, the patterned fluidic features may comprise a regular period or a random layout in their arrangement on the fluidic device surface.

[0267] For embodiments whereby the patterned fluidic features comprise a patterned topology, examples of patterned topologies include: open channels (isolated, connected, networked), pits, pillars, cones, corners, chevrons, branches, obstacles, pinch-points, forks, or intersections. In some embodiments, the cross-sectional profile of the topology may be square, rectangular, polygon, triangular, rounded, semi-oval, or semi-circular.

[0268] For embodiments whereby the patterned fluidic features comprise a patterned surface region, examples of pattern surface regions include: lines, rectangles, circles, hexagons, polygons, squares, open channel walls, open channel floors, open channel edges, open channel corners, pit walls, pit floors, pit edges, pillar bases, pillar walls, pillar roofs, pillar edges, cone walls, cone spikes. In some embodiments the surface region may be flat, or contoured, or have corners, or have edges.

[0269] The patterned fluidic feature may comprise an at least one confining dimension that is approximately 25% the length of said macromolecule’s axes, or is approximately 30% the length of said macromolecule’s axes, or is approximately 35% the length of said macromolecule’s axes, or is approximately 40% the length of said macromolecule’s axes, or is approximately 45% the length of said macromolecule’s axes, or is approximately 50% the length of said macromolecule’s axes, or is approximately 55% the length of saidmacromolecule’s axes, or is approximately 60% the length of said macromolecule’s axes, or is approximately 65% the length of said macromolecule’s axes, or is approximately 70% the length of said macromolecule’s axes, or is approximately 75% the length of said macromolecule’s axes, or is approximately 80% the length of said macromolecule’s axes, or is approximately 85% the length of said macromolecule’s axes, or is approximately 90% the length of said macromolecule’s axes, or is approximately 95% the length of said macromolecule’s axes, or is approximately 100% the length of said macromolecule’s axes, or is approximately 105% the length of said macromolecule’s axes, or is approximately 110% the length of said macromolecule’s axes, or is approximately 115% the length of said macromolecule’s axes, or is approximately 120% the length of said macromolecule’s axes, or is approximately 125% the length of said macromolecule’s axes, or is approximately 130% the length of said macromolecule’s axes, or is approximately 135% the length of said macromolecule’s axes, or is approximately 140% the length of said macromolecule’s axes, or is approximately 145% the length of said macromolecule’s axes, or is approximately 150% the length of said macromolecule’s axes, or is approximately 160% the length of said macromolecule’s axes, or is approximately 170% the length of said macromolecule’s axes, or is approximately 180% the length of said macromolecule’s axes, or is approximately 190% the length of said macromolecule’s axes, or is approximately 200% the length of said macromolecule’s axes, or is approximately 250% the length of said macromolecule’s axes, or is approximately 300% the length of said macromolecule’s axes, or is approximately 500% the length of said macromolecule’s axes.

[0270] The patterned fluidic feature may comprise an at least one confining dimension that is approximately 0.1 -0.2 microns, or 0.2-0.3 microns, or 0.3-0.4 microns, or 0.4-0.5 microns, or 0.5-0.6 microns, or 0.6-0.7 microns, or 0.7-0.8 microns, or 0.8-0.9 microns, or 0.9-1.0 microns, or, 1.0-1.1 microns, or 1.1-1.2 microns, or 1.2-1.3 microns, or 1.3-1.4 microns, or 1.4-1.5 microns, or 1.5-1.6 microns, or 1.6-1.7 microns, or 1.7-1.8 microns, or 1.8-1.9 microns, or 1.9-2.0 microns, or 2.0-2.2 microns, or 2.2-2.4 microns, or 2.4-2.6 microns, or 2.6-2.8 microns, or 2.8-3.0 microns, or 3.0-3.5 microns, or 3.5-4.0 microns, or 4.0-4.5 microns, or 4.5-5.0 microns, or 5.0-6.0 microns, or 6.0-7.0 microns, or 7.0-8.0 microns, or 8.0-9.0 microns, or 9.0-10 microns.

[0271] In some embodiments, the patterned fluidic features may comprise a multitude of patterned topologies or patterned surface energy regions, wherein a variety or range of different confining dimensions are suitably sized to accommodate a variety or range of differently sized macromolecules.

[0272] In the embodiments whereby the at least two macromolecules comprise chromosomes, or more preferably meta-phase or pro-phase human chromosomes, the at least one confining dimension is between 0.25 to 10 microns, or more preferably between 0.5 to 5 microns. For example, a single human meta-phase chromosome that is approximately 1.5 micron in width, 1.5 micron in height, and 10 microns in length could be accommodated in an open channel that is 2 microns wide, 2 micron deep, and 50 microns in length.

[0273] In some embodiments, the patterned fluidic features may comprise patterned topologies or patterned surface energy regions that are patterned of a size to accommodate multiple macromolecules in a single-file fashion, and thus comprise at least one non- confining dimension that is between 2-10000 microns, or more preferably, 5-1000 microns. For example, a single-file row of human metaphase or prophase chromosomes, aligned along their respective major axis would require an open channel approximately 500 microns in length to accommodate all 46 chromosomes from a single cell.

[0274] In some embodiments, the patterned fluidic features may comprise of patterned topologies or patterned surface energy regions that are patterned of size to accommodate a macromolecule or a long nucleic acid molecule in at least a partially elongated state, and thus comprises at least one confining dimension that is between 5-1000 nm, or more preferably, 10-500 nm. In some embodiments, the patterned fluidic features may comprise of patterned topologies or patterned surface energy regions that are patterned of a size to accommodate a macromolecule or a long nucleic acid molecule in at least a partially elongated state, and thus comprises at least one non-confining dimension that is between 10-5000 microns, or more preferably, 25-2000 microns. For example, a single 5 mega-base length long nucleic acid molecule fully elongated can be accommodated in a single open channel of length 2000 microns.

[0275] Figure 16 is a cross-section SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise 2 micron wide channels that are 1 micron deep and separated from each other by 2 microns of silicon. In the preferred embodiment, the surface region separating the channels is made hydrophobic or anti-fouling via chemical modification of the surface.

[0276] Figure 17 is a cross-section SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise silicon pillars that are 250 nm wide, and 1 micron tall. In the preferred embodiment, the surface region of the pillar roofs are made hydrophobic or anti-fouling via chemical modification of the surface.

[0277] Figure 18 is a top-down SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise an array of channels etched in borofloat glass that vary in width along the length of the channels. In this particular embodiment, the wider portion of the channel is 2 microns wide, and sized to accommodate a single human chromosome. The narrower portion of the channel, is 0.8 micron wide, and sized to block the entry of a human chromosome into the channel. The channel is 0.5 microns deep. In the preferred embodiment, the surface region separating the channels is made hydrophobic or anti-fouling via chemical modification of the surface.

[0278] Figure 19 is a top-down SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise an array of 0.8 micron wide pits in silicon that are 1 micron deep. In the preferred embodiment, the surface region separating the pits is made hydrophobic or anti-fouling via chemical modification of the surface.

[0279] Figure 20 is a top-down SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise an array of 0.3 micron wide pits in silicon that are 1 micron deep. In the preferred embodiment, the surface region separating the pits is made hydrophobic or anti-fouling via chemical modification of the surface.

[0280] Figure 21 is a top-down SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise an array of 0.3 micron wide pillars in silicon that are 1 micron deep. In the preferred embodiment, the surface region of the pillar roofs are made hydrophobic or anti-fouling via chemical modification of the surface.

[0281] Figure 22 is a top-down SEM image demonstrating an embodiment of an open fluidic device with fluidic features that comprise a circular pit 20 microns in diameter that is connected to channels that are 1.5 micron wide and radiate outwards 25 microns in length. In the preferred embodiment, the surface region separating the pits and channels is made hydrophobic or anti-fouling via chemical modification of the surface.

[0282] In some embodiments, the patterned fluidic features may comprise of patterned topologies or patterned surface energy regions that are patterned of size to accommodate at least one ecDNA, and thus comprise at least one confining dimension that is between 0.1 to 5.0 microns, or more preferably 0.2 to 2.0 microns. For example, a 5 megabase ecDNA in a compact circular state may range in size from 0.01 to 2.0 micron in diameter, more typically from 0.1 to 1.0 micron in diameter.

[0283] In some embodiments, the patterned fluidic features may comprise patterned topologies or patterned surface energy regions that are patterned with at least one confining dimension suitably sized to exclude at least a single macromolecule with a physicaldimension that lies within a certain range or set of values. In some embodiments, the at least one confining dimension may comprise a length dimension, a width dimension, a depth dimension, a height dimension, cross-sectional dimension, a radius dimension, a diameter dimension, or an average distance between two planes dimension. In some embodiments, the physical dimension may comprise a width, a length, a diameter, a mass, a volume, a density, a cross-sectional area. For example, an open fluidic channel with a width of 0.2 micron such that a human meta-phase chromosome with a 2 micron cross-sectional width is unable to be physically accommodated by the channel.

[0284] In some embodiments, the patterned fluidic features may comprise patterned topologies or patterned surface energy regions that are patterned with at least one confining dimension suitably sized to exclude at least a single macromolecule of type A, and accommodate at least a single macromolecule of type B, wherein type A and type B are macromolecules which have a physical dimension that is different from each other. In some embodiments, the at least one confining dimension may comprise a length dimension, a width dimension, a depth dimension, a height dimension, cross-sectional dimension, a radius dimension, a diameter dimension, or an average distance between two planes dimension. In some embodiments, the physical dimension may comprise a width, a length, a diameter, a mass, a volume, a density, a cross-sectional area. For example, an open fluidic channel with a width of 0.2 micron such that a human meta-phase chromosome with a 2 micron cross- section width is unable to be physically accommodated by the channel, however an ecDNA with a 0.1 nm cross-section width is able to be accommodated by the channel. In another example, a fluidic feature may be designed with physical dimensions to accommodate a smaller chromosome, such as human Chromosome 22, while excluding larger chromosomes, such as human Chromosome 1.

[0285] In some embodiments, the patterned fluidic features may comprise a plurality of patterned topologies of different characteristic dimensions, with or without patterned surface energy regions. A fluidic device can contain large scale topological features that influence the positioning of packages prior to or during the package rupture process, whether to influence the density of packages, the distance between adjacent packages, encourage or discourage the placement of multiple packages within a common radius or adjust the fine positioning of packages with respect to additional topological features. In some embodiments, the large scale features coexist with smaller scale features that direct the flow of the package contents when the package is ruptured, and in further embodiments the larger scale topologies coexist with finer scale topological features that orient and or confinemacromolecules. In further embodiments, large scale topological features are combined with microscopic roughness of the surface present uniformly over the fluidic device or specifically applied to portions of the fluidic device such that portions of the device have different roughness.

[0286] In some embodiments, the fluidic device may comprise at least one filter element. In some embodiments, the filter element may comprise a fluidic feature, a patterned topology, or a patterned surface energy. In the preferred embodiment, the filter element is suitably sized to hinder at least a single macromolecule of type A from transit through, and to allow at least a single macromolecule of type B from transit through, wherein type A and type B are macromolecules that have at least one physical property that is different from each other. In some embodiments, the filter element may comprise a channel, a corner, a pit, a pillar, a trench, an edge, a branch, a gel, a polymer, or a porous material.

[0287] Figure 23 is a fluorescent image demonstrating an embodiment of a fluidic feature designed to capture or block the transit of a suitably sized macromolecule, in this case, a human chromosome. In this particular example, a cell has been ruptured on the surface of an open fluidic device, releasing the cell’s fluid and chromosomes into the hydrophilic channels, such that the fluid and chromosomes flow in the channels in a direction shown by the arrows (2303), towards the filter element (2301). In this particular example, the filter element is a branch that splits the 3 micron wide channel into two smaller 1 micron wide channels. The human chromosome (2302) upon reaching the filter element comes to a rest, and is unable to progress into the smaller 1 micron wide channels of the branch filter element. In other embodiments whereby the ruptured cell contains smaller macromolecules such as ecDNA, the ecDNA is able to pass through the branch.

[0288] Figure 24 is a fluorescent image demonstrating an embodiment of limiting the outwards growth of a chromosome spread by using branched filter elements previously described in the description of Figure 23 to block the movement of chromosomes in the fluidic channels after rupture. In this particular example, the cell ruptured on the surface of the fluidic device somewhere between where the vertical line of filter elements are located, as indicated by the white arrows (2401). After the rupture, as the chromosomes flowed into the fluidic features of the device with the cell fluid, the chromosomes moving along the x-axis were blocked from progressing into a typical oval spread shape by the presence of the filter elements, and thus instead forming a rectangle shaped spread. Such an embodiment can be very effective in controlling or limiting the size and shapes of spreads, or separating various macromolecules of different sizes that originate from the package.

[0289] In some embodiments, the filter elements designed to capture, exclude, or accommodate at least a single macromolecule of type A is used to further analyze or process the at least a single macromolecule of type A. In some embodiments, the analysis may comprise interrogation. In some embodiments, the analysis may comprise interaction with a contact probe. In some embodiments, the analysis may comprise interaction with a dispensing system. In some embodiments, the processing may comprise a chemical reaction with at least one reagent, or at least one enzyme. In some embodiments, the interrogation may comprise an analysis of the number or genomic state of the macromolecule.

[0290] For example, Figure 25 is a fluorescent image that demonstrates an open fluidic channel with a width of 2 microns and a depth of 2 microns whereby human metaphase chromosomes (2503) and ecDNA (2501) originating from a single cell are physically accommodated by the channels (2502). Here, the smaller ecDNA are further separated from the chromosomes and isolated along the bottom corners of the channels due to the surface tension force of the evaporating package fluid in the channel, while the larger chromosomes experience surface tension forces from both bottom corners of the channels, and thus remain centered within the channels.

[0291] In another example, Figure 26 is a fluorescent image that demonstrates a chromosome spread whereby the chromosomes (2602) and the ecDNA (2601) that originated from the same package have been physically separated from each other. Such an embodiment is highly effective at generating ecDNA population statistics from a single package without the chromosomes obfuscating the ecDNA signal during interrogation. In one embodiment, the method of separation of the two populations is a fluid flow within the fluidic features after the spread has been generated, whereby the ecDNA and the chromosomes have a different mobility to said fluid flow. In some embodiments, the chromosomes are mostly immobilized and so their mobility is zero, or substantially less than the ecDNA mobility. In another embodiment, the ecDNAs are mostly immobilized and so their mobility is zero, or substantially less than the chromosome mobility.

[0292] In the preferred device embodiment, the fluidic features comprise regions of different surface energies. In particular, in the preferred device embodiment, the most elevated regions comprise a surface energy that is relatively hydrophobic with respect to the most depreciated regions. In another preferred device embodiment, the most elevated regions comprise a surface energy that is relatively antifouling with respect to the most depreciated regions.

[0293] In some embodiments, the channels are separated from adjacent and substantially parallel channels with a distance that can range from 0.2 to 100 microns. In someembodiments, the channels are patterned at fixed period. In some embodiments, the channels are patterned randomly. In some embodiments, the patterned features are at least partially comprising of pits or pillars. In some embodiments, the depth of the patterned features, or the height of the patterned features can range from 0 nm to 10 microns. (0 depth for embodiments where-by the channel is defined only by surface energy features.) In some embodiments, the patterned features are grouped in sets, in which they are physically co- located on the surface of the fluidic device. In some embodiments, the sets of features are defined to physically accommodate at least a portion of the macromolecules or long nucleic acid molecules that originate from at least one package. In some embodiments, the sets of features are defined to physically accommodate at least a portion of the macromolecules or long nucleic acid molecules that originate from only one package. In some embodiments, the sets are physically separated from each other sufficiently far that a single package cannot physically contact two adjacent sets. In some embodiments the physical distance between sets varies from 2 to 500 microns.

[0294] In some embodiments whereby the fluidic device comprises a channel or chamber with a porous roof, the porous roof may comprise silicon oxide, or silicon, or silicon nitride, or ITO, or glass, or quartz, or spin-on glass, or metal, or an oxidized metal, or a gel, or a polymer, or a cross-linked polymer. In some embodiments, the porous roof is manufactured with a process that comprises partially etching through a roof material until a certain level of porosity is achieved. Etching methods may comprise wet etching, acid etching, chemical etching, plasma etching, ion beam etching, reactive ion etching, inductively coupled plasma etching, sand blasting, focused ion beam etching. In some embodiments, the porous roof is manufactured with a process that comprises partially dissolving the roof material in a solvent. In some embodiments, the porous roof is manufactured by selectively removing a sacrificial material under the porous roof to form a channel or chamber under said roof. In the preferred embodiment, the sacrificial material is etched away with a solution, or dissolved away with a solvent. In some embodiments, the porous roof is manufactured with a process that comprises a degree of self-assembly. For example: self-assembly of nano particles, or self-assembly of micro particles, or self-assembly of nanorods or nanotubes, or self-assembly of beads, or self- assembly of polymer cross-linking. In some embodiments, the porous roof is manufactured by selectively controlling the cross-linking density of a polymer, such that the regions of low cross-linking density are porous, and the regions of high cross-linking density are non- porous. In some embodiments, the porous roof is manufactured by depositing or growing material over a channel or chamber and accumulating sufficient material to form a porousstructure over the chamber or channel. For example, Figure 27 shows an embodiment whereby a chamber comprises pillars with nickel seeds on the roof, on which additional nickel material can be grown via electroplating. In some embodiments the deposition process comprises sputtering, or evaporation, or thermal evaporation, or electron-gun evaporation, or electro-chemical growth, or spray coating, or spin coating, or lamination. In some embodiments, the porous roof comprises an adhesive film with porous properties.

[0295] Figure 27 is a schematic that demonstrates a particular embodiment of fabricating a fluidic device with a porous roof, whereby the porous roof comprises grown nickel deposits from a pattern array of seeds. In this drawn embodiment, a substrate is first patterned (i) with channels (2714), in which there are pillars (2713). Between the channels (2712) and on top of the pillars (2715) there is a seed layer a material to support the deposition of Nickel via electroless growth. After the deposition of nickel (ii), nickel films have formed (2722, 2725), growing up and out from the seeds. The result is nickel blobs on top of the pillars (2723), however the growth is stopped before completely sealing the roof, resulting in pores (2726) between the nickel blobs which allow for the formation of a porous roof over the channel (2724) of the fluidic device (2721). For a review on nickel plating micro-nanostructures, see [Ahn 2020]. In some embodiments the deposition process for generating the porous roof comprises sputtering, or evaporation, or thermal evaporation, or electron-gun evaporation, or electro-chemical growth, or spray coating, or spin coating, or lamination. In some embodiments, the porous roof comprises an adhesive film with porous properties. In some embodiments, the surface of the porous roof is further modified. In some embodiments the modification comprises functionalization, or a modification in surface energy, or a modification in the surface’s anti-fouling properties, or a modification in charge density.

[0296] In one preferred embodiment, the open fluidic device is compatible with a traditional cytogenetic workflow for generating cells spreads on glass slides as used in a cytogenetic lab, including reagents, sample preparation methods, sample dispensing, liquid dispensing, automated handling, imaging systems, or analysis software. In one preferred embodiment, the form-factor of the open fluidic device is compatible with standard microscope slide 25 mm by 75 mm in size. In some embodiments, the device is composed of an optically transparent material, for example plastic, glass, or quartz. In some embodiments, the device is composed of COC, PMMA, PC. In some embodiments, the device is composed of a non-transparent material, including silicon, non-transparent glass, or non-transparent plastic. In some embodiments, a coverslip is added by the user, while in other embodiments, the coverslip is integrated into the device.

[0297] In some embodiments the open fluidic device can include fiducial markers or alignment markers that can be used to enable visual alignment of the device either manually or with automated imaging systems. In some embodiments, there are multiple zones on the fluidic device, with each zone designed to physically isolate different cells or samples. In some embodiments, there are fiducial markers on the device that guide the user or automated dispensing system where to dispense a solution. In some embodiments there are fiducial markings or physical reference points on or in the device that guide the user or automated data acquisition imaging system as to the orientation of the device or interrogation region(s). Physical reference points could include notched or rounded corner(s). In some embodiments at least one well or topologically recessed regions is integrated onto the surface of the device to aid in solution containment during dispensing, or to provide guidance.

[0298] In some embodiments, the fluidic device has the form factor of a standard glass slide, and is approximately 25 mm x 75 mm in length and width. In some embodiments, the fluidic device comprises a barcode, or a label. In some embodiments, the fluidic device comprises at least one interrogation region. In some embodiments, the fluidic device has physical or hydrophobic barriers separating at least two interrogation regions. In some embodiments, cells from different samples are applied to different interrogation regions. In some embodiments, cells from different patients are applied to different interrogation regions.

[0299] In some embodiments, the fluidic device comprises at least 100 spreads, or at least 250 spreads, or at least 500 spreads, or at least 1,000 spreads, or at least 2,500 spreads, or at least 5,000 spreads, or at least 10,000 spreads, or at least 25,000 spreads. In some embodiments, the fluidic device comprises a yield of at least 50 useful spreads, or a yield of at least 100 useful spreads, or a yield of at least 250 useful spreads, or a yield of at least 500 useful spreads, or a yield of at least 1,000 useful spreads, or a yield of at least 2,500 useful spreads, or a yield of at least 5,000 useful spreads, or a yield of at least 10,000 useful spreads, or a yield of at least 25,000 useful spreads.

[0300] In some embodiments, the fluidic device comprises at least 1 useful spread per square millimeter, or at least 2 useful spreads per square millimeter, or at least 5 useful spreads per square millimeter, or at least 10 useful spreads per square millimeter, or at least 20 useful spreads per square millimeter, or at least 50 useful spreads per square millimeter, or at least 100 useful spreads per square millimeter, or at least 200 useful spreads per square millimeter. Positioning Macromolecules

[0301] In some embodiments, a package containing a package fluid and at least two macromolecules is ruptured on the surface of an open fluidic device, with said devicecomprising fluidic features, such that a fluid comprising the package fluid flows within the fluidic features, and positions the at least two macromolecules within the fluidic features such that the probability of the at least two macromolecules being in physical contact with each other is reduced when compared to a similar package that has been ruptured on a glass slide with traditional cytogenetic methods.

[0302] In some embodiments, two packages A and B, each containing a package fluid and at least one macromolecule are ruptured on the surface of an open fluidic device, with said device comprising fluidic features, such that a fluid comprising the package fluid from package A flows within the fluidic features and positions package A’s at least one macromolecule within the fluidic features, and a fluid comprising the package fluid from package B flows within the fluidic features and positions package B’s at least one macromolecule within the fluidic features such that the probability of two macromolecules, one originating from package A and the other from package B, being in physical contact is reduced when compared to a similar pair of packages that have been ruptured on a glass slide with traditional cytogenetic methods.

[0303] In some embodiments, the probability of the at least two macromolecules being in physical contact when positioned within the fluidic features of the open fluidic device is reduced when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods. In some embodiments, at least 10% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 20% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 30% of ruptured packages on the surface of the open fluidic device generate spreads with non- contacting macromolecules. In some embodiments, at least 40% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 50% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 60% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 70% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 80% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules. In some embodiments, at least 90% of ruptured packages on the surface of the open fluidic device generate spreads withnon-contacting macromolecules. In some embodiments, at least 95% of ruptured packages on the surface of the open fluidic device generate spreads with non-contacting macromolecules.

[0304] In some embodiments, the probability of the at least two macromolecules overlapping each other when positioned within the fluidic features of the open fluidic device is reduced when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods. In some embodiments, at least 10% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 20% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 30% of ruptured packages on the surface of the open fluidic device generate spreads with non- overlapping macromolecules. In some embodiments, at least 40% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 50% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 60% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 70% of ruptured packages on the surface of the open fluidic device generate spreads with non- overlapping macromolecules. In some embodiments, at least 80% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 90% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules. In some embodiments, at least 95% of ruptured packages on the surface of the open fluidic device generate spreads with non-overlapping macromolecules.

[0305] In some embodiments, the probability of the at least two macromolecules originating from a single ruptured package physically separating from each other beyond a maximum spread diameter when positioned within the fluidic features is reduced when compared to a similar package being ruptured on a glass slide with traditional cytogenetic methods. In some embodiments, the orientation of the maximum spread diameter is determined at least in part by the fluidic features. In some embodiments, the maximum spread diameter is 500 microns or less, or 400 microns or less, or 300 microns or less, or 200 microns or less, or 150 microns or less, or 140 microns or less, or 130 microns or less, or 120 microns or less, or 110 microns or less, or 100 microns or less, or 90 microns or less, or 80 microns or less, or 70 microns or less, or 60 microns or less, or 50 microns or less.

[0306] In some embodiments, the maximum separation distance of nearest-neighbor macromolecules originating from a single ruptured package is no more than 1 micron, or no more than 2 micron, or no more than 5 micron, or no more than 10 micron, or no more than 15 micron, or no more than 20 micron, or no more than 30 micron, or no more than 40 micron, or no more than 50 micron, or no more than 70 micron, or no more than 100 micron, or no more than 125 micron, or no more than 150 micron, or no more than 200 micron, or no more than 250 micron.

[0307] In some embodiments, the probability of the at least two macromolecules having a substantially similar orientation with respect to each molecule’s major axis when positioned within the fluidic features is increased when compared to a similar package being ruptured on a glass slide using traditional cytogenetic methods. Figure 28 is a fluorescent image that demonstrates an example whereby more than 90% of the human metaphase chromosomes originating from a single cell align their major axis along the channels of the fluidic device in the horizontal direction. In some embodiments, the at least two macromolecules’ respective major axis have a relative angle that is at least in part determined by the physical layout of the fluidic features. In another example, shown in Figure 29, the fluidic features can be modified to allow for greater degrees of freedom for direction selection of the molecule’s major axis. In this example, the fluidic features allow for the molecule’s major axis to predominantly align either in the vertical or horizontal direction. A multiple of different designs are feasible. For example, macromolecule axial alignment may be on a radial array, on a device similar to that shown in Figures 30 and 31.

[0308] In some embodiments wherein the at least two macromolecules comprise chromosomes, the probability of the at least two chromosomes having a similar nucleic acid density along the length of the each molecule’s major axis when positioned within the fluidic features is increased when compared to a similar package being ruptured on a glass slide using traditional cytogenetic methods. In the preferred embodiment, the linear nucleic acid density along the length of the macromolecule’s major axis is at least in part controlled by at least one confining dimension of the fluidic feature in which said molecule is positioned within. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 100-50 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 80-40 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 60-30 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s majoraxis is between 50-25 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 40-20 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 30-15 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 20-10 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 16-8 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 10-5 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 6-3 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 4-2 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 2-1 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 1-0.5 mega-bp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 800-400 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 500-250 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 400-200 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 300-150 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 200-100 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 150-75 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 100-50 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 80-40 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 50-25 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 40-20 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 30- 15 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 20-10 kbp per micron. In some embodiments, thelinear nucleic acid density along the length of the macromolecule’s major axis is between 15- 7.5 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 10-5 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 8-4 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 6-3 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 4-2 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 2-1 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 1.5-0.75 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 1-0.5 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 0.8-0.4 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 0.6-0.3 kbp per micron. In some embodiments, the linear nucleic acid density along the length of the macromolecule’s major axis is between 0.4-0.2 kbp per micron.

[0309] In the preferred embodiments, the linear density of the macromolecule is at least in part determined by at least one constraining dimension of a fluidic feature in which the macromolecule is at least partially within. In the preferred embodiment, the constraining dimension is the width of an open fluidic channel. In some embodiments, the channel width is approximately 3.0 microns, or approximately 2.8 microns, or approximately 2.6 microns, 2.5 microns, or approximately 2.4 microns, or approximately 2.3 microns, or approximately 2.2 microns, or approximately 2.1 microns, or approximately 2.0 microns, or approximately 1.9 microns, or approximately 1.8 microns, or approximately 1.7 microns, or approximately 16 microns, or approximately 1.5 microns, or approximately 1.4 microns, or approximately 1.3 microns, or approximately 1.2 microns, or approximately 1.1 microns, or approximately 1.0 microns, or approximately 0.9 microns, or approximately 0.8 microns, or approximately 0.7 microns, or approximately 0.6 microns, or approximately 0.5 microns.

[0310] Figure 32 is a fluorescent image that demonstrates the ability of manipulating the length of a chromosome, and stretching it beyond the chromosome’s natural length when at rest. In this particular example, a cell has been ruptured on a fluidic device comprising hydrophilic channels that are approximately 0.5 microns in width, below this human chromosome’s approximate normal 2 micron wide width. To accommodate the narrowchannels, the chromosomes are stretched lengthwise about twice their normal length. Such an embodiment allows for greater resolution of a patterned physical map along the length of the macromolecule. The challenge of generating long chromosomes on a bare glass slide to achieve high-resolution karyotyping has been a difficult problem for several decades, and although several elegant cell biological and biochemical means to prepare long chromosomes exist, they exhibit high variability and frequent low yields across different sample preparations and can require considerable biochemical expertise to master, making such methods impractical or extremely expensive for routine analysis. This embodiment enables a simple method for high-resolution cytogenetic analysis.

[0311] In some embodiments whereby the at least two macromolecules are chromosomes, a pair of chromatids from at least one of the chromosomes’s arm are separated from each other by a fixed distance. In some embodiments, the minimum distance is determined at least in part by at least one constraining dimension of a fluidic feature in which the macromolecule is at least partially within. In the preferred embodiments the fixed distance is between 3-2 microns, or between 2.5-1.5 microns, or between 2-1.5 microns, or between 1.5-1 microns, or between 1-0.5 microns, or between 0.75-0.25 microns. In some embodiments, the arm is a q arm. In some embodiments, the arm is a p arm.

[0312] In some embodiments, centromeres are made easier to identify during interrogation by positioning of chromosomes inside channels with channel widths larger than the natural width of a chromosome, as demonstrated in Figure 33, resulting in a pronounced X pattern around the centromere. The centromere is further emphasized by the fact that chromatin approaching the centromere no longer has the straight border that chromatids have when they are in direct contact with the walls. The stretching force of the channels also results in lower density of chromatin around the centromere, providing a reduction in stain density in the centromere region, further enhancing its signal.

[0313] In some embodiments, the width of the channels is larger than the natural separation between sister chromatids, and chromatids are pulled apart as illustrated in the fluorescent image Figure 34. Such an embodiment enables more efficient identification of the centromere of a chromosome, or multiple centromeres in the case of aberrant chromosomes with duplicated centromeres, as is demonstrated by the dicentric chromosome (3401). Cell biological techniques and biochemical techniques to lengthen chromosomes often make it difficult to determine the exact position of the centromere, leading to uncertainty in the interpretation of standard karyograms, but that problem is lessened by suitable channel design.

[0314] Chromosome identification procedures as used in clinical aneuploidy detection start with the classification of chromosomes according to centromere position as described in [Arsham 2017], sorting chromosomes according to whether they are metacentric (centromere in the middle), submetacentric (centromere a little away from the middle so that one arm is longer than the other), acrocentric (centromere close to one end), or telocentric (centromere right at the end).

[0315] In some embodiments, the chromosomal elements include but are not limited to centromere, telomere, chromatid, P-arm, Q-arm, satellite DNA, heterochromatin, or kinetochore are preferentially situated on some parts of fluidic features. Applications

[0316] In some embodiments, the addition, removal, diluting, rising, washing, suspending or washing of a sample, a spread, a macromolecule, a chromosome, a reagent, a labeling body, a gel, a polymer, a film, a fluidic feature, a well, a pit, a channel, from or on the surface of the fluidic device is performed with at least one manual, semi-automated, or automated solution handling process. In some embodiments, the solution handing process may comprising dispensing, coating, spin-coating, flowing, or dipping. In the preferred embodiments, these solution handling process are similar or compatible to traditional cytogenetic preparation protocols for preparing chromosome spreads on standard microscope slides. In some embodiments, the solution is applied to the entire surface of the fluidic device, or to at least a single region, or to at least one interrogation region, or to at least one spread, or to at least one sample, or to at least one macromolecule, or to at least a portion of at least one macromolecule.

[0317] In addition to condensed pro- or metaphase chromosomes, chromosomes of different phases are also possible. For example, interphase chromosomes for use with FISH applications. Furthermore, the nucleic acid being positioned may be a portion of a chromosome such as circular polynucleotides, or extra-chromosomal DNA (ecDNA), or nucleic acid originating from a source other than chromosomes such as mitochondrial DNA, an artificial mini-chromosomes, or microbial originated DNA, or circular DNA. The nucleic acid may be in chromatin form. The nucleic acid may be a long nucleic fragment, thus enabling fiber-FISH applications. Much like DNA-combing methods, long nucleic fragments will be aligned and elongated onto a surface to enable cytogenetic applications. However here, due to the patterned surface features or differential entropic energies, the long nucleic acid fragments will be aligned, arranged or elongated in a controlled manner rather than a purely random process. In addition, unlike combing that requires dipping a slide into a poolof sample, here the input sample can be dispensed via a pipette or probe in small quantities of 10s of microliters or less, and thus the input sample volume requirements are considerably less.

[0318] Described within are a multitude of different device embodiments. However, most embodiments share some basic similarities. First, an input sample comprising of suspended packages is introduced to the surface of an open fluidic device via the dispensing of a delivery fluid. Second, the package contents of macromolecules are ruptured on the surface of the fluidic device generating a spread of macromolecules from each package. Third, the macromolecules are prepared for interrogation, typically via exchange of reagent solutions, rinses, and additional processing. Forth, the macromolecules are interrogated.

[0319] The reagent materials and solutions may include any commonly used by someone trained in the art of performing cytogenetic analysis. These reagents may include various stains, dyes, fixatives, and solvents. Additional reagents may include various dyes for physical mapping, FISH-probes, labelling bodies, methylation dyes, non-methylation dyes. Figure 35 is a fluorescent image that demonstrates a FISH probe labeling of chromosome spreads that have been previously positioned in a fluidic device, whereby 3501 indicates an example of 4 bright FISH probes hybridized to the ends of a chromosome.

[0320] In some embodiments, prior to dispensing onto the fluidic device, or after dispensing onto the fluidic device, macromolecules are labelled with a labelling body to generate a physical map. In some embodiments, the physical map is a karyotype. In some embodiments, the physical map is a linear physical map. In the preferred embodiment, the physical map is an AT / CG density linear physical map.

[0321] In some embodiments, the surface of at least one of the boundary walls of the fluidic device that constitutes the interrogation region are modified to change the surface energy or add functionalization to promote nucleic acid molecule immobilization with the said surface. In some embodiments, a specific region of functionalization on the device surface is designed to immobilize a specific target of macromolecule. In some embodiments, the specific target is a type of chromosome. In some embodiments, the functionalization is designed to promote cell lysing or rupture, such as chip surface treated with chemical or enzymatic coating activated upon wetting or by co-factors in sample loading to be in contact with target cells or molecules.

[0322] In some embodiments, at least one portion of one chromosome is isolated for analysis or further processing. In some embodiments, the processing comprises an isolation. In some embodiments, the processing comprises a reaction with a reagent. In some embodiments thereagent comprises an enzyme. In some embodiments the further processing comprises amplification. In some embodiments the further processing comprises sequencing. In some embodiments the further processing comprises capture into a droplet. In some embodiments, the further processing comprises exposure to a solution or gel dispensed from a dispensing system. In some embodiments, the further processing comprises further interrogation by a contact probe system, or physical manipulation by a contact probe system. In some embodiments, the further processing comprises exposure to at least environmental condition. In some embodiments, the chromosomes are positioned within the fluidic features of an open microfluidic device that includes patterning of topological and / or surface energy modifications to form wells on the surface of the device, so as to physically contain a dispensed solution within said wells.

[0323] In addition to traditional cytogenetic methods and procedures of interrogation via optical imaging, this set of embodiment devices and methods in which the nucleic acid molecules are arranged in a more controlled manner on the device surface is highly amendable to other forms of interrogation and processing, either separately from optical imaging, or in conjunction with optical imaging. In one embodiment, the optical imaging is used to select specific macromolecules or long nucleic acid molecules for specific additional interrogation and processing. Non limiting examples of interrogation and processing methods that can be performed on the macromolecules include: interrogation with a contact probe system, an Atomic Force Microscopy (AFM), a Scanning Tunnelling Microscopy (STM), a Scanning Probe Microscopy (SPM), a Transmission Electron Microscopy (TEM), a Scanning Electron Microscopy (SEM), capture and removal from the surface with a contact probe system, or submergence in solution dispensed from a dispending system, PCR amplification or Sequencing. In a further extension of the embodiment, the at least one nucleic acid molecule in a drop of solution could be resuspended into the drop solution, and in another additional embodiment, said drop containing the at least one molecule can be extracted from the surface.

[0324] For all embodiment devices and methods described, the interrogation of the at least one portion of at least one macromolecules may be performed while the at least one portion of at least one macromolecule is at least partially elongated.

[0325] In some embodiments, the device or method facilitates the interrogation of a native state chromatin, a structural variation, a higher order structure, or any genomic information in a macromolecule, a chromosome, a long nucleic acid molecule, or ecDNA.

[0326] The devices and methods disclosed herein can enable improved quality of physical maps, for example karyotyping or FISH spreads. In some device and method embodiments, the sample input cell quantity is substantially reduced over traditional methods, thus allowing for a reduction in time required to multiply the cells via growth; or allowing direct interrogation of primary cells without culture, or scarce or rare samples, such as CFC (circulating fetal cells), CTC ( circulating tumor cells), forensic samples, environmental samples, archaeological samples (frozen tundra), extraterrestrial samples.

[0327] In some device and method embodiments, the macromolecules are presented, with improved physical alignment to a desired orientation, with reduced physical contact among the macromolecules within the spread, or improved elongation consistency, when compared to spreads prepared from similar packages by traditional cytogenetic methods. This disclosure demonstrates improved resolution of identifying genomic features within the chromosomes, thus reducing ambiguities in the image data, and allowing for greater automation, improved machine learning and artificial intelligent analysis, and reduced manual curation, when compared to spreads prepared from similar packages by traditional cytogenetic methods. This disclosure will enable more accurate data analysis, faster turnaround time and more precise and more conclusive diagnosis and prognosis report generation, ultimately leading to less patient suffering, in time and duration of sample acquisition, waiting time ( less so-called “Diagnostic Odyssey” that could last up to decades or life time), more efficient clinic practice, less clinician and staff exhaustion, and overall tremendous saving in medical cost and burden to society).

[0328] In some device and method embodiments, package macromolecule contents are manipulated at least in part by the fluidic device such that at least one region of interest within at least one macromolecule may be interrogated with at least one interrogation method or process, including a process that may vary dynamically in space or time, to un-obfuscate any information contained within the macromolecule, including any higher order nucleic acid structure, structural variation, or genomic information. In some device and method embodiments, the information to be interrogated may compromise dynamic genomic and epigenomic transformation (for example: degradation or reconstruction), structural arrangements (for example: chromatin domain, kinks or nodules, folds), epigenomic content (for example: TADs, regulatory binding complexes, methylation patterns), and genomic content (for example: sequencing or mapping, Cis or trans- regulatory functional elements, intro or exon boundaries, initiation, termination sites). In some embodiments, the information contained within the macromolecule is interrogated with some degree of native context, forexample: within the analytes originating from the macromolecule’s package, or the same sample, or the same individual or patient.

[0329] In some device and methods embodiments, in at least one portion of at least one macromolecule, a physical map is present. In some embodiments, the portion of the at least one macromolecule that comprises a physical map is elongated. In some embodiments, an interrogation of the physical map is used to identify the location, type of de novo or inherited change, pinpoint variant loci influencing or altering the biological or pathological impact. In the preferred embodiment, the identification is performed with the macromolecule in physical state that allows for the interrogation of additional contextual information, including functional, regulatory, or structural genomic information. In some embodiments, information generated by interrogation is used for diagnostic, prognostic, or identifying an actional drug target for a therapeutic agent.

[0330] For example, with a translation like bcr / abl, a means to disrupt the sequence order and in frame coding to create a new fusion kinase oncogene to drive tumorigenesis would be of interest. In another example, genomic repeat expansion or contraction ( Fragile X or FSHD ) related genetic disease. A device or method described in this disclosure may interrogate the macromolecule in a top-down manner and can allow for the further isolation and dissemination of the variant location, exact fusion point for diagnosis.

[0331] In another example, a retrovirus such as HIV, or a herpes virus such as EBV, could be integrated into a host genome in specific or random location or orientation, to disrupt the sequence order, coding framing, distances of functional elements, foreign sequence elements, within the host genome. In such an example, the devices or methods as described in this disclosure may enable the further isolation and dissemination of the insertion location, orientation , exact boundary sequence or fusion point, to determine if it is an in frame coding or out of frame coding sequence, to cause early protein truncation, or a new fusion protein, or if a strong viral promoter were inserted to drive an oncogene previously were expressed at lower level.

[0332] In another example, the devices or methods as described in this disclosure may have the ability to precisely identify in the genome through physically mapping, the location, type of de novo or genetic targeted large changes intractable by sequencing alone, and especially to pinpoint the intended locus target for gene editing or gene therapy in methods such as viral vector delivery (AAV type) or CRISPR-type, if an off target integration or modification sites due to errant or non-optimal recombination sites or guide RNA sequence probe design, it might have unintended detrimental short or long term genetic consequences to the patient. Itwould be of great medical value to quality control and proof test the vector or probe design by assessing the off target rate, and locations in the context of whole genome and its consequential biological or pathological outcome.

[0333] In another example, the devices or methods as described in this disclosure may have the ability to precisely identify in the genome through physically mapping, the location, type of de novo or genetic engineered large changes intractable by sequencing alone, and especially to pinpoint the intended locus target for gene editing in methods such as genetic engineering. It would be of great utility value to be able to visualize and monitor the engineered modification such as transfection or knock-in or knock-out processes, in the context of whole genome and its consequential biological trait improvement in molecular breeding. For example, a desired trait locus could be enhanced by introducing a much more robust promotor or enhancer, in the right location and orientation to the target gene, to increase its expression level or but knocking out a suppressive regulatory mechanism in the genome.

[0334] In some embodiments a package comprising a package fluid and at least one macromolecule is ruptured on the surface of an open fluidic device, positioning said at least one macromolecule within the fluidic features of said open fluidic device to generate a spread, and then identifying a region of interest (ROI) within the at least one macromolecule. In some embodiments the ROI comprises an entire macromolecule. In some embodiments the ROI comprises a portion of the macromolecule. In some embodiments, the ROI comprises at least two portions of the macromolecule.

[0335] In some embodiments, the ROI identification comprises interrogation of said macromolecule. In some embodiments, the ROI identification comprises an analysis of the macromolecule’s physical map. In some embodiments the macromolecule’s physical map is a karyotype banding pattern or an AT / CG density profile. In some embodiments, the analysis of the physical map comprises a comparison to a reference.

[0336] In some embodiments, the identified ROI is isolated for analysis or further processing. In some embodiments, the processing comprises an isolation. In some embodiments, the processing comprises a reaction with a reagent. In some embodiments the reagent comprises an enzyme. In some embodiments the further processing comprises amplification. In some embodiments the further processing comprises sequencing. In some embodiments the further processing comprises capture into a droplet.

[0337] Figure 36 is a schematic that demonstrates an embodiment of targeting an ROI within a macromolecule with a solution containing a reagent. In this particular example, an ROI(3603) is first identified (i) within a macromolecule 3605 positioned on the surface of the open fluidic device 3606. Here, the positioning of the ROI is determined by an analysis of the macromolecule’s physical map: the shaded banding along the length of the macromolecule. The ROI is then targeted with a dispensed volume of liquid 3604 from a dispenser 3601. In the preferred embodiment, the liquid solution contains at least one reagent 3602 which can engage directly or indirectly in a binding or enzymatic reaction with the ROI. Once dispensed (ii), the drop 3612 containing the at least one reagent 3613 has sufficient volume of solution to submerge the ROI (3611). In some embodiments, an oil is then dispensed on the fluidic device, covering the drop, with said drop maintaining contact with the device surface and ROI.

[0338] In some embodiments, the binding and / or enzymatic reaction takes place in the drop of solution that is dispensed with the reagent. In the preferred embodiment, the environmental conditions that the drop is contained in while the drop is in contact with the ROI are controlled to minimize evaporation. In the preferred embodiment, the volume of reagent solution dispensed is controlled to minimize exposure of the solution to non-ROI regions. In some embodiments, the amount dispensed may a single drop of reagent solution, or multiple drops of reagent solution. In some embodiments, there may be multiple, different reagent solutions dispensed on a single ROI.

[0339] In some embodiments, the reagent solution(s) are allowed to dry on the ROI, such that the reagents are physically localized in proximity to the ROI. In some embodiments, at least one reaction involving at least one reagent occurs in a solution that is dispensed or applied without said reagent. For example, a series of different reagents may be dispensed on the ROI(s) and allowed to dry. After drying, another solution not containing reagents is dispensed or applied on the surface of the fluidic device over an area that substantially exceeds the ROI boundaries. In this embodiment, cross-talk of reagent interaction with non- ROI regions may be controlled by limiting the opportunity of diffusion of reagents to non- ROI regions via time limited process, after which the regents may be rinsed.

[0340] Figure 37 is a schematic that demonstrates an embodiment of targeting multiple ROIs within a macromolecule, whereby each ROI is targeted with a specific solution. In this particular example, multiple ROIs (3704, 3705, 3706, 3708) are first identified (i) within a macromolecule (3707) positioned on the surface of the open fluidic device (3709). Here, the positioning of the ROIs is determined by an analysis of the macromolecule’s physical map: the shaded banding along the length of the macromolecule. Here shown is one dispenser 3701 capable of dispensing a solution containing at least one reagent 3703 on a desired ROI, and atleast a second dispenser (not shown) capable of dispensing at least a second solution with at least a second type of reagent on a desired ROI. By adjusting combination of solution at dispensed at each ROI, in addition to the volume of each solution dispensed at each ROI, the result is (ii) a desired reagent mixture can be selected for each ROI as desired (3721, 3722, 3723, 3724).

[0341] In some embodiments, the macromolecules are positioned within the fluidic features of an open microfluidic device that includes patterning of topological or surface energy modifications to form wells on the surface of the device, so as to physically contain the dispensed solution within said wells. Figure 38 is a schematic that demonstrates and embodiment of an open fluidic device 3801 with fluid capture wells 3803 onto which macromolecules (3802 and 3807) are positioned and then interrogated to identify ROI(s). In this particular example, the macromolecules are contained within a single well. In other embodiments, a macromolecule may span two or more wells. (i) A dispenser 3806 dispenses a solution of at least one reagent 3804 onto the selected ROI, which in this example is an entire macromolecule (3807), such that (ii) the selected ROI (3817) becomes submerged by the dispensed drop (3814) contained within the well. In the preferred embodiment, the size and density of patterning of patterning of the wells is such that the smallest ROI can be contained within a single well. In some embodiments an ROI may span multiple wells, requiring multiple dispensing events, at least one for each well. In one embodiment, the boundary of the wells are hydrophilic, while the regions between the wells is hydrophobic, such that a volume of liquid that can be dispensed into the well can exceeds the well’s volume, while still being physically constrained by the well boundaries. In some embodiments, an oil is then dispensed on the fluidic device, covering the drop, with said drop maintaining contact with the ROI and the device surface inside the well. In some embodiments, the bottom of the wells includes immobilized reagents on the surface of the well that can be re-suspended in the drop solution. In some embodiments, the reagents on the surface of the well are bound to the well surface by a cleavable linker, preferably a photocleavable linker. In some embodiments, the solution dispensed on the ROI contains no reagents, as the reagents originate from the well surface.

[0342] In the following set of embodiments, the macromolecules are positioned along the fluidic features of an open fluidic device and interrogated to identify ROI(s), and then target said ROI(s) for segregation or capture. In this set of embodiments, the ROI(s) to be targeted are on the surface of the open fluidic device, or are contained within a thin porous film on the surface of the device, or a combination there-of, and thus the ROI(s) are accessible to directinteraction with an applied or dispensed solution, a photon, or contact probing. In the preferred set of embodiments, the process of interrogating the macromolecule generates a coordinate map of the surface of the open fluidic device in which the molecules and their respective ROI(s) are located within said coordinate map. Employing such a map, the targeting of focused photons, dispensed solution, or a contact probe can be guided to the desired ROI(s) on the surface. In the preferred embodiment, the open fluidic device physically engages with a control instrument that interrogates the macromolecule, is the same instrument that directs the targeting of focused photons, dispensed solution, or contacting probing, such that all electrical mechanical systems within the instrument can share the same coordinate space to target molecules and ROIs within the coordinate map.

[0343] In some embodiments, the targeting is performed on a different instrument from the interrogation instrument, and fiducials on / within the open fluidic device are used to register the coordinate map.

[0344] Figure 39 is a schematic that demonstrates an embodiment where-by a macromolecule (3902) positioned on an open fluidic device (3901) has an ROI (3903) identified for capture. In this particular embodiment, the ROI is identified by a process that comprises an analysis of the molecule’s physical map: the shaded regions along the length of the molecule. In this embodiment, first (i) the identified ROI (3903) is segregated via photo-cleaving (3904) of the boundaries of the ROI. Once segregated, the contact probe (3905) with a functionalized point (3906) is lowered and positioned to contact the ROI using the previously registered ROI coordinates on the surface of the fluidic device. The desired ROI is then removed by (ii) contacting the contact probe functionalized point with the ROI under conditions that allow the ROI portion of the molecule to bind to the functionalized point (3912), such that the contract probe can retract from the surface with the ROI portion of the molecule (3911). In some embodiments whereby the ROI comprises the entire macromolecule, or a significant portion of the macromolecule, the contact probe may bind and remove the entire macromolecule without the need for photo cleaving.

[0345] In some embodiments, a polymer or gel is dispensed on at least one non-ROI portion of at least one macromolecule. In some embodiment, a polymer or gel is dispensed on at least one ROI portion of at least one macromolecule. In some embodiments, the dispensed polymer or gel is used to facilitate a chemical reaction. In some embodiments, the dispensed polymer is used to hinder a chemical reaction. In some embodiments, the polymer or gel is used promote the separation of at least a portion of a macromolecule from the surface of the fluidic device, in particular when said polymer or gel is brought into contact with a solution.In some embodiments, the polymer or gel is used to maintain the immobilization of at least a portion of a macromolecule on the surface of the fluidic device, in particular when said polymer or gel is brought into contact with a solution. In some embodiments, the polymer or gel is dispensed from a dispenser. In some embodiments, the polymer or gel comprises at least one reagent. In some embodiments, the polymer or gel can be cross-linked with the exposure to at least one wavelength of light. In some embodiments, the process of cross- linking results in a polymer or gel that is more resistive to being dissolved by a solvent, or less permeable to a reagent or solvent, or improve adherence to the fluidic device surface or macromolecule. In some embodiments, the polymer or gel can have bonds within its structure broken by the exposure to at least one wavelength of light. In some embodiments, the process of bond-breaking results in a polymer or gel that is less resistive to being dissolved by a solvent, or more permeable to a reagent or solvent, or reduced adherence to the fluidic device surface or macromolecule.

[0346] In some embodiments the output of interrogation of the spreads on the surface of the fluidic device comprises a karyotype. In some embodiments, the karyotype generated by the interrogation is used to diagnose chromosomal abnormalities. In some embodiments, these abnormalities may comprise structural variations. In some embodiments, these abnormalities may comprise Wolf-Hirschhorn syndrome, or Jacobsen syndrome, or Charcot-Marie-Tooth disease. In some embodiments, the abnormalities may comprise deletions, duplications, inversions, insertions, translocations, ring-formation, isochromosome-formation, and chromosome instability.

[0347] IN some embodiments, the “abnormal” chromosome is identified by a difference to a “normal” control chromosome. In some embodiments, the difference may comprise a genomic difference, a genomic variation, an epigenetic difference. In some embodiments, the “abnormal” chromosomes may not be identifiable with respect to patient’s “normal” chromosome, but instead may originate from a different organism from the patient, such as a bacteria or parasite. In some embodiments, the rare event flag is used to identify early stage cancer, or the recurrence of cancer that previous was previously in remission, or to assess or monitor the progress of a cancer therapy, or to assess or monitor molecular residual disease (MRD). In some embodiments, the rare event flag is used to assess or monitor the genetic or epigenetic health of a patient, in particular, a patient who is at risk of random genomic or epigenetic aberrations from exposure to certain conditions, for example: chemical exposure, radiation exposure, extreme temperature exposure, toxin exposure, pollution exposure.

[0348] In some embodiments, an assessment or monitoring of the population of ecDNA in a sample is determined. In some embodiments, single cell ecDNA population statistics are generated. In some embodiments, the number or faction of cells with ecDNA is determined. In some embodiments, the number or faction of cells with an ecDNA population above or below a threshold is determined. In some embodiments, the number or faction of cells with a particular type of ecDNA population above or below a threshold is determined. In some embodiments, the particular type of ecDNA comprises a size greater than a threshold, or lower than a threshold. In some embodiments, the particular type of ecDNA comprises the topology. In some embodiments, the particular type of ecDNA comprises the presence of at least one sequence. In some embodiments, the presence of the at least one sequence is confirmed by the binding or hybridizing of a labeling body to said sequence, or complement of said sequence.

[0349] In some embodiments, nucleic acids, chromatin or chromosomes are probed using a plurality of labeling bodies sensitive to sequences, loci or chromatin domains per molecule such that multiple labeling bodies are present per chromosome, molecule, or macromolecule, and observation of multiple probes provides phasing linkages between the labeling body targets. In further embodiments, a plurality of chromosome types are each probed with a plurality of labeling bodies, the labeling bodies not necessarily being identical across different chromosomes. The labeling bodies include without limitation FISH probes, FISH probes to repeating regions, FISH probes to hypervariable regions, in situ PCR, single nucleotide polymorphism probes including without limitation labeled CAS-9 or catalytically inactive CAS-9, repeated CRISPR-CAS9 cutting followed by ligation, nick translation or other polymerase based incorporation of labelled nucleotides, most preferably fluorescently labeled nucleotides, restriction endonuclease based detection by nick translation or binding of labelled catalytically impaired restriction endonucleases, methyltransferase labeling, binding of labelled transcription factors, immunohistological or antibody-based recognition of DNA binding proteins including without limitation histones, transcription factors, enhancers, polymerases, cohesins, condensins, Ki-67 and enyzmes associated with DNA repair. In further embodiments, the same set of phasing probes are used to analyze parents and siblings to refine phasing data.

[0350] In some embodiments, non-invasive prenatal diagnosis (NIPT) is performed without enrichment for fetal cells, with cell types including lymphoid progenitors, hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, leukocytes, nucleated red blood cells and extravillous cytotrophoblasts.

[0351] In some embodiments, non-invasive prenatal diagnosis (NIPT) is performed with enrichment for fetal cells. Enrichment techniques are not limited to those described in [Gussin 2002] and [Sabbatinelli 2021] and all references therein, including culture with low levels of erythropoietin, immunodepletion, immunoaffinity, fluorescence activated cell sorting, magnetic activated cell sorting, buoyant particle based cell sorting, dielectrophoretic particle separation, dielectrophoretic particle separation using immunolabeled particles, density gradient centrifugal separation, high molecular filtration, charge flow separation, lateral magnetophoresis, microfluidic methods, hyperaggregation, immunoaffinity chromatography, lectin induced separation, soybean agglutinin, selective lysis, selective lysis exploiting differential carbonic anhydrase activity and paramagnetic selection by the conversion of hemoglobin (Hb) into methemoglobin. Antibodies for recognition include antibodies with affinity for CD71, CD45, CD19, i-antigens, I-antigens, CD34, CD38, vimentin, fibronectin, vascular cell adhesion molecule, CD14, CD147, HLA antigens, zeta and epsilon chains of embryonic hemaglobin, gamma chain of fetal hemoglobin, 4B9, Gly A, CGB17, GB25, cytokeratins, CD105, human leukocyte G antigen, CD141.

[0352] In some embodiments, non-invasive prenatal diagnosis (NIPT) is performed in concert with metabolic labeling to differentiate maternal and fetal cells on the basis of Thymidine Kinase 1 levels. A thymidine analogue, preferably EdU, is added to cells prior to harvest, preferably a pulse of 1 hour followed by a 3 hr washout followed by 1 hour colcemid. Chromosomes are enriched and positioned in spreads on the surface of a fluidic device, preferably fixed, then stained with click chemistry and a labeling body, preferably a fluorophore, most preferably Cy5. Candidate fetal chromosomes are identified on the basis of enhanced labeling from the click conjugated label. In further embodiments, the thymidine analogue is introduced to cells that are not currently in S phase, including cells that are blocked with cell cycle inhibitors including nocodazole and cells that are in stationary phase or are exhibiting slow growth due to sub-optimal conditions not limited to crowding and depletion of nutrients in cell growth media. In further embodiments, the mixture of maternal and fetal cells are challenged with a low dose of ammonium chloride and potassium bicarbonate in the presence of an inhibitor of carbonic anhydrase II, acetazolamide. Only maternal cells contain carbonic anhydrase I, and will undergo osmotic stress, disrupting growth or lysing the cells [deGraaf 1999]. In this case the replication marker is added during or after the period of cell stress.

[0353] In some embodiments, non-invasive prenatal diagnosis (NIPT) is performed in concert with quantitative telomere labeling to differentiate maternal and fetal chromosomes, with fetal chromosomes displaying a greater length of telomeres.

[0354] In some embodiments, non-invasive prenatal diagnosis (NIPT) is performed in concert with FISH labeling to differentiate maternal and fetal content, whereby the FISH probe is selected for a gene known to be present or suspected to be present in fetal DNA such as Y chromosomal DNA in the case of a male fetus.

[0355] In some embodiments, non-invasive prenatal diagnosis (NIPT) is performed using a pair of maternal samples, one from before the pregnancy and one during the pregnancy. The samples are screened for structural and numerical aberrations and concerns are raised if the second sample contains aberrations not present in the first sample.

[0356] Sequence Out of Place Analysis (SOoPA): In some embodiments, chromosomes that are positioned on a surface and probed for known sequences, preferably using a multiplicity of FISH probes, most preferably using sequential rounds of hybridization, clearing and rehybridization of a set of FISH probes, each with a different fluorescent label. In further embodiments, chromosomes are also analyzed to determine their identity by methods including without limitation morphological examination of chromosome shape, g-banding, arm length, centromere position, telomere position, in situ PCR and all other labeling and staining methods discussed herein. Interrogation results from the sequence FISH probing are analyzed by themselves or preferably analyzed as a collection of some or all FISH probes together, or most preferably combined with all FISH probe data and the interrogation results from chromosome identification to determine whether any sequences are present in unexpected locations, for example as a result of a chromosomal translocation.

[0357] In some embodiments, SOoPA is used to screen oncogenes to determine if oncogene relocation has occurred and to provide a means of screening against new, current, treated or relapsed cancer or to assess the degree of minimally resistant disease.

[0358] In some embodiments, a plurality of pachytene spermatocytes or oocytes are induced to the mitotic phase using, without limitation, Okadaic acid or Calyculin A and chromosomes are positioned on a fluidic substrate. Chromosomes are variously analyzed for aneuploidy, structural variations, epigenetic marks, viral integration events including HIV, telomere length, frequency with which two or more sequence variants, repeats, satellites, indels legions, other genomic feature, viral integration events including HIV appear on the same chromosome. In further applications spermatocyte screening is used to assess reproductive health of one or more human or non-human individuals, as a standalone sample of as asequence exploring the effects of environmental perturbations on reproductive health, including without limitation exposure to chemicals, radiation, diet, food additives, microbiome, temperature, temperature of gonads and physiological exertion. Numbered Embodiments

[0359] The disclosure is further understood through the following number embodiment. A method of preparing the spread of macromolecules originating from a package, said method comprising: rupturing a package containing a package fluid and at least two macromolecules on the surface of an open fluidic device, with said device comprising at least one fluidic feature, such that a fluid comprising the package fluid flows within, among, or along the at least one fluidic feature, and positions the at least two macromolecules within, among, or along the at least one fluidic feature. 2. The method of any previous embodiment such as 1, wherein at least one of the at least two macromolecules comprises a chromosome. 3. The method of any previous embodiment such as 1, wherein at least one of the at least two macromolecules comprises an ecDNA. 4. The method of any previous embodiment such as 1, wherein at least one of the at least two macromolecules comprises a circular polynucleotide. 5. The method of any previous embodiment such as 1, wherein the package comprises a cell. 6. The method of any previous embodiment such as 1, wherein the package comprises a nucleosome. 7. The method of any previous embodiment such as 1, wherein the package comprises a droplet. 8. The method of any previous embodiment such as 1, wherein the at least one fluidic feature comprises a channel. 9. The method of any previous embodiment such as 8, wherein the channel comprises a dimension between 10 and 0.1 microns. 10. The method of any previous embodiment such as 9, wherein the dimension comprises a width. 11. The method of any previous embodiment such as 9, wherein the dimension comprises a length. 12. The method of any previous embodiment such as 9, wherein the dimension comprises a depth. 13. The method of any previous embodiment such as 9, wherein the dimension comprises a height. 14. The method of any previous embodiment such as 9, wherein the dimension comprises a cross-section. 15. The method of any previous embodiment such as 1, wherein the at least one fluidic feature comprises a pillar. 16. The method of any previous embodiment such as 15, wherein the pillar comprises a dimension between 10 and 0.1 microns. 17. The method of any previous embodiment such as 16, wherein the dimension comprises a separation distance to an adjacent pillar. 18. The method of any previous embodiment such as 16, wherein the dimension comprises a depth. 19. The method of any previous embodiment such as 16, wherein the dimension comprises a height. 20. The method of any previous embodiment such as 16, wherein the dimension comprises awidth. 21. The method of any previou...

Claims

CLAIMS What is claimed:

1. A method of preparing the spread of macromolecules originating from a package, said method comprising: rupturing a package containing a package fluid and at least two macromolecules on the surface of an open fluidic device, with said device comprising at least one fluidic feature, such that a fluid comprising the package fluid flows within, among, or along the at least one fluidic feature, and positions the at least two macromolecules within, among, or along the at least one fluidic feature.

2. The method of claim 1, wherein at least one of the at least two macromolecules comprises a chromosome.

3. The method of claim 1, wherein at least one of the at least two macromolecules comprises an ecDNA.

4. The method of claim 1, wherein at least one of the at least two macromolecules comprises a circular polynucleotide.

5. The method of claim 1, wherein the package comprises a cell.

6. The method of claim 1, wherein the package comprises a nucleosome.

7. The method of claim 1, wherein the package comprises a droplet.

8. The method of claim 1, wherein the at least one fluidic feature comprises a channel.

9. The method of claim 8, wherein the channel comprises a dimension between 10 and 0.1 microns.

10. The method of claim 9, wherein the dimension comprises a width.

11. The method of claim 9, wherein the dimension comprises a length.

12. The method of claim 9, wherein the dimension comprises a depth.

13. The method of claim 9, wherein the dimension comprises a height.

14. The method of claim 9, wherein the dimension comprises a cross-section.

15. The method of claim 1, wherein the at least one fluidic feature comprises a pillar.

16. The method of claim 15, wherein the pillar comprises a dimension between 10 and 0.1 microns.

17. The method of claim 16, wherein the dimension comprises a separation distance to an adjacent pillar.

18. The method of claim 16, wherein the dimension comprises a depth.

19. The method of claim 16, wherein the dimension comprises a height.

20. The method of claim 16, wherein the dimension comprises a width.

21. The method of claim 16, wherein the dimension comprises a length. Page 1^^ of 13122. The method of claim 16, wherein the dimension comprises a cross-section.

23. The method of claim 16, wherein the dimension comprises a diameter.

24. The method of claim 1, wherein the at least one fluidic feature comprises a region with a surface property.

25. The method of claim 24, wherein the surface property comprises hydrophobicity.

26. The method of claim 24, wherein the surface property comprises hydrophilicity.

27. The method of claim 24, wherein the surface property comprises anti-fouling capability.

28. The method of claim 24, wherein the surface property comprises a roughness greater than 50 nm rms.

29. The method of claim 24, wherein the surface property comprises a roughness less than 50 nm rms.

30. The method of claim 1, wherein the at least one fluidic feature comprises a fluidic channel with a porous roof or a chamber with a porous roof.

31. The method of claim 30, wherein at least one macromolecule of the at least two macromolecules can transit through the porous roof.

32. The method of claim 30, wherein at least one macromolecule of the at least two macromolecules cannot transit through the porous roof.

33. The method of claim 1, wherein the package comprises a package boundary that is weakened or partially ruptured prior to rupturing.

34. The method of claim 1, wherein the package comprises a hypotonic state.

35. The method of claim 1, wherein the package comprises a hypertonic state.

36. The method of claim 1, wherein the package comprises an isotonic state.

37. The method of claim 1, wherein the package comprises a state of osmotic pressure.

38. The method of claim 1, wherein the package is delivered to the surface of the open fluidic device in a delivery fluid.

39. The method of claim 38, wherein the delivery process comprises wetting at least a portion of the open fluidic device with the delivery fluid.

40. The method of claim 39, wherein the wetting process comprises dispensing the delivery fluid onto the surface of the open fluidic device.

41. The method of claim 39, wherein the wetting process comprises dipping the open fluidic device into a reservoir containing the delivery fluid.

42. The method of claim 39, wherein the wetting process comprises blade coating the delivery fluid onto the surface of the open fluidic device. Page 1^^ of 13143. The method of claim 39, wherein the wetting process comprises flowing the delivery solution onto the surface of the open fluidic device.

44. The method of claim 43, wherein the flow comprises capillary flow.

45. The method of claim 43, wherein the flow comprises pressure flow.

46. The method of claim 39, wherein the wetting process comprises contacting the surface of the open fluidic device with a substrate previously wetted with the delivery fluid.

47. The method of claim 38, wherein the delivery process comprises removing said delivery fluid from at least a portion of the surface of the fluidic device.

48. The method of claim 47, wherein the package is immobilized on at least a portion of the at least a portion of the surface of the fluidic device.

49. The method of claim 47, wherein the removal process comprises evaporation.

50. The method of claim 47, wherein the removal process comprises a capillary flow.

51. The method of claim 38, wherein the delivery process comprises controlling an environmental condition.

52. The method of claim 51, wherein the controlling process comprises maintaining the environmental condition at an approximate value for at least a period of time.

53. The method of claim 51, wherein the controlling process comprises changing the environmental condition from one approximate value to a second approximate value over a period of time.

54. The method of claim 51, wherein the environmental condition comprises temperature.

55. The method of claim 51, wherein the environmental condition comprises humidity.

56. The method of claim 38, wherein the delivery fluid comprises a reagent.

57. The method of claim 38, wherein the delivery fluid comprises methanol.

58. The method of claim 38, wherein the delivery fluid comprises a fixative.

59. The method of claim 38, wherein the delivery fluid comprises a volatile liquid.

60. The method of claim 38, wherein the delivery fluid comprises an enzyme.

61. The method of claim 1, wherein the rupturing process comprises a shear force between the package boundary and the fluidic features.

62. The method of claim 1, wherein the rupturing process comprises the package adhering to the fluidic features.

63. The method of claim 1, wherein the rupturing process comprises a tension in the package boundary.

64. The method of claim 1, wherein the rupturing process comprises a strain in the package boundary. Page 1^^ of 13165. The method of claim 1, wherein the rupturing process comprises a stress in the package boundary.

66. The method of claim 1, wherein the rupturing process comprises a force on the package directed towards the fluidic features.

67. The method of claim 1, wherein the rupturing process comprises controlling an environmental condition.

68. The method of claim 67, wherein the controlling process comprises maintaining the environmental condition at an approximate value for at least a period of time.

69. The method of claim 67, wherein the controlling process comprises changing the environmental condition from one approximate value to a second approximate value over a period of time.

70. The method of claim 67, wherein the environmental condition comprises temperature.

71. The method of claim 67, wherein the environmental condition comprises humidity.

72. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a capillary flow.

73. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises an evaporative flow.

74. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a gravity driven flow.

75. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a centrifugal driven flow.

76. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a thermal flow.

77. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a convection flow.

78. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a surface-tension flow.

79. The method of claim 1, wherein the package fluid flow within, among, or along the at least one fluidic feature comprises a flow oriented in a direction defined at least in part by the fluidic features.

80. The method of claim 1, wherein the probability of the at least two positioned macromolecules being in physical contact is less than the probability of a similar package rupturing on the surface of a glass slide with traditional cytogenetic methods. Page 12^ of 13181. The method of claim 1, wherein the probability of the at least two positioned macromolecules overlapping each other is less than the probability of a similar package rupturing on the surface of a glass slide with traditional cytogenetic methods.

82. The method of claim 1, wherein the probability of the at least two positioned macromolecules maintaining a minimum separation distance is less than the probability of a similar package rupturing on the surface of a glass slide with traditional cytogenetic methods.

83. The method of claim 1, wherein the probability of the at least two positioned macromolecules maintaining a maximum separation distance is less than the probability of a similar package rupturing on the surface of a glass slide with traditional cytogenetic methods.

84. The method of claim 1, wherein the probability of the at least two positioned macromolecules maintaining a minimum separation distance is less than the probability of a similar package rupturing on the surface of a glass slide with traditional cytogenetic methods.

85. The method of claim 1, wherein the at least two positioned macromolecules have their major axis aligned along a set of orientations defined by the fluidic features.

86. The method of claim 85, wherein the set of orientations comprises at least one orientation.

87. The method of claim 85, wherein the set of orientations comprises at least two orientations.

88. The method of claim 85, wherein the set of orientations comprises a collection of radial orientations from a center point.

89. The method of claim 1, wherein the at least two positioned macromolecules are elongated along the length of their major axis.

90. The method of claim 89, wherein the degree of elongation is 1.5x or more the average elongation of a similar macromolecule on the surface of a glass slide prepared by traditional cytogenetic methods.

91. The method of claim 89, wherein the degree of elongation is 2x or more the average elongation of a similar macromolecule on the surface of a glass slide prepared by traditional cytogenetic methods.

92. The method of claim 89, wherein the degree of elongation is 3x or more the average elongation of a similar macromolecule on the surface of a glass slide prepared by traditional cytogenetic methods.

93. The method of claim 89, wherein the degree of elongation is 5x or more the average elongation of a similar macromolecule on the surface of a glass slide prepared by traditional cytogenetic methods. Page 12^ of 13194. The method of claim 89, wherein the degree of elongation is at least partially controlled by a confining dimension property of the at least one fluidic feature.

95. The method of claim 94, wherein the confining dimension property is a channel width.

96. The method of claim 94, wherein the confining dimension property is a channel cross- section.

97. The method of claim 94, wherein the confining dimension property is a channel depth.

98. The method of claim 94, wherein the confining dimension property is a channel height.

99. The method of claim 94, wherein the confining dimension property is a pillar cross- section.

100. The method of claim 94, wherein the confining dimension property is a pillar height.

101. The method of claim 94, wherein the confining dimension property is a pillar depth.

102. The method of claim 94, wherein the confining dimension property is a pillar separation distance.

103. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules is in an elongated state.

104. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules comprises a physical map.

105. The method of claim 1, wherein the at least two positioned macromolecules are interrogated.

106. The method of claim 105, wherein the interrogation is done with an interrogation system.

107. The method of claim 105, wherein the interrogation comprises determining the number of macromolecules originating from the package.

108. The method of claim 105, wherein the interrogation comprises identifying a bound labeling body associated with at least one of the at least two macromolecules.

109. The method of claim 108, wherein the bound labeling body is a FISH probe.

110. The method of claim 108, wherein the bound labeling body is a fluorescent label.

111. The method of claim 108, wherein the bound labeling body is a quantum dot.

112. The method of claim 105, wherein the interrogation comprises identifying a property associated with the at least one of the at least two macromolecules. Page 12^ of 131113. The method of claim 112, wherein the property comprises a classification of the molecule.

114. The method of claim 113, wherein the classification comprises an ecDNA.

115. The method of claim 113, wherein the classification comprises a chromosome.

116. The method of claim 113, wherein the classification comprises a foreign body.

117. The method of claim 113, wherein the classification comprises a virus.

118. The method of claim 113, wherein the classification comprises a mitochondrion.

119. The method of claim 112, wherein the property comprises a centromere count.

120. The method of claim 112, wherein the property comprises an ROI.

121. The method of claim 112, wherein the property comprises a physical map.

122. The method of claim 112, wherein the property comprises a structural variation.

123. The method of claim 112, wherein the property comprises a higher order structure.

124. The method of claim 112, wherein the property comprises a chromosome number.

125. The method of claim 112, wherein the property comprises a chromosome type.

126. The method of claim 112, wherein the property comprises a length of a q arm.

127. The method of claim 112, wherein the property comprises a length of a p arm.

128. The method of claim 112, wherein the property comprises a length of chromatid.

129. The method of claim 112, wherein the property comprises genomic information.

130. The method of claim 112, wherein the property comprises a trait associated with a disease.

131. The method of claim 130, wherein the disease is cancer.

132. The method of claim 130, wherein the disease is trisomy 21 (Down syndrome).

133. The method of claim 130, wherein the disease is trisomy 18 (Edwards syndrome).

134. The method of claim 130, wherein the disease is trisomy 13 (Patau syndrome).

135. The method of claim 130, wherein the disease is Kilnefelter syndrome.

136. The method of claim 130, wherein the disease is Turner syndrome.

137. The method of claim 1, wherein at least one property of the spread is at least partially controlled by the at least one fluidic feature.

138. The method of claim 137, wherein the at least one property of the spread comprises a physical boundary.

139. The method of claim 137, wherein the at least one property of the spread comprises a physical area.

140. The method of claim 137, wherein the at least one property of the spread comprises an area density of the at least two macromolecules within the spread. Page 12^^of 131141. The method of claim 137, wherein the at least one property of the spread comprises a boundary minor axis length or a boundary minor axis orientation.

142. The method of claim 137, wherein the at least one property of the spread comprises a boundary major axis length or a boundary major axis orientation.

143. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules are brought into contact with a contract probe system.

144. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules are brought into contact with solution from a dispensing system.

145. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules are brought into contact with a reagent.

146. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules are brought into contact with a labeling body.

147. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules is removed from the surface.

148. The method of claim 147, wherein the removal process comprises removal by contact probe system.

149. The method of claim 147, wherein the removal process comprises re-suspension in a solution.

150. The method of claim 147, wherein the removal process comprises placement inside a droplet.

151. The method of claim 1, wherein at least a portion of at least one of the at least two positioned macromolecules is further processed.

152. The method of claim 151, wherein the processing comprises amplification.

153. The method of claim 151, wherein the processing comprises sequencing.

154. The method of claim 151, wherein the processing comprises confirming the presence of a certain sequence is present or absent from the at least a portion.

155. The method of claim 151, wherein the processing comprises hybridization.

156. The method of claim 151, wherein the processing comprises genotyping.

157. An open fluidic device for preparing the spread of macromolecules originating from a package, said device comprising fluidic features, such that a fluid comprising the package fluid flows from the ruptured package within, along, among the fluidic features, and positions the at least two macromolecules within, along, among the fluidic features.

158. A method of positioning packages on the surface of an open fluidic device comprising at least one fluidic feature, said method comprising: Delivering a delivery solution Page 12^ of 131comprising at least one package to the surface of the device, allowing the at least one package to physically interact with fluidic features, and immobilizing said at least one package at a defined at least one capture point.

159. The method of claim 158, wherein the at least one package ruptures at said capture point.

160. The method of claim 158, wherein the at least one package comprises a macromolecule.

161. The method of claim 160, wherein the macromolecule comprises a chromosome.

162. The method of claim 158, wherein the at least one package comprises a cell.

163. The method of claim 158, wherein the at least one package comprises a nucleosome.

164. The method of claim 158, wherein the at least one package comprises a droplet.

165. The method of claim 158, wherein the at least one fluidic feature comprises a channel.

166. The method of claim 165, wherein the channel comprises a dimension between 10 and 0.1 microns.

167. The method of claim 166, wherein the dimension comprises a width.

168. The method of claim 166, wherein the dimension comprises a length.

169. The method of claim 166, wherein the dimension comprises a depth.

170. The method of claim 166, wherein the dimension comprises a height.

171. The method of claim 166, wherein the dimension comprises a cross-section.

172. The method of claim 158, wherein the at least one fluidic feature comprises pillars.

173. The method of claim 172, wherein the pillar comprises a dimension between 10 and 0.1 microns.

174. The method of claim 173, wherein the dimension comprises a separation distance to an adjacent pillar.

175. The method of claim 173, wherein the dimension comprises a depth.

176. The method of claim 173, wherein the dimension comprises a height.

177. The method of claim 173, wherein the dimension comprises a width.

178. The method of claim 173, wherein the dimension comprises a length.

179. The method of claim 173, wherein the dimension comprises a cross-section.

180. The method of claim 173, wherein the dimension comprises a diameter.

181. The method of claim 158, wherein the at least one fluidic feature comprises a region with a surface property.

182. The method of claim 181, wherein the surface property comprises hydrophobicity.

183. The method of claim 181, wherein the surface property comprises hydrophilicity. Page 12^ of 131184. The method of claim 181, wherein the surface property comprises anti-fouling capability.

185. The method of claim 181, wherein the surface property comprises a roughness greater than 50 nm rms.

186. The method of claim 181, wherein the surface property comprises a roughness less than 50 nm rms.

187. An open fluidic device for positioning packages on the surface comprising at least one fluidic feature and at least one capture point, such that a delivery fluid comprising at least one package will position the at least one package in the at least one capture point. Page 1^^ of 131

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