Continuous stage scanning instrument for imaging and light-induced tagging of biological samples

Abstract

A system is described that leverages the advantages of TDI scanning by combining it with an innovative illumination methodology. The instrument may include a continuous stage scanning system moving the biological sample in one direction, a sample imaging system with an optical axis perpendicular to the stage scanning direction, and an optical system with an optical axis perpendicular to the stage scanning direction for applying a dynamic light pattern to induce a photochemical or a photophysical modification of the sample, wherein the photochemical or photophysical modification is used as one parameter in a subsequent analysis or processing decision on the same instrument or on a different instrument.

Description

CONTINUOUS STAGE SCANNING INSTRUMENT FOR IMAGING AND LIGHT-INDUCED TAGGING OF BIOLOGICAL SAMPLESCROSS REFERENCE TO RELATED APPLICATIONSThis application claims priority to US Provisional Application Serial No. 63 / 614,655, filed Dec. 25, 2023. This prior application is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH Not applicable.STATEMENT REGARDING MICROFICHE APPENDIX Not applicable.FIELD OF THE INVENTION

[0001] The present invention relates to light-induced tagging of biological samples in an instrument.BACKGROUND

[0002] A continuous line scanning microscope based on time-delayed-integration (TDI) camera detection offers significantly higher throughput compared to traditional stop-and-stare approaches. The TDI-based detection is therefore a favorable candidate for various existing imaging systems, as well as for the next generation biological imaging systems, that require high-throughput imaging, where optionally additional optical sectioning and ability to image volume samples such as tissue sections, organoids or whole organs are highly desired. To enable high-throughput microscopy, high illumination intensities are required. Though high intensity illumination can be introduced by illumination with a single diffraction-limited point focus or line focus, this approach comes with many disadvantages since expensive and bulky laser instrumentation is required and scanning speed is either restricted by low fluorescence, luminescence, or phosphorescence signal from the small illuminated area, and / or local intensities have to be extremely high, potentially leading to sample damage.

[0003] Other manipulations of biological samples may include optical manipulation, Cell activation, gene activation, light-induced apoptosis, cell damage / DNA damage, selective tagging, and the like. Tagging may also be performed for other biological processes in addition to simply finding the cell later in downstream analysis.

[0004] One application in which speed is particularly helpful is genetic sequencing. Throughput in next generation sequencing (NGS) by synthesis as well as spatial multi-omicsapproaches (e.g. spatial proteomics, spatial transcriptomics, spatial genomics or a combination thereof) rely on the speed of the optical microscope. A continuous stage scanning concept together with detection by a multi-channel TDI camera setup was developed, requiring multi-color, high power, flat line, sharp edge illumination to the sample. For most efficient use of available light power, the line aspect ratio has to be up to x:y = 32: 1 or higher (depending on the camera sensor geometry) at a line width of 1 mm or more while not exceeding a line height of 32 pm. These numbers are examples for a TDI camera featuring a detector array of 128 pixels in scan direction and 4000 pixels perpendicular to the scan direction, a pixel width and height of 5 pm, respectively, and a microscope that has a 20x magnification. Additionally, following features of the illumination system are desired: multicolor excitation, spectrally separated excitation for suppression of cross-talk between fluorescent channels, cost efficiency, modularity, low maintenance effort, to name a few.SUMMARY

[0005] A continuous line scanning microscope based on time-delayed-integration (TDI) camera detection offers significantly higher throughput compared to traditional stop and-stare approaches. The TDI-based detection is therefore a favorable candidate for various existing imaging systems, as well as for the next generation biological imaging systems, where additional optical sectioning and ability to image volume samples such as tissue sections, organoids or whole organs are highly desired. A cost effective way on combining a continuous line scanning microscope based on time-delayed-integration (TDI) camera detection system with a system allowing to precisely depositing light on to fixed cells or tissue sample in 2D and / or 3D is further described.

[0006] Precisely depositing light to cells can be used to activate or deactivate molecules. Light can also be used to activate the incorporation of a synthesized nucleotide enabling writing molecular codes into each cell. Accordingly, a system is described that leverages the advantages of TDI scanning by combining it with an innovative illumination methodology.

[0007] The instrument may include a continuous stage scanning system moving the biological sample in one direction, a sample imaging system with an optical axis perpendicular to the stage scanning direction, and an optical system with an optical axis perpendicular to the stage scanning direction for applying a dynamic light pattern to induce a photochemical or a photophysical modification of the sample, wherein the photochemical orphotophysical modification is used as one parameter in a subsequent analysis or processing decision on the same instrument or on a different instrument.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Various exemplary details are described with reference to the accompanying drawings which should not be taken to limit the invention to the specific embodiments shown but are for explanation and understanding only.

[0009] Fig. 1 is a simplified schematic drawing of the components of the system;

[0010] Fig. 2 is an exemplary workflow;

[0011] Fig. 3 is a simplified overview of the systems in which the instrument may be used;.

[0012] Fig. 4 is a simplified overview on how a continues time delayed integration (TDI) based imaging system can be combined with a polygon scanner concept which is being used for cell tagging.

[0013] Fig. 5 is a simplified overview on how a continues time delayed integration (TDI) based imaging system can be combined with a galvanometric or resonant scanner concept which is being used for cell tagging.

[0014] Fig. 6 is a simplified view of the herein described optical path showing the focal plan, objective lens and pupil plane.

[0015] Fig. 7 is a schematic diagram illustrating the UNIT-DNA system.

[0016] It should be understood that the drawings are not necessarily to scale, and that like numbers maybe may refer to like features.DETAILED DESCRIPTION

[0017] Fig. 1 is a simplified schematic drawing of the components of the system.] It should be understood that Fig. 1 is exemplary only. And that systems are envisioned which have fewer or more components. Shown in Fig. 1 is a control and analysis unit 12. This unit 12 may be a microprocessor-driven device that executes a set of software instructions. This unit may control and operate the system as described below.

[0018] In the left hand portion of Fig.1 is the illumination leg that provides the excitation to the sample. The control and analysis unit 12 may be in communication with a light source 32 that will illuminate the sample as described below. The light source 32 may further include a light modulating element such as a moving shutter of pattern of opaque and transmissive elements. The light source 32 may further comprise an optical system, 52, whichmay include a plurality of optical elements such as lenses, filters and mirrors that route the light to the sample.

[0019] The right hand leg in Fig. 1 is the detection leg. This leg may include a fluorescence microscope 62 that gathers and images the fluorescence generated by the sample upon excitation. The fluorescence microscope 62 may further include an objective lens that images the sample, as is well known in the art of microscopy. The biological sample is the source of the fluorescence signal which is being imaged by the microscope 62. The biological sample 82 may be mounted on a moving stage 92, which moves the sample relative to the objective lens 72, such that different areas of the sample are imaged at different times. This element enables the TDI scanning as described further below.

[0020] Both legs may be managed by one or more controllers or processors 12, executing a set of software instructions, according to the details of the workflow.

[0021] Fig 2 shows an exemplary workflow, using the system of Fig. 1. It should be understood that all steps may not be required, and that the steps may also be performed in an order different than that shown in Fig. 2. The exemplary method starts in step SI 00. The remaining steps are described in detail below.S200: -tissue block, fresh frozen or FFPE-cut block on microtome and transfer tissue slices on to microscopeS300:-tissue block, fresh frozen or FFPE-cut block on microtome and transfer tissue slices on to microscope cover glass-process sample on the lab bench and do antigen retrieval stepS400:-scan microscope cover glass with fiducial marks on it-scan entire tissue (DAPI stain)-identify region of interest-add necessary molecular chemistry to enable molecule activation / deactivation with lightS500: -calculate the offset between fiducial marks and location of tissue-bring light to identified region of interest-repeat step of tagging for n cycle at various locations-record x and y location on the tissue where light was deposited;S600: -collect all cells and remove cells form microscope cover glass, -lyse cells;S700: -sequencing library preparation;S800: -sequencing target region of interest including the light tagged barcodeS900: -match sequenced target region with light tagged barcode with the recorded x and y location from step S500;-visualize the sequence information per cell with the tissue overview image from step S400.The process ends in step S950.

[0022] Fig. 3 shows a simplified exemplary flow chart that illustrates an number of applications to which the instrument may be applied. The workflow may include a process for spatial proteomics or in-situ sequencing or spatial transcriptomics. The sample may then bee tagged. Both of these steps may use the continuous stage scanning instrument as described. Subsequent to tagging, the sample may be lysed or dissociated. After separation, the sample may undergo bulk sequencing, flow cytometry, soring or other means of separation. The

[0023] Fig. 3 should be understood to mean that any combinations of, or all options may be used, either sequentially or in parallel. These options are not mean to be exhausting, but rather to illustrate how the instrument may be used in a variety of systems, methods or work flows.

[0024] Fig. 4 is a simplified diagram showing how a sample may be tagged with the polygon scanner. The figure is intended to illustrate the orthogonal nature, or perpendicularity, of the stage scanning direction relative to the laser scanning direction. Fig. 4 also illustrates the areas tagged while scanning.

[0025] Fig. 5 is a simplified diagram of another embodiment of the instrument using instead a galvanometric or resonant scanner. As with Fig. 4, the figure is intended to illustrate the orthogonal nature, or perpendicularity, of the stage scanning direction relative to the laser scanning direction. However, in this embodiment, the timing of the movements result in the the pattern shown in Fig. 5 Details of this embodiment are also provided below.

[0026] Fig. 6 shows is a simplified view of the herein described optical path showing the focal plan, objective lens and pupil plane. Fig. 6 illustrates the orientation of the focalplane of the relative to the objective lens and pupil plane. Details of the focal plane, objective lens and pupoil plane are provided in the description below.

[0027] Light induced tagging in an instrument that contains a continuously moving stage while the tagging is performed allows for significantly higher throughput compared to stopping the stage for the tagging.

[0028] Light induced tagging can overcome throughput limitations of an instrument for chemical, physical or biological, image-based analysis of samples in genomic, transcriptomic, proteomic or combined applications.

[0029] Image based analysis methods have a limited optical resolution and, in most cases, the detected objects are optically too crowded. Therefore, it is not possible to properly separate them. The direct light tagging of cells circumvents the limitation of optical crowding. Regions of interest are tagged and then further downstream analyzed in additional instruments.

[0030] Image based methods can analyze samples in their spatial context which gets lost when the sample is dissociated or lysed. Such dissociation may be performed after image based analysis to perform DNA-sequencing, RNA-sequencing, separation, sorting, further analysis workflows or a combination thereof. To partly or fully maintain the spatial context in dissociated samples, light induced tagging may be performed based on an image based analysis before the dissociation into subcellular compartments, single cells or multicellular regions of the sample which are selectively tagged.

[0031] Image based methods can use fluorescence, Raman-spectrum, transmitted and / or reflected light contrast, transmitted and / or reflected light phase, transmitted and / or reflected light spectrum, other light microscopy methods or a combination of aforementioned methods for one-, two-, or three-dimensional imaging of the sample.

[0032] High throughput of the imaging procedure may be achieved by time-delayed- integration (TDI) microscopy at significantly higher throughput compared to traditional stop- and-stare approaches. The TDI-based detection is therefore a favorable candidate for a high throughput imaging system.

[0033] Continuous motion allows to acquire images and perform tagging with very short delay. This allows the tagging of cells that move or change quickly on large sample areas.

[0034] Optionally, TDI-based detection can be combined with optical sectioning to enhance the image quality and thus quality of the image based sample analysis in tissue sections, organoids or whole organs.

[0035] Optionally, TDI-based detection can be combined with a line illumination of the sample preferably restricted to the actively imaged sample. Line illumination may be achieved by directly imaging the output of a two-dimensional light source, such as the output of a rectangular optical multi-mode fiber, to the sample plane at different magnifications for x and y.

[0036] For combination of an image based analysis system and light-induced tagging, different beam paths sharing a common sample plane can be combined and share the same stage which is moved both during the imaging process and the tagging process of the sample.

[0037] The tagging process can be induced by photochemical and / or photophysical modification of the sample.

[0038] During the continuous movement of the stage, the light pattern may be dynamically updated to enable a high spatial resolution and / or high contrast of the tagging pattern.

[0039] For temporal and / or spatial synchronization of the continuously moving stage and the optical systems, the stage and / or the tagging system may be equipped with encoders detecting lateral and / or angular movement and generating appropriate trigger signals.

[0040] The beam path of the tagging system can be equipped with various active optical elements or a combination thereof to enable the dynamic update of the light distribution in the sample which induces the tagging.

[0041] The light distribution may be varying in one, two, or three dimensions.

[0042] Active optical elements enabling the dynamic light distribution in the sample plane or a volume around the sample plane may be placed in a position which is optically conjugate to the sample plane or optically conjugate to the pupil plane of the microscope objective lens or close to such planes, respectively.

[0043] Non-limiting examples of an element placed optically conjugate close to the sample plane contain a digital mirror devices (DMD), an output-modulated ID or 2D fiber bundle array, a polarization-modulating spatial light modulator (SLM) in combination with a polarizing filter or a combination of the aforementioned elements.

[0044] Non-limiting examples of elements placed optically conjugate close to the pupil plane contain a holographic SLM, a galvanometric mirror scanner in combination with a temporal modulation of the light power, a resonant mirror scanner in combination with atemporal modulation of the light power, a polygon scanner in combination with a temporal modulation of the light power, a microelectronic mechanical system (MEMS) mirror, a grating light valve (GLV).

[0045] Non-limiting examples to achieve temporal modulation of the light power are the use of an acousto optic tunable filter (AOTF), the use of a temporally modulated light source or a combination thereof.

[0046] Non-limiting example of a light source creating the illumination beam are a laser, an array of lasers, an optical fiber, an array of optical fibers, a laser diode, an array of laser diodes, an LED, an array of LEDs, an incandescent lamp, an array of incandescent lamps, a gas-discharge lamp, and an array of gas-discharge lamps or a combination thereof.

[0047] In some embodiments, the light distribution is varied laterally in the direction orthogonal to the continuous movement of the stage.

[0048] In some embodiments, the lateral variation is achieved by a polygon scanner in combination with a temporally modulated output of a laser source where the angular, onedimensional deflection of laser beam by the rotating polygon is translated into a onedimensional scanning of the laser spot in the sample.

[0049] In some embodiments, the variation along the direction of the continuous motion of the stage is achieved by a polygon scanner. Fig. 4 is a simplified overview on how a continues time delayed integration (TDI) based imaging system can be combined with a polygon scanner concept which is being used for cell tagging. In other embodiments, the projection of a 2D display may be used, projected onto the continuously moving sample, synchronized with the motion.

[0050] In some embodiments, the laser beam is collimated at the surface of the polygon and imaged via a relay lens system to the pupil plane of the objective lens.

[0051] In some embodiments, the laser beam illuminates a diffraction limited spot in the sample.

[0052] In some embodiments, the laser beam illuminates an area in the sample that is bigger than the diffraction limit.

[0053] A simultaneously illuminated area at the size of a diffraction-limited spot may be preferred for high optical resolution of the tagged pattern. A simultaneously illuminated area at a size bigger than the diffraction limit may be preferred for high throughput of the tagging procedure.

[0054] In some embodiments, light sources other than a laser may be used interchangeably.

[0055] In some embodiments, the one-dimensional movement of the laser beam in the sample is orthogonal to the continuous movement of the stage.

[0056] In some embodiments, the modulated light source is turned on when the moving laser beam in the sample hits an area that is supposed to be tagged.

[0057] In some embodiments using a polygon scanner, the laser beam is scanned from one side to other in the sample for each scanning cycle and the light source is turned on accordingly when an area is crossed that is supposed to be tagged.

[0058] In some embodiments using a galvanometric or resonantly scanned mirror, the laser beam is scanned back and forth between both sides in the sample for each scanning cycle and the light source is turned on accordingly when an area is crossed that is supposed to be tagged. As mentioned previously, Fig. 5 is a simplified overview on how a continues time delayed integration (TDI) based imaging system can be combined with a galvanometric or resonant scanner concept which is being used for cell tagging.

[0059] In some embodiments using a 2D varying element as for instance a DMD, the active pattern on the element corresponds to the momentary light distribution in the focal plane of the objective lens or a magnified or a demagnified version thereof.

[0060] To efficiently combine a 2D varying element with an orthogonally continuously moving stage, the pattern has to be updated at the frequency of the stage velocity divided by the pixel pitch in the movement direction and divided by the optical system magnification from the element to the sample.

[0061] Alternatively, the motion of the projected 2D pattern can be synchronized with the stage motion. In this case, the pattern has to be updated at the frequency of the stage velocity divided by the dimension of the image along the scan direction.

[0062] In some embodiments, the photochemical or photophysical modification of the sample is preferably achieved in areas where two or more different light illumination wavelengths are present simultaneously or in temporal short sequence.

[0063] Temporal short sequences may be achieved by alternated switching of two or more different light sources.

[0064] Simultaneous illumination by two or more wavelengths may be achieved by homogeneous illumination of a first set of one or multiple wavelengths and patterned illumination of the tagged area only for a second set of one or multiple wavelengths.

[0065] Alternatively, when using a scanning device that is positioned close to a plane conjugate to the objective lens focal plane, dividing the pupil into two distinct regions that are illuminated exclusively either by a first set of one or multiple wavelengths and a second set ofone or multiple wavelengths achieves simultaneous presence of the first and second set of wavelengths exclusively in the focal point of the imaging system. As mentioned previously, Fig. 6 is a simplified view of the herein described optical path showing the focal plan, objective lens and pupil plane.

[0066] In some embodiments, simultaneous presence of the first and second set of wavelengths exclusively in the focus of the imaging system may be used for three- dimensional tagging of the sample.

[0067] In some embodiments, three-dimensional tagging may be achieved for different planes in axial direction which are tagged subsequently by subsequently moving the sample or the objective lens to the corresponding z-positions.

[0068] When light-induced tagging is combined with a system that enables the changing of the chemical environment of the sample, light-induced tagging and chemical changes of the environment can be iteratively performed to imprint barcodes in a spatial pattern to the sample

[0069] Non-limiting examples for changing the chemical environment of the sample are a pipetting system, a microfluidic system or a combination thereof.

[0070] In some embodiments, the barcode can be read out by different methods after removing the sample from the instrument.

[0071] In some embodiments, the imprinted barcode can be a nucleotide sequence.

[0072] In some embodiments, the nucleotide can be read in a sequencing application.

[0073] In some embodiments, the sequencing can be performed by the imaging system of the same instrument.

[0074] In some embodiments, tagging induces one or multiple photophysical modifications of the sample.

[0075] Non-limiting examples for photophysical modifications contain fluorescence excitation, phosphorescence excitation, stimulated emission from an excited singlet state, stimulated emission from an excited triplet state, photoinduced intersystem crossing.

[0076] In some embodiments, tagging induces one or more chemical or biochemical modifications of the sample.

[0077] Non-limiting examples of chemical or biochemical modifications of the sample are irreversible protein denaturation, light-induced activation or deactivation of enzymes (e.g. polymerase?), (see also, e.g. https: / / www.degruyter.eom / document / doi / l 0.1515 / psr-2022- 0109 / html and reviews referenced therein)

[0078] Irreversible protein denaturation may be used in a downstream sorting application, where fluorescently labeled moieties preferably bind to un-denatured parts of the sample and the sorting decision is made based on the presence of the labeled moieties.

[0079] Irreversible protein denaturation may be used in a downstream separation application, where magnetically labeled moieties preferably bind to un-denatured parts of the sample and the separation mechanism is based on the presence of magnetically labeled moieties.

[0080] Non-limiting examples for fluorescently and / or magnetically labeled moieties are antibodies, nanobodies, Fab-fragment and aptamers.

[0081] Description of photochemical modifications, e.g. chemistry used, nucleotide barcoding chemistry.

[0082] Light-triggered chemical reactions can provide excellent tools to investigate the fundamental mechanisms important in biology. Light is easily applicable and orthogonal to most cellular events, and its dose and locality can be controlled in tissues and cells. Light- induced conversion of photochemical groups installed on small molecules, proteins, andoligonucleotidescanaltertheirfunctionalstatesandthusthe ensuing biological events.

[0083] Recently, photochemical control of DNA / RNA structure and function has garnered attention thanks to the rapidly expanding photochemistry used in diverse biological applications. Photo convertible groups can be incorporated in the backbone, ribose, and nucleobase of an oligonucleotide to undergo various irreversible and reversible light-induced reactions such as cleavage, crosslinking, isomerization, and intramolecular cyclization reactions. There are four different broad categories:• Photocleavage irreversible• Intermolecular photo crosslinking reversible• Cis-transphoto isomerization reversible• Intramolecular photocyclization reversible(Reference: Photochemical modifications for DNA / RNA oligonucleotides , Royal society of chemistry, Amirrasoul Tavakoli and Jung-Hyun Min, 2022).

[0084] What follows is an exemplary embodiment showing how technology disclosed here can be used in an application Unit-DNA.

[0085] Spatial sequencing is a collective term for methods that allow direct sequencing of the mRNA content of a cell in tissue context. These methods can on the one hand serve to analyze mRNA expression profiles of cells in a kind of highly multiplexed fluorescence in situ hybridization (FISH) assay.

[0086] On the other hand, in situ sequencing can also enable the read-out of mRNA sequence information, using specific mRNA-binding probes, which can take up a copy of predefined portion of specific mRNA or cDNA sequence (“Gap-fill padlock probes”, Ke et al., Nature Methods 2013, doi: 10.1038 / nmeth.2563). Recently also in situ genome sequencing (IGS) approaches were published (In situ genome sequencing resolves DNA sequence and structure in intact biological samples”, A. C. Payne et al., Science 10.1126 / science.aay3446 (2020)). All in situ sequencing methods require a signal amplification step, which is in most cases performed by circularization of mRNA- or cDNA-binding probes or gDNA insert circularization by hairpin ligation and subsequent rolling circle amplification (RCA), creating a DNA molecule containing multiple copies of the probe and / or target sequence, the so called Nanoballs, Rolonies or Rolling circle amplification products (RCPs). As these are large molecules with size in nm or pm scale, the number of rolonies that can be formed within one cell is strictly limited by the size of this cell.

[0087] Furthermore, if the density of rolonies within cells is too high, discrimination of single mRNA signals during the optical detection step of the sequencing procedure is strongly impaired. As this is a major drawback of the technology, various techniques have been developed to circumvent this, e.g. design of smaller rolonies or generation and clearing of tissue-hydrogel complexes (Asp et al., BioEssays 2020, DOI: 10.1002 / bies.201900221) or to expand the cellular target termed expansion sequencing (Alon et al., Science 371, eaax2656 (2021)).

[0088] However, these methods do still not fully evade the inherent spatial limitations of in situ sequencing. Another approach avoids in situ signal amplification: In situ capturing relies on the transfer of mRNA molecules from tissue onto a surface coated with spots of barcoded primers, allowing backtracking of the ex situ gained sequence information to the specific tissue region the sequenced mRNA was extracted from. Nevertheless, this method is also limited, as RNA capture efficiency is restricted and resolution is poor (no single-cellanalysis) due to the relatively large size of the barcoded capturing spots (Asp et al., BioEssays 2020, DOI: 10.1002 / bies.201900221).

[0089] This embodiment is directed to a composition which uses optical methods to insert a genetic code into a sequence. This code can be used to retrieve the position at which the coding was carried out. The aim here is that the limitations of existing in situ sequencing methods with regard to the number of measurable mRNA sequences in situ and also the expression dynamics are largely overcome. Optical coding can have a resolution in the range of one pm and a variability of the code that is sufficient for each cell to receive its own code in tissue sections of typical size. The proposed coding and decoding workflow is depicted in Fig. 7.

[0090] The basic principle as disclosed herein is based on the composition for spatial barcoding of nucleic acids by universal template directed DNA synthesis. The composition will subsequently be referred to as UNIT-DNA (Universal Template DNA). Fig. 7 uses the following reference numbers:001 Tissue donor002 Stained tissue section003 Cell004 Cell nucleus005 mRNA in cytoplasm006 mRNA (linked to UNIT-5 DNA composition)100 Imaging101 Segmentation or cluster analysis, calculation of masks for the structured illumination102 Photo-treatment for UNIT-DNA Code generation103 Single Cell Encapsulation104 Sequencing105 Cyclic barcoding106 Sequence analysis200 UNIT-DNA composition before coding201 UNIT-DNA composition after coding202 Single Cell Indexing reagents203 UNIT-DNA composition H204 Linearized Template switched cDNA with spatial barcode205 Sequencing Library derived from cDNAUNIT-DNA composition for spatial barcoding

[0091] The UNIT-DNA composition consist of a double stranded nucleic acid with at least one 5 'overhang, where the 5' overhang includes at least one universal base and the recessed 3' end has a free 3'-OH. Figure 2 shows an example of the UNIT-DNA composition for a 9 bp double stranded nucleic acid and for a 6 universal base 5 'overhang.

[0092] The direct light tagging of cells circumvents the limitation of optical crowding. Cells are tagged and then further downstream the transcriptome is sequenced In Fig The number of bp for the double stranded nucleic acid or the number of universal bases for single stranded 5' overhang may vary in length. Universal base designs have been described in the literature mainly as part of degenerate primers or probes due to their property to pair with all natural bases (e.G, Loakes, Nucleic Acid Research, 2001, Vol. 29, No. 122437-2447). Figure 3 shows the basic principle of coding by the UNIT-DNA composition. With the support of a polymerase (not shown) the recessed free 3'-OH of the composition is incorporating the nucleotide provided (shown for G). Thereby the recessed 3' -OH strand is extended and coded by the first nucleotide. Any nucleotide provided will pair with the composition as the 5' overhang is including universal bases which support the universal template directed DNA synthesis. The recessed 3' -OH is only extended by one nucleotide as the 3' end of the nucleotide is blocked. As a next step the blocked 3'-OH is unblocked by a cleave reagent which allows the next coding cycle to occur. The incorporation of fluorescently labeled 3'- OH blocked nucleotides which are later unblocked by a cleave reagent is also known from Sequencing by Synthesis (Chen et al., Genomics, Proteomics & Bioinformatics, Volume 11, Issue 1, February 2013, Pages 34-40). Opposite to Sequencing by Synthesis, the UNIT-DNA composition is used to write a DNA code and not to read a DNA code.

[0093] In order to write the spatial DNA barcode using the UNIT-DNA composition, structured illumination is used as part of the coding workflow which was already conceptually introduced by Figure 1. How the structured illumination of individual cells by light (e.G. UV light) is chemically releasing the cleave reagent (e.G. TCEP) spatially is detailed in Figure 4. The chemical reaction to release TCEP after illumination of Cy5-TCEP conjugate by UV lighthas been described before (Vaughan et al., J Am Chem Soc., 2013 Jan 30, 135(4), 1197- 1200).

[0094] In summary, the spatial code written by the UNIT-DNA composition depends on the order of the nucleotides provided and the spatial activation of the cleave reagent by light. The total number of spatial codes which can be written by the UNIT-DNA depends on the number universal bases within the 5 'overhang which allow nucleotide incorporation (e.G. 10 universal bases would translate to ~1 million codes (410)). The spatial resolution of the coding principle is dependent on the resolution of light used for illumination (~300nm for UVB) and the local reaction kinetics of the released cleave reagent and is therefore easily achieving a cellular (~10pm) or subcellular (~lpm) resolution level.

[0095] After the coding has been completed, all cells (or nuclei and organells) are isolated from the tissue sample and are subjected to single cell sequencing. In principle, the method is not limited in terms of the number of cells examined simultaneously. The number of cells examined individually at the same time is dependent on the number of universal bases within the 5 'overhang of the UNIT-DNA composition to provide a unique spatial barcode. The real limitation is eventually only in the capacity and throughput of the sequencer.UNIT-DNA derivatives for spatial barcoding

[0096] UNIT-DNA derivatives for spatial barcoding are known. The core functional elements of the UNIT-DNA composition is maintained. The derivatives listed receive additional functionality as introduced by modifications of the 3' and 5 'ends. The additional derivate functionality combines the core UNIT-DNA composition for spatial barcoding with further nucleic acid manipulation workflows. There is also UNIT-DNA derivate H which is used as part of the template switch oligonucleotide process within the single cell sequencing workflow. After generation of the spatial DNA barcode by the UNIT-DNA, the resulting nucleic acid can be further analyzed by sequencing using the unique molecular identifier (UMI) for error correction. It is worth to mention that the TSO does not include a Cell Identifier. The UNIT-DNA composition provides the spatial barcode which can serve as a cell identifier in case resolution of structured illumination was chosen to be aligned with the cellular resolution level. The coding and decoding workflow is for use of the UNIT-DNA derivate H composition. After coding a sequencing library is prepared and the spatial barcode as well as the target nucleic acid is sequenced. As the spatial barcode is physically linked to the target nucleic acid, the spatial information of the target sequence can be derived by in vitro sequencing and the relation of the results to the initial sample source is provided.

[0097] The UNIT-DNA composition H for spatial barcoding of the target nucleic acid can also be used within a padlock workflow leading to a circularized ssDNA or within a targeted DNA workflow. After coding the resulting nucleic acid would be subjected to sequencing in order to determine the spatial barcode and the linked target nucleic acid.

[0098] Depending on the molecular workflow different UNIT-DNA derivates may be used to combine the spatial coding with the sequencing and decoding workflow. The UNIT- DNA derivatives for coding as shown in Figure 5 may also be used for multimodal targeted RNA and DNA workflows or solid support workflows (not shown).

[0099] Accordingly, described above is An instrument for analysis and modification of a biological sample that contains a continuous stage scanning system moving the biological sample in one direction, a sample imaging system with an optical axis perpendicular to the stage scanning direction, and an optical system with an optical axis perpendicular to the stage scanning direction for applying a dynamic light pattern to induce a photochemical or a photophysical modification of the sample. This instrument, the photochemical or photophysical modification may be used as one parameter in a subsequent analysis or processing decision on the same instrument or on a different instrument.

[0100] While various details have been described in conjunction with the exemplary implementations outlined above, various alternatives, modifications, variations, improvements, and / or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent upon reviewing the foregoing disclosure. Accordingly, the exemplary implementations set forth above, are intended to be illustrative, not limiting.

Claims

WHAT IS CLAIMED IS:

1. An instrument for analysis and modification of a biological sample comprising: a continuous stage scanning system moving the biological sample in one direction; a sample imaging system with an optical axis perpendicular to the stage scanning direction; and an optical system with an optical axis perpendicular to the stage scanning direction for applying a dynamic light pattern to induce a photochemical or a photophysical modification of the sample, wherein the photochemical or photophysical modification is used as one parameter in a subsequent analysis or processing decision on the same instrument or on a different instrument.

2. The instrument of claim 1, wherein the photochemical or photophysical modification is based on an analysis of data recorded by the sample imaging system.

3. The instrument of claim 1, wherein the modification contains a chemical, biological or physical cleaving reaction; a chemical, biological or physical linking reaction; a chemical, biological or physical transformation from a non-fluorescent to a fluorescent state; a chemical, biological or physical transformation from a fluorescent to a non-fluorescent state; a chemical, biological or physical transformation that changes a fluorescence absorption and / or fluorescence emission spectrum.

4. The instrument of claim 1, wherein the photochemical or photophysical modification is induced by a single wavelength.

5. The instrument of claim 1, wherein the photochemical or photophysical modification is preferably induced by multiple wavelengths.

6. The instrument of claim 1, wherein the multiple wavelengths are present simultaneously only in the focal plane of the optical system.

7. The instrument of claim 1, wherein the dynamic light pattern is created by an element placed in an optical plane conjugate to the pupil plane of the microscope objective lens.

8. The instrument of claim 1, wherein the dynamic light pattern is created the combination of an optical scanning and laser power modulation while the stage is moving9. The instrument of claim 1, wherein the dynamic light pattern is one dimensional with the dimension being perpendicular to the stage movement direction.

10. The instrument of claim 1, wherein the optical scanning is achieved by at least one of a polygon scanner, galvanometric mirror, resonantly scanned mirror, MEMS scanner, holographic SLM, and a grated light valve.

11. The instrument of claim 1, wherein the dynamic light pattern is created by an element placed in an optical plane conjugate to the focal plane of the microscope12. The instrument of claim 1, wherein the dynamic light pattern is a two- dimensional light distribution.

13. The instrument of claim 1, wherein the dynamic light pattern is created by a digital mirror device, a polarization modulating SLM.

14. The instrument of claim 1, wherein the imaging system is a fluorescence microscope.

15. The instrument of claim 1, further comprising a static illumination pattern with respect to the optical system and a TDI camera for detection.

16. The instrument of claim 1, further comprising a scanned line in combination with a rolling shutter camera.

17. The instrument of claim 1 used with at least one of a cell sorting application, a cell separation application, a downstream analysis, a DNA sequencing system, an RNA sequencing system, and a system for performing spatial transcriptomics.

18. The instrument of claim 17, wherein a cell sorting decision is based on the photochemical or photophysical modification.

19. The combination of the instrument of claiml with a single-cell analysis application.

20. The instrument of claim 17, wherein the downstream analysis uses at least one of a moiety preferably reacting with photochemically or photo physically modified parts of the sample, and a moiety which preferably reacts with photochemically or photo physically unmodified parts of the sample.

21. A method for analysis of a continuously moving biological sample, comprising: imaging the sample; processing the data recorded during the imaging to make a modification decision; using the modification decision to apply a dynamic light pattern; inducing a photochemical or a photophysical modification of the sample during continuous movement of the sample; and using the photochemical or photophysical modification as one parameter in a subsequent analysis or processing decision.

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