Kits, methods, and systems for cellular profiling

Abstract

Embodiments herein provide kits, systems, and methods for cellular profiling. Some embodiments utilize probes that limit crosstalk between imaging filters. Additional embodiments image a multi-well plate in multiple planes to provide high throughput analysis with three dimensional (3D) analysis.

Description

Atty Dkt: ALTI-739WOKITS, METHODS, AND SYSTEMS FOR CELLULAR PROFILINGCROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63 / 715,294 filed on November 1 , 2024, which application is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to cellular imaging, more specifically, kits and probes for high-throughput profiling of cellular structure and function.INTRODUCTION

[0003] Cellular profiling is an important tool for studying cells and samples to help ascertain cellular function and / or differences between samples or across time. Current methods profile cells based on morphological features (i.e. morphological profiling). In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. Current methods of cellular profiling require complex procedures. Additionally, these methods typically balance throughput against imaging detail. Thus, there is a need in the field for kits and methods that simplify procedures and while providing both high throughput and detailed imagery.SUMMARY

[0004] Embodiments herein provide kits, systems, and methods for cellular profiling, including multiplexed cellular profiling. Some embodiments utilize probes that limit crosstalk between imaging filters. Additional embodiments image a multi-well plate in multiple planes to provide high throughput analysis with three dimensional (3D) imaging.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The description and claims will be more fully understood with reference to the following FIGS. and data graphs, which are presented as exemplary embodiments of the invention and should not be construed as a complete recitation of the scope of the invention.Atty Dkt: ALTI-739WO

[0006] FIG. 1 illustrates an example of structural profiling of a cell across six imaging channels and five Z-positions along with a composite image overlaying the six imaging channels in accordance with various embodiments.

[0007] FIGS. 2A-2B illustrate exemplary images of adherent A549 cells (FIG. 2A) and suspension CD4 T cells (FIG. 2B) in accordance with various embodiments.

[0008] FIGS. 3A-3B illustrate exemplary images obtained from adherent (FIG. 3A) and suspension (FIG. 3B) cells following structural and functional imaging in accordance with various embodiments.

[0009] FIGS. 4A-4B illustrate exemplary images matching functional imaging (FIG. 4A) with structural profiling (FIG.4B) in accordance with various embodiments.

[0010] FIG. 5 illustrates an exemplary plot of mitochondrial intensity of various cell lines in accordance with various embodiments.

[0011] FIG. 6 illustrates exemplary images of two cell lines, where some cells were treated with a WEE1 TALE repressor, while the other cells were controls showing WEE1 expression in accordance with various embodiments.DETAILED DESCRIPTION

[0012] The embodiments of the invention described herein are not intended to be exhaustive or to limit the invention to precise forms disclosed. Rather, the embodiments selected for description have been chosen to enable one skilled in the art to practice the invention.

[0013] Turning now to the drawings, many embodiments are directed to kits, methods, and systems for cellular profiling. Many embodiments provide a selection of probes that can be used simultaneously with limited crosstalk (e.g., overlap in excitation and / or emission spectra of a fluorophore) to identify targets. Certain embodiments provide methods to profile one or more cells based on imaging. In some embodiments, profiling includes structural profiling and functional profiling, where functional profiling includes gene expression information. Further embodiments allow for pooling cells from multiple samples or donors into a single well and deconvolving the source. Such pooling can be used to avoid spatial or positional effects that may occur during sample processing and labeling.Atty Dkt: ALTI-739WODEFINITIONS

[0014] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the kits, methods, and systems for cellular profiling. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the kits, methods, and systems for cellular profiling, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the kits, methods, and systems for cellular profiling.

[0015] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the kits, methods, and systems for cellular profiling belong. Although any kits, methods, and systems for cellular profiling similar or equivalent to those described herein can also be used in the practice or testing of the kits, methods, and systems for cellular profiling, representative illustrative kits, methods, and systems for cellular profiling are now described.

[0017] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present kits, methods, andAtty Dkt: ALTI-739WO systems for cellular profiling are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0018] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0019] It is appreciated that certain features of the kits, methods, and systems for cellular profiling, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the kits, methods, and systems for cellular profiling, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present kits, methods, and systems for cellular profiling and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0020] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0021] As used herein, the term “nucleotide” refers to those moieties that contain not only the naturally occurring purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. InAtty Dkt: ALTI-739WO addition, the term “nucleotide” includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

[0022] As used herein, the term “oligonucleotide” refers to a single-stranded multimer of nucleotides from 2 to 500 nucleotides, e.g., 2 to 200 nucleotides. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are 5 to 50 nucleotides in length (e.g., 9 to 50 nucleotides in length). Oligonucleotides may contain ribonucleotide monomers (i.e. , may be oligoribonucleotides or “RNA oligonucleotides”) or deoxyribonucleotide monomers (i.e., may be oligodeoxyribonucleotides or “DNA oligonucleotides”). Oligonucleotides may be 5 to 9, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500 or more nucleotides in length, for example.

[0023] As used herein, the terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1 ,000,000, up to about 1010 or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., PNA as described in U.S. Patent No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally-occurring nucleotides include guanine, cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2-aminoethyl)- glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylenecarbonyl bonds. A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon.Atty Dkt: ALTI-739WOThe bridge “locks” the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid,” or “UNA,” is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G' residue and a C residue, where these residues correspond to non-naturally occurring forms, i.e. , analogs, of G and C that base pair with each other with reduced stability but retain an ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acid is described in US20050233340, which is incorporated by reference herein for disclosure of UNA.CELLULAR PROFILING

[0024] The cellular response to perturbations (e.g., physical, chemical, genetic, etc.) can cause phenotypic changes (e.g., structural changes) in cells due to the response. High throughput screening of cells can be a powerful way to determine and / or identify these phenotypic effects. While certain dyes exist to identify various organelles, the protocols are complex, have low resolution, are not compatible with all cell types, and / or have system error due to imaging channel crosstalk (e.g., dyes with overlapping excitation spectra causing emission from an off-target fluorophore).

[0025] Various embodiments solve this problem using one or more markers (including two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, a plurality, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, etc.), where each marker is specific for a subpart of a cell (e.g., membrane, DNA, RNA, organelle, cytoskeleton, other structure). The various subparts that can be marked include a nucleus, a nucleolus, an endoplasmic reticulum, a Golgi apparatus, a mitochondria, a cytoskeleton, a lysosome, a chloroplast, a ribosome, a peroxisome, a vacuole, a cell wall, a membrane (e.g., plasma membrane, nuclear envelope, etc.) and / or any other applicable organelle or structure for a cell or cell type. In some preferred embodiments, the markers are specific to one or more of (including combinations and sub-combinations thereof) of a nucleus, a nucleolus, an endoplasmic reticulum, a Golgi apparatus, a mitochondria, and a cytoskeleton.Atty Dkt: ALTI-739WO

[0026] Each marker can be a dye, stain, probe, antibody and / or other composition that binds to a particular component. Certain markers include a fluorophore conjugate, to provide a fluorescent response in response to an excitation wavelength. Each marker that is specific for a particular structure possesses a different emission maximum — e.g., a marker for the nucleus has an emission maximum different from a marker for the nucleolus, etc. In many embodiments, one or both of the excitation spectrum and the emission spectrum of each marker diverge sufficiently to avoid crosstalk between each marker.

[0027] In many embodiments, a first marker is used to stain the nucleus. In such embodiments, a marker that can identify DNA is used to stain the nucleus, as the nucleus holds the genomic DNA of a cell. Various embodiments select a marker specific for DNA from one or more of DAPI (4',6-diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, and Hoechst 34580.

[0028] Additional embodiments identify the nucleolus of the cell. A nucleolus is a round structure residing within the nucleus and is responsible for the production of ribosomes. As such, the nucleolus is rich in RNA, due to the production (e.g., transcription) of ribosomal RNA (rRNA) from the genome. As such, various embodiments utilize an RNA- specific marker to identify the nucleolus. Various RNA-specific dyes, stains, or other markers are available in the art, including certain SYTO dyes, thiazole orange, RiboGreen, acridine orange, SYBR Green II, among others. Some embodiments utilize SYTO 14 as a marker specific for RNA.

[0029] Some embodiments use a dye to identify mitochondria in the cells to be imaged. In various instances, the mitochondria are labeled using a MitoTracker dye. Various MitoTracker dyes are available and may be selected from one or more of MitoTracker Green FM, MitoTracker Deep Red FM, MitoTracker Red CMXRos, MitoTracker Red FM, MitoTracker Red CM-H2Xros, and MitoTracker Orange CMTMRos. The particular MitoTracker dye may be selected based on preferred emission and / or excitation spectra. In some preferred embodiments, MitoTracker Deep Red FM is preferred based on its emission and / or excitation spectra. Various instances incubate a cell with a marker to identify mitochondria for a time sufficient to stain mitochondria — such information may be comprised in the packaging materials or manufacturer’s instructions.Atty Dkt: ALTI-739WO

[0030] Further embodiments identify the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton using markers conjugated to a fluorophore. Such markers can include antibodies, proteins, small molecules, and / or other molecules. Some preferred embodiments use lectins as the molecule used in the marker. Lectins are a class of proteins with the ability to bind to carbohydrates. Such lectins include concanavalin A (con-A) and wheat germ agglutinin (WGA). Both con-A and WGA have the ability to bind specific sugars, glycoproteins, and glycolipids. Based on these specific properties, con-A can be used to identify the endoplasmic reticulum, while WGA can be used to identify the Golgi apparatus. Additional embodiments identify the cytoskeleton using phalloidin. Phalloidin is a toxin formed as a bicyclic peptide and has known ability to bind actin filaments, thus phalloidin can be used as a marker for the cytoskeleton.

[0031] In various embodiments, phalloidin, con-A, and WGA are conjugated to a fluorophore to be able to image and identify each component. Various fluorophores are known in the art and can be used identify each of these cellular components, depending on preferred emission and / or excitation spectra, available filters, etc. As with other markers, the emission and / or excitation spectra of fluorophores can diverge sufficiently to avoid crosstalk between each fluorescent marker in the plurality of markers. Such fluorophores can include one or more of fluorescein isothiocyanate (FITS or fluorescein), rhodamine (e.g., rhodamine B, rhodamine 6G), Texas Red, Cy3, Cy5, Cy5.5, Cy7, green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, mCherry, phycoerythrin (PE), allophycocyanin (APO), tetramethylrhodamine isothiocyanate (TRITC), Alexa Fluor dyes (e.g., Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 647, etc.). Some preferred embodiments use Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 750 as fluorophore conjugates to con-A, WGA, and phalloidin.

[0032] Some embodiments provide a plurality of fluorescent markers, where each fluorescent marker in the plurality of fluorescent markers is specific for a subpart of a cell, where each fluorescent marker has a different emission maximum, and where one or both of the excitation spectrum and the emission spectrum of each fluorescent marker diverge sufficiently to avoid crosstalk between each fluorescent marker in the plurality of fluorescent markers. In various embodiments, the panel comprises at least two fluorescent markers, at least three fluorescent markers, at least four fluorescent markers,Atty Dkt: ALTI-739WO at least five fluorescent markers, at least six fluorescent markers, or at least seven fluorescent markers. In various embodiments, each fluorescent marker is specific for one of the following: a nucleus, a nucleolus, an endoplasmic reticulum, a Golgi apparatus, a mitochondria, and a cytoskeleton.

[0033] In some embodiments, the fluorescent markers are selected from: one of Hoechst 33342, Hoechst 33258, Hoechst 34580, or DAPI, con-A conjugated to Alexa Fluor 488, SYTO 14, WGA conjugated to Alexa Fluor 594, MitoTracker Deep Red FM, and phalloidin conjugated to Alexa Fluor 750.FUNCTIONAL IMAGING

[0034] Additional embodiments provide for functional imaging of a cell, such as through fluorescence in situ hybridization (FISH), including RNA FISH to examine gene expression levels and location (e.g., within nucleus, cytoplasm, or other cellular organelle or region) . In some embodiments, such functional imaging occurs after cellular profiling as described herein . In many instances, functional profiling includes hybridizing a nucleic acid probe to a target nucleic acid molecule or sequence. In many instances, the nucleic acid probe is complimentary to the target nucleic acid.

[0035] In many embodiments, RNA FISH occurs via any available methodology, including single molecule FISH (e.g., smRNA FISH), branched DNA FISH (e.g., bDNA based smRNA FISH), viewRNA, and / or any other applicable method to identify RNA sequences in situ. Fluorescent markers, dyes, or probes may utilize a fluorescent label as described above (e.g., FITC, TRITC, Alexa Fluor dye (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 750), etc.). Certain instances may utilize a secondary antibody to amplify or enhance a signal. In such instances, a nucleic acid probe may be labeled with an antigen or other conjugate which is the target of a second molecule (e.g., an antibody). Such secondary labeling can include a biotinstreptavidin system (e.g., nucleic acid probe is conjugated to biotin, and streptavidin is conjugated to a fluorophore) and / or various antigen-antibody couplings, where the nucleic acid is conjugated to an antigen, and the targeting antibody is conjugated to a fluorophore. Alternatively or additionally, some embodiments utilize an in situ amplification reaction to amplify a signal.Atty Dkt: ALTI-739WOSAMPLE IDENTIFICATION

[0036] Within a multi-well plate, each well or position on a plate can introduce technical error, as created by timing, uneven heating, or other effects. As such, pooling multiple samples within a single well may control for these positional (or well-dependent) effects. To identify individual sources, certain a probe targeting a surface antigen, where each sample had a differently colored fluorophore attached to it. Such source-identifying markers can be an antibody or other dye or probe that is removable or reversible. In such embodiments, a first imaging occurs to identify sample origins of particular cells based on the antibodies. Then, the source-identifying markers are removed, stripped, or otherwise eliminated, then additional markers are added to identify structures, followed by subsequent structural profiling. In various instances, the source-identifying markers occur after inclusion or incorporation of a marker that requires living organelles or cells — for example, MitoTracker dyes are incorporated by function of the mitochondria. Thus, the source-identifying markers may have an emission and / or excitation spectrum that diverges from a MitoTracker dye.MULTIPLEXING

[0037] Certain embodiments combine one or more of functional imaging, structural profiling, and sample identification into a single imaging strategy. In some instances, a single panel of probes can label one or more cellular components, one or more RNA locations, and / or identify samples. In some instances, each panel of probes may be separate, such that multiple rounds of labeling and imaging may be performed. For example, a first imaging round may identify sample sources (e.g., from pooling), then a second round to identify a structural profile, followed by a third round to functionally image the cells — it should be noted that this is merely exemplary and each type of imagine (e.g., functional, structural, etc.) may be performed in a different order (e.g., sample identification, then functional, then structural, etc.). Alternatively, as noted previously, one or more round of imaging may include one or more probes from multiple types ofAtty Dkt: ALTI-739WO imaging — as a non-limiting example: a first round of structural profiling followed by a second round for RNA-FISH and sample identification.

[0038] In instances, where multiple rounds of labeling and imaging occur, probes may be stripped or removed from the cells prior to introducing the second round of probes. For example, one or more structural probes may be stripped or removed prior to introduction of one or more nucleic acid probes. Stripping the probes can include using a chemical agent that disrupts the molecular interaction between a marker and its target subpart (e.g., disrupts the interaction between con-A and the endoplasmic reticulum) or the marker can be stripped by removing the fluorescent moiety, such as by using a cleavable linker between the fluorophore and the conjugated molecule. Stripping can occur using one or more of a de-ionizing agent, a denaturing agent, a solvent, a hydrolyzing agent, and / or any other agent capable of stripping,. Various embodiments may select the particular stripping agent from one or more of formamide, ethanol, acetone, isopropyl alcohol (isopropanol), an enzyme, and / or other stripping agent. Some instances may photobleach a fluorophore to reduce its signal. Photobleaching may comprise exposing the labeled cells to its excitation wavelength for a period of time sufficient to photobleach the fluorophore. In some instances, a broad spectrum of light may be used for photobleaching multiple fluorophores simultaneously — for example, a white light may be used to photobleach multiple fluorophores simultaneously.

[0039] In certain instances, not all of a signal may be stripped, which can interfere with a subsequent functional imaging. In such instances, the fluorescent label for a subsequent round of labeling and imaging may be selected based on which labels do not strip — e.g., MitoTracker dyes may not strip easily, thus subsequent probes may avoid the emission and / or excitation wavelengths / spectra of the MitoTracker dye.KITS

[0040] Various embodiments are directed to kits that include one or more of the markers described above, including kits comprised of one or markers for structural profiling, one or more markers for functional imaging, and / or one or more markers for pooling samples (e.g., marking cellular sources). Some embodiments comprise a substrate for imaging, such as a slide, a petri dish, and / or a multi-well plate (e.g., 6-well,Atty Dkt: ALTI-739WO12-well, 24-well, 48-well, 96-well, 192-well, 384-well, 1536-well, etc.). Such substrate may be opaque, translucent, or have some translucent areas and opaque areas (e.g., a translucent top and / or bottom to allow light to pass through a sample, while having opaque walls or sides, such that light does not bleed through from or to other wells, to minimize background or stray illumination. Plates of some embodiments are poly-lysine coated (e.g., poly-L-lysine and / or poly-D-lysine). Certain embodiments may include a sealing mechanism for the substrate, such as a film or cover.

[0041] Certain embodiments include buffers or reagents for sample preparation, processing, and / or any other purpose. Such buffers and reagents may be packaged in liquid form and / or solid (e.g., powdered, desiccated, tabletized, etc.) for reconstitution at a later time point.

[0042] Additional embodiments may comprise an instruction manual, material safety data sheet, or other form of literature. Various embodiments include packaging, such as baggies, boxes, vials, tubes, etc. as necessary for protection and / or storage. Certain kits may package certain components in separate boxes or outer containers, such as when certain components should be kept refrigerated (e.g., approximately 4°C) or frozen (e.g., approximately -20°C and / or approximately -80°C).METHODS

[0043] Various embodiments are directed to methods of cell painting. In various instances, one or more cells are cultured in the appropriate media (e.g., liquid culture and / or solid media). In some instances, adherent cells are cultured directly on a plate or substrate for imaging (e.g., a multi-well plate, a slide, etc.) For suspension cells, the cells may be collected (e.g., via sedimentation, centrifugation, etc.) then seeded onto (or into) the imaging plate or substrate. In many instances, the one or more cells are fixed to the substrate and / or permeabilized to increase or allow marker perfusion.

[0044] The one or more cells can then be mixed with one or more markers (e.g., fluorescent markers), such as described previously — e.g., where each marker is specific for a subpart of a cell, where each marker has a different emission maximum, and where one or both of the excitation spectrum and the emission spectrum of each marker diverge sufficiently to avoid crosstalk between each marker.Atty Dkt: ALTI-739WO

[0045] As noted above, certain markers may be introduced via cellular activity (e.g., MitoTracker dyes), thus such dyes may be introduced and incubated prior to fixing, permeabilizing, seeding and / or adding additional markers. For example, some methods may provide a marker specific for mitochondria to the one or more cells; incubate the one or more cells and the marker specific for mitochondria for a time sufficient to stain mitochondria; wash the one or more cells to remove excess of the fluorescent marker specific for mitochondria; fix the one or more cells; permeabilize the one or more cells; and provide the remaining markers to the one or more cells. The incubation step for a marker specific for mitochondria may occur at any applicable temperature or time for sufficient staining. For example, the timing may be approximately 15-60 minutes, such as approximately 15 minutes, approximately 20 minutes, approximately 30 minutes, approximately 45 minutes, or approximately 60 minutes. Temperature can occur at an ambient (e.g., “room”) temperature or approximately 20°C, or within a range of approximately 15°C-25°C.

[0046] The one or more cells can then be imaged to obtain a morphological profile of the one or more cells. Imaging can occur at a single vertical position or a multiple Z- positions (or Z-slices) (e.g., 5-15 Z-slices). In some preferred embodiments, the one or more cells are imaged at multiple Z-slices to be able to generate a 3D image of the cell. Such Z-slices may be taken at any applicable spacing (e.g., 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 50 pm, etc.). Any number of Z-slices may be obtained, such as 1 slice (e.g., single Z-position), 2 slices, 3 slices, 5 slices, 10 slices, 15 slices, 20 slices, 25 slices, or more. In various instances, the number of Z-slices and distance between these Z-slices are determined based on approximate cell size, desired resolution, total time, total amount of data, processing speed, etc. For example, fewer Z-slices may allow for faster imaging but sacrifice resolution. Similarly, the particular objective may be altered based on the resolution and / or speed — e.g., a 100X objective may have finer resolution, but it has a lower field of view. Objectives can be any applicable type, including water immersion, oil immersion, etc.

[0047] In various instances, imaging comprises utilizing an appropriate filter set to allow excitation and / or detect emission of the markers. In various instances, the combination of a particular filter set and the markers may assist in preventing or avoidingAtty Dkt: ALTI-739WO crosstalk. In some embodiments, each filter in the filter set is paired to one fluorescent marker and / or each fluorescent marker is paired to one filter in the filter set. In some instances, each filter is paired with one fluorescent marker in the mixture of fluorescent markers such that a combination of the emission spectrum of each fluorescent marker and the emission band of its paired emission filter minimize fluorescence cross talk between each filter. In some instances, the filter set may include filters commonly used in blue, green, yellow, orange, red, near-infrared, and infrared regions of the light spectrum. In some embodiments, the filters are named based on common fluorophores or fluorescent dyes, such as DAPI, FITC, YFP, TRITC, Texas Red, Cy5, and Cy7. Some embodiments may utilize filters with similar excitation and emission excitation and emission spectra (e.g., maximum and bandwidth) as the foregoing fluorophores and dyes. In some embodiments, the filter set is selected from: excitation maximum 405 nm, bandwidth 20 nm; emission maximum 452 nm, bandwidth 45 nm; excitation maximum 468 nm, bandwidth 21 nm; emission maximum 520 nm, bandwidth 28 nm; excitation maximum 520 nm, bandwidth 10 nm; emission maximum 549 nm, bandwidth 12 nm; excitation maximum approximately 555 nm; emission maximum 582 nm, bandwidth 15 nm; excitation maximum approximately 555 nm; emission maximum 630 nm, bandwidth 20 nm; excitation maximum 638 nm, bandwidth 17 nm; emission maximum 692 nm, bandwidth 40 nm; and excitation maximum 725 nm, bandwidth 20 nm; emission maximum 794 nm, bandwidth 32 nm.Functional Imaging

[0048] Various embodiments may follow cellular profiling by functionally imaging gene expression, such as by components described herein. In such instances, markers for structural profiling may be stripped or otherwise removed. Such stripping can includeAtty Dkt: ALTI-739WO chemically removing the markers, such as by the use of a formamide solution (e.g., 25% formamide, 50% formamide, 75% formamide, 100% formamide), an alcohol solution (e.g., ethanol, isopropanol, etc.), a ketone solution (e.g., acetone), and / or any other applicable method that can disrupt or otherwise strip markers from their target subpart.

[0049] Some embodiments can optionally image the one or more cells after stripping for a determination of stripping efficiency, background signal, or other aspect that may cause interference with downstream imaging or profiling. Based on any residual signal, probes for functional profiling (e.g., RNA FISH) may be selected to avoid any interference or background noise that may arise from incomplete stripping of structural probes.

[0050] Once the structural profiling markers are stripped from one or more cells, a nucleic acid probe (or multiple nucleic acid probes) may be added to the one or more cell to allow imaging of a particular nucleic acid sequence. In many instances, functional profiling includes producing a reaction mixture comprising a cell (e.g., cell from morphological profiling) and a nucleic acid probe. In many instances, the nucleic acid probe is complimentary to the target nucleic acid. Such nucleic acid probes are described elsewhere herein, and such FISH processes may proceed according to the applicable protocol, such as for an amplification-based FISH method, addition of secondary antibodies, etc.

[0051] After introduction of a nucleic acid probe, Some may provide one or more counterstains to identify a structure within the cell. Some counterstains may bind a nucleic acid (e.g., RNA and / or DNA) in a sequence-nonspecific manner. For example, DAPI or a Hoechst stain as indicated above to identify the nucleus within each of the one or more cells. Alternatively or additionally, the one or more cells may be imaged again to obtain expression data including location of the sequences within the cell and possible quantification of expression via signal intensity (e.g., brighter signal may indicate a higher level of expression). As with the structural profiling, imaging may occur at different Z- positions (or Z-slices) to further provide 3D positioning and / or 3D expression data of the RNA molecules targeted via the probes.Pooling Samples

[0052] Different positions within a plate (e.g., imaging plate, culturing plate, etc.) may have positional effects, creating variation in sample preparation. Such positional effectsAtty Dkt: ALTI-739WO may be caused by unequal temperature distribution (e.g., heating during incubation such that positions on the edges may reach ideal temperatures prior to more internal positions), time differences (e.g., differences between imaging a first well and a final well, time differences created by adding reagents to wells, etc.), and various other phenomena that cause differences between wells or positions within a plate. Combining samples within a well can compensate for these effects. However, simply combining samples may prevent the ability to identify individual samples from each other.

[0053] Many embodiments are capable of minimizing positional effects by pooling samples and identifying which cells arise from each sample. To accomplish this task, prior to seeding wells, removable or reversible probes are mixed with cells from each sample, where each probe has a unique fluorophore or marker — e.g., each fluorophore differs in excitation and / or emission spectra. In some instances, probes may be combined to increase the number of samples that can be used — as a non-limiting example, two probes may be used to identify three samples, where a first sample receives the first probe, the second sample receives the second probe, and the third sample receives both the first and second probes. One of skill in the art will understand the various combinations that can be created using multiple probes (e.g., 3 probes can identify 7 samples, 4 probes can identify 14 samples, etc.).

[0054] Such probes may be a peptide (e.g., antibody, protein, etc.), carbohydrate, fatty acid, any combination of any of the foregoing, and / or any other compound that can affiliate with a cell. In many embodiments, the probe has the same molecule (e.g., peptide, carbohydrate, etc.), so that every probe should act similarly. In many embodiments, a probe is conjugated or otherwise linked to a marker (such as a fluorophore or other marker noted herein). Such markers may reversibly linked to the probe (e.g., where a linking moiety is cleavable, breakable, hydrolysable, etc.). In certain embodiments, the probe attaches to a surface of a cell (e.g., membrane, surface antigen, etc.). Surface attachment may assist with stripping or removing the probe or its marker.

[0055] Once each sample is mixed with a probe to identify the cell, the cells can be seeded and fixed to a plate, such as described above. Certain embodiments may utilize one or more additional counterstains (e.g., DAPI, Hoechst, etc.) to identify particularAtty Dkt: ALTI-739WO landmarks — however, such a step can be optional, such as when a MitoTracker dye is used identify mitochondria.

[0056] The cells can be imaged to identify which cells arise from which sample. Such imaging can occur across as many channels that are appropriate for the number of used probes. When combining probes to be able to image multiple samples, image overlays may be used to identify cells that have multiple probes.

[0057] Once the cells have been identified based on sample, the probes may be stripped and / or removed from the cells. As mentioned previously, some probes may be linked to the fluorophore or other marker via a cleavable linker. In such instances, the appropriate reactant, enzyme, or other reagent that breaks the linkage. Other embodiments may strip the probe using a stripping reagent, such as formamide, alcohol, or other stripping agent.General Considerations

[0058] The various methods, processes, kits, etc. may be used individually or in combination with each other. For example, some embodiments may perform sample identification, structural profiling, functional imaging, and any combination thereof. When performing multiple such procedures, various embodiments perform image registration. Such image registration notes the position of one or more cells on such a plate. By performing such registration, a subsequent imaging step can overlay the data (e.g., sample of origin and structural profiling; structural profiling and functional imaging, etc.) with minimal variation on position. As cells are microscopic, small changes in position may cause an additional level of variation between each imaging cycle. Thus, image registration may benefit from precision alignment for each round. To perform such positioning, various embodiments align an imaging slide or plate using precision guides, robotic arms, and / or other feature that allows the positioning to be the same during each imaging step. In many instances, the alignment allows for a functional profile to occur at the same coordinates as a morphological profile and / or pooled sample. By obtaining images at the same coordinates, the images may be superimposed upon each other between imaging cycles. In some instances, the aligning an imaging plate or slide comprises utilizing a robotic arm.Atty Dkt: ALTI-739WOASPECTS OF THE INVENTION

[0059] For purposes of completeness, various aspects of the present disclosure are set out in the following numbered clauses.Aspect 1 . A method for painting cells based on morphology, comprising: producing a reaction solution comprising: a cell, and a mixture of at least six fluorescent markers, wherein each fluorescent marker is specific for a subpart of the cell, selected from a nucleus, a nucleolus, an endoplasmic reticulum, a Golgi apparatus, a mitochondrion, and a cytoskeleton, and wherein each fluorescent marker has a different emission spectrum; and imaging the cell across a set of filters to obtain a morphological profile of the cell, wherein each filter is paired with one fluorescent marker in the mixture of fluorescent markers such that a combination of the emission spectrum of each fluorescent marker and the emission band of its paired emission filter minimize fluorescence cross talk between each filter.Aspect 2. The method of Aspect 1 , wherein the set of filters are selected from: a) excitation maximum 405 nm, bandwidth 20 nm; emission maximum 452 nm, bandwidth 45 nm; b) excitation maximum 468 nm, bandwidth 21 nm; emission maximum 520 nm, bandwidth 28 nm; c) excitation maximum 520 nm, bandwidth 10 nm; emission maximum 549 nm, bandwidth 12 nm; d) excitation maximum approximately 555 nm; emission maximum 582 nm, bandwidth 15 nm; e) excitation maximum approximately 555 nm; emission maximum 630 nm, bandwidth 20 nm; f) excitation maximum 638 nm, bandwidth 17 nm; emission maximum 692 nm, bandwidth 40 nm; andAtty Dkt: ALTI-739WO g) excitation maximum 725 nm, bandwidth 20 nm; emission maximum 794 nm, bandwidth 32 nm.Aspect 3. The method of Aspect 1 or 2, wherein the mixture of fluorescent markers comprise: a) one of Hoechst 33342, Hoechst 33258, or Hoechst 34580; b) con-A conjugated to Alexa Fluor 488; c) SYTO 14; d) WGA conjugated to Alexa Fluor 594; e) MitoTracker Deep Red FM; and f) phal loidin conjugated to Alexa Fluor 750.Aspect 4. The method of any one of Aspects 1 -3, wherein producing a reaction mixture comprises: incubating the cell with a fluorescent marker specific for mitochondria for a time sufficient to stain mitochondria; and providing the remaining fluorescent markers from the plurality of fluorescent markers to the cell.Aspect 5. The method of any one of Aspects 1 -3, wherein producing a reaction mixture comprises: providing a fluorescent marker specific for mitochondria to the cell; incubating the cell and the fluorescent marker specific for mitochondria for a time sufficient to stain mitochondria; washing the cell to remove excess of the fluorescent marker specific for mitochondria; fixing the cell; permeabilizing the cell; and providing the remaining fluorescent markers from the plurality of fluorescent markers to the cell.Aspect 6. The method of Aspect 4 or 5, wherein incubating occurs at an ambient temperature and wherein the time is 15-60 minutes.Aspect 7. The method of Aspect 6, wherein the ambient temperature is approximately 20°C.Atty Dkt: ALTI-739WOAspect 8. The method of Aspect 6, wherein the ambient temperature is 15-25°C.Aspect 9. The method of any of Aspects 1 -8, wherein the cell comprises a cell culture comprising a plurality of cells.Aspect 10 The method of Aspect 9, wherein the cell culture comprises one or both adherent cells and suspended cells.Aspect 11. The method of any of Aspects 1 -10, wherein imaging the reaction mixture comprises obtaining a plurality of images, wherein each image captures an X-Y plane at a different Z-position.Aspect 12. The method of Aspect 11 , wherein the plurality of images comprises 5-15 images.Aspect 13. The method of Aspect 1 1 , wherein the plurality of images comprises 10 images.Aspect 14. The method of any of Aspects 1 -13, wherein imaging occurs with a 40x objective.Aspect 15. The method of any of Aspects 1 -14, wherein imaging occurs with at least 400x magnification.Aspect 16. The method of any of Aspects 1 -15, further comprising removing a signal from at least one of the fluorescent markers.Aspect 17. The method of Aspect 16, wherein removing the signal comprises photobleaching or stripping a marker.Aspect 18. The method of Aspect 17, wherein stripping the marker comprises contacting the cell with one or more of formamide, ethanol, acetone, and isopropyl alcohol.Aspect 19. The method of any one of Aspects 16-18, further comprising: producing a second reaction mixture comprising: the cell, and a nucleic acid probe that is complimentary to a target nucleic acid molecule within the cell; and imaging the cell across the set of filters to obtain a functional profile of the cell.Aspect 20. The method of Aspect 19, wherein the nucleic acid probe utilizes an amplification reaction.Atty Dkt: ALTI-739WOAspect 21. The method of Aspect 19 or 20, wherein the nucleic acid probe is conjugated to a fluorescent dye.Aspect 22. The method of any one of Aspects 19-21 , wherein the nucleic acid probe comprises a plurality of nucleic acid probes, each of which is specific to a different target nucleic acid within the cell.Aspect 23. The method of any one of Aspects 19-22, wherein the nucleic acid probe is a ViewRNA probe system.Aspect 24. The method of any one of Aspects 19-23, further comprising aligning an imaging plate, such that imaging the cell to obtain a morphological profile and imaging the cell to obtain a functional profile occur at the same coordinates as to allow superimposition of the acquired images.Aspect 25. The method of Aspect 24, wherein aligning the imaging plate comprises utilizing a robotic arm.Aspect 26. A method for profiling a cell based on functional details, comprising: producing a reaction solution comprising: a cell, a mixture of at least three nucleic acid probes, wherein each nucleic acid probe is complimentary to a different target nucleic acid molecule within the cell, and wherein each nucleic acid probe is conjugated to a fluorescent moiety, wherein each fluorescent moiety has a different emission spectrum, and a fluorescent dye that binds to one or both of RNA and DNA in a sequence-nonspecific manner, wherein the fluorescent dye has an emission spectrum that differs from the fluorescent moieties conjugated to the nucleic acid probes. imaging the reaction solution across a set of filters to obtain a functional profile of the cell, wherein each filter is paired with one fluorescent moiety or the fluorescent dye that a combination of the emission spectrum of each fluorescent moiety or dye and the emission band of its paired emission filterAtty Dkt: ALTI-739WO minimize fluorescence cross talk between each filter.Aspect 27. A method for multiplexed cell morphology imaging, comprising: providing a first fluorescently labeled antibody to a first cell and a second fluorescently labeled antibody to a second cell, wherein the first and second fluorescently labeled antibodies are specific to a surface antigen on each cell and the first and second fluorescently labeled antibodies possess different emission spectra; fixing the first cell and the second cell to a well of an imaging plate; imaging the well across multiple filters to identify a position of the first cell and a position of the second cell; removing a signal from the first and second fluorescently labeled antibodies in accordance with any one of Aspects 16-18; and painting the first and second cells via the method of any one of Aspects 1 -26.Aspect 28. The method of Aspect 27, further comprising: providing both the first and second fluorescently labeled antibodies to a third cell; fixing the third cell to the well of the imaging plate; wherein imaging the well across multiple filters further identifies a position of the third; wherein painting the first and second cells via the method of any one of Aspects1 -24 also paints the third cell.Aspect 29. The method of Aspect 27 or 28, wherein the first cell, the second cell, and the third cell are from different sources.Aspect 30. A kit comprising the mixture of fluorescent markers listed Aspect 3.Aspect 31. The kit of Aspect 30, further comprising a stripping agent, optionally selected from the agents according to Aspect 18.Aspect 32. The kit of Aspect 30 or 31 , further comprising one or more filters selected from the filters listed in Aspect 2.EXEMPLARY EMBODIMENTS

[0060] Although the following embodiments provide details on certain embodiments of the inventions, it should be understood that these are only exemplary in nature and are not intended to limit the scope of the invention.Atty Dkt: ALTI-739WOExample 1 : Three Dimensional Cellular Imaging

[0061] Background: Using multiple dyes when imaging can cause crosstalk, due to overlap in excitation wavelength of certain dyes. This embodiment provides an alternative dye set that reduces such crosstalk.

[0062] Methods: Cells were cultured in the appropriate media for growth — adherent cells were grown directly in a multi-well plate coated with poly-L-lysine. MitoT racker Deep Red FM was provided to the cells according to the manufacturer's instructions to allow incorporation and identification of mitochondria. Suspension cells were collected then seeded into a multi-well plate coated with poly-L-lysine. The cells were then fixed with formaldehyde and permeabilized. The permeabilization step included adding additional dyes to identify additional cellular components, as listed below. Hoechst (to identify the nucleus)- Sytol 4 (to identify the nucleoli)Concanavalin A conjugated to Alexa Fluor 488 (to identify the endoplasmic reticulum)- Wheat germ agglutinin conjugated to Alexa Fluor 594 (to identify the Golgi apparatus)- Phalloidin conjugated to Alexa Fluor 750 (to identify the cytoskeleton)Combining permeabilization and the other dyes allows for more rapid and simpler protocol. The cells were then washed to remove excess dye.

[0063] The plate was imaged using a 40X water-immersion lens using a Z-stack of 10 images with each channel.

[0064] Results: FIG. 1 illustrates an array of images obtained from a cell at various Z- stack depths, where each row represents a layer in the Z-stack, and each column illustrates the image obtained from each channel, as indicated by the target at the top. The seventh column provides a composite image or overlay of all six channels, which shows the ability to image a cell three dimensionally over several fluorescence channels.

[0065] FIGS. 2A-2B illustrate the ability for the embodiment to work with adherent A549 cells (FIG. 2A) and suspension CD4 T cells (FIG. 2B). As seen by these figures, the combination of probes and protocol are capable of imaging both adherent and suspensionAtty Dkt: ALTI-739WO cells. Additionally, both sets of images show high resolution of the structures within each channel.

[0066] Conclusions: The protocol and probe panel provides high resolution imaging of cells in three dimensions across cell types.Example 2: Sample Pooling

[0067] Background: Within a multi-well plate, each well or position on a plate can introduce an error factor, as created by timing, uneven heating, or other effects. As such, pooling multiple samples within a single well may control for these positional (or welldependent) effects. To identify individual sources, this embodiment introduced a probe targeting a surface antigen, where each sample had a differently colored fluorophore attached to it. Such probes can be an antibody or other dye or probe that is removable or reversible.

[0068] Methods:. This embodiment grew each sample (adherent cells and suspension cells) independently and apart from the multi-well plate. A MitoTracker dye was added according to manufacturer’s instructions. Cells were then incubated with an antibody to identify that sample. Cells were then collected and seeded into wells, where each well included cells from one or more samples, where multi-sample wells included samples with differently colored labels. The plates were then imaged to identify which cells were from which sample. The labels were then stripped from the cells to remove potential cross talk from additional dyes or probes (e.g., dyes / probes from Example 1 ). Once the labels were stripped, the cells were permeabilized and the additional dyes (e.g., form Example 1 ) were added. The remaining protocol from Example 1 was repeated in order to stain the cells, which were then imaged using a Z-stack (as in Example 1 ).

[0069] Results: The cells were able to be identified based on sample as well as identify the individual structures as described in Example 1 .Example 3: Functional Imaging of Cells

[0070] Background: While structural cellular identification is important, the underlying understanding of gene expression in a positional manner can identify additional characteristics of the cellsAtty Dkt: ALTI-739WO

[0071] Methods: Following structural imaging according to Either Example 1 or Example 2, the dyes were stripped from the cells. Nucleic acid probes for RNA-FISH were added according to manufacturer instructions or other relevant protocol. In this example, the probe was specific to RPS18 RNA. The plates were then re-imaged to identify the positional expression of the gene.

[0072] Results: FIGS. 3A and 3B illustrate the images obtained from adherent (FIG. 3A) and suspension (FIG. 3B) cells following structural and functional imaging. The six initial channels used for structural imaging (top row) are illustrated to show the applicable structures identified in each channel. The middle row illustrates cellular imaging in each channel after removal of the dyes to indicate potential artifacts remaining after stripping the structural dyes. The bottom row illustrates the RNA-FISH images obtained from DAPI staining of the nuclei and the applicable channel for the fluorophore used to identify RPS18. A seventh imaging channel was used to image the RPS18 probe. The remaining channels were imaged to show any remaining artifacts after stripping and FISH probing.

[0073] Cells were then matched based on structure and function as illustrated in FIGS. 4A-4B. As seen in FIG. 4A, the cells along the top row have a higher expression level than cells along the bottom row. FIG. 4B provides images of the commensurate structural imaging for each of the cells in FIG. 4A.

[0074] Results: This embodiment illustrates the ability to image cells based on both structure and function.Example 4: Cellular health assessment

[0075] Various cells were imaged according to Example 1. The cells had differing levels of health or viability based on the gene expression. FIG. 5 illustrates a plot of mitochondrial intensity of various cell lines. The WEE1 line had the lowest cellular health of the other lines and had the lowest intensity signal from mitochondria. This data indicates how functional imaging (e.g., RNA-FISH) can be connected to the morphological or structural imaging. Combining these methods of functional and structural imaging allows for assessment of how gene expression affects cellular morphology.Atty Dkt: ALTI-739WOExample 5: Gene Expression

[0076] Embodiments can identify gene expression in cells. In this example, certain cells were treated with a WEE1 TALE repressor. FIG.6 illustrates two cell lines, where some cells were treated with a WEE1 TALE repressor, while the other cells were controls showing WEE1 expression.

[0077] These results show that embodiments are capable of differentiating gene expression within a cell (e.g., location of the RNA) from nuclear or genomic copies of the genes.DOCTRINE OF EQUIVALENTS

[0078] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, a number of well- known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.

[0079] Those skilled in the art will appreciate that the foregoing examples and descriptions of various preferred embodiments of the present invention are merely illustrative of the invention as a whole, and that variations in the components or steps of the present invention may be made within the spirit and scope of the invention. Accordingly, the present invention is not limited to the specific embodiments described herein, but, rather, is defined by the scope of the appended claims.

Claims

Atty Dkt: ALTI-739WOWHAT IS CLAIMED IS:1 . A method for painting cells based on morphology, comprising: producing a reaction solution comprising: a cell, and a mixture of at least six fluorescent markers, wherein each fluorescent marker is specific for a subpart of the cell, selected from a nucleus, a nucleolus, an endoplasmic reticulum, a Golgi apparatus, a mitochondrion, and a cytoskeleton, and wherein each fluorescent marker has a different emission spectrum; and imaging the cell across a set of filters to obtain a morphological profile of the cell, wherein each filter is paired with one fluorescent marker in the mixture of fluorescent markers such that a combination of the emission spectrum of each fluorescent marker and the emission band of its paired emission filter minimize fluorescence cross talk between each filter.

2. The method of claim 1 , wherein the set of filters are selected from: a) excitation maximum 405 nm, bandwidth 20 nm; emission maximum 452 nm, bandwidth 45 nm; b) excitation maximum 468 nm, bandwidth 21 nm; emission maximum 520 nm, bandwidth 28 nm; c) excitation maximum 520 nm, bandwidth 10 nm; emission maximum 549 nm, bandwidth 12 nm; d) excitation maximum approximately 555 nm; emission maximum 582 nm, bandwidth 15 nm; e) excitation maximum approximately 555 nm; emission maximum 630 nm, bandwidth 20 nm; f) excitation maximum 638 nm, bandwidth 17 nm; emission maximum 692 nm, bandwidth 40 nm; andAtty Dkt: ALTI-739WO g) excitation maximum 725 nm, bandwidth 20 nm; emission maximum 794 nm, bandwidth 32 nm.

3. The method of claim 1 or 2, wherein the mixture of fluorescent markers comprise: a) one of Hoechst 33342, Hoechst 33258, or Hoechst 34580; b) con-A conjugated to Alexa Fluor 488; c) SYTO 14; d) WGA conjugated to Alexa Fluor 594; e) MitoTracker Deep Red FM; and f) phal loidin conjugated to Alexa Fluor 750.

4. The method of any one of claims 1 -3, wherein producing a reaction mixture comprises: incubating the cell with a fluorescent marker specific for mitochondria for a time sufficient to stain mitochondria; and providing the remaining fluorescent markers from the plurality of fluorescent markers to the cell.

5. The method of any one of claims 1 -3, wherein producing a reaction mixture comprises: providing a fluorescent marker specific for mitochondria to the cell; incubating the cell and the fluorescent marker specific for mitochondria for a time sufficient to stain mitochondria; washing the cell to remove excess of the fluorescent marker specific for mitochondria; fixing the cell; permeabilizing the cell; and providing the remaining fluorescent markers from the plurality of fluorescent markers to the cell.

6. The method of claim 4 or 5, wherein incubating occurs at an ambient temperatureAtty Dkt: ALTI-739WO and wherein the time is 15-60 minutes.

7. The method of claim 6, wherein the ambient temperature is approximately 20°C.

8. The method of claim 6, wherein the ambient temperature is 15-25°C.

9. The method of any of claims 1 -8, wherein the cell comprises a cell culture comprising a plurality of cells.10 The method of claim 9, wherein the cell culture comprises one or both adherent cells and suspended cells.1 1 . The method of any of claims 1 -10, wherein imaging the reaction mixture comprises obtaining a plurality of images, wherein each image captures an X-Y plane at a different Z-position.

12. The method of claim 11 , wherein the plurality of images comprises 5-15 images.

13. The method of claim 11 , wherein the plurality of images comprises 10 images.

14. The method of any of claims 1 -13, wherein imaging occurs with a 40x objective.

15. The method of any of claims 1 -14, wherein imaging occurs with at least 400x magnification.

16. The method of any of claims 1 -15, further comprising removing a signal from at least one of the fluorescent markers.

17. The method of claim 16, wherein removing the signal comprises photobleaching or stripping a marker.Atty Dkt: ALTI-739WO18. The method of claim 17, wherein stripping the marker comprises contacting the cell with one or more of formamide, ethanol, acetone, and isopropyl alcohol.

19. The method of any one of claims 16-18, further comprising: producing a second reaction mixture comprising: the cell, and a nucleic acid probe that is complimentary to a target nucleic acid molecule within the cell; and imaging the cell across the set of filters to obtain a functional profile of the cell.

20. The method of claim 19, wherein the nucleic acid probe utilizes an amplification reaction.

21. The method of claim 19 or 20, wherein the nucleic acid probe is conjugated to a fluorescent dye.

22. The method of any one of claims 19-21 , wherein the nucleic acid probe comprises a plurality of nucleic acid probes, each of which is specific to a different target nucleic acid within the cell.

23. The method of any one of claims 19-22, wherein the nucleic acid probe is a ViewRNA probe system.

24. The method of any one of claims 19-23, further comprising aligning an imaging plate, such that imaging the cell to obtain a morphological profile and imaging the cell to obtain a functional profile occur at the same coordinates as to allow superimposition of the acquired images.

25. The method of claim 24, wherein aligning the imaging plate comprises utilizing a robotic arm.Atty Dkt: ALTI-739WO26. A method for profiling a cell based on functional details, comprising: producing a reaction solution comprising: a cell, a mixture of at least three nucleic acid probes, wherein each nucleic acid probe is complimentary to a different target nucleic acid molecule within the cell, and wherein each nucleic acid probe is conjugated to a fluorescent moiety, wherein each fluorescent moiety has a different emission spectrum, and a fluorescent dye that binds to one or both of RNA and DNA in a sequence-nonspecific manner, wherein the fluorescent dye has an emission spectrum that differs from the fluorescent moieties conjugated to the nucleic acid probes. imaging the reaction solution across a set of filters to obtain a functional profile of the cell, wherein each filter is paired with one fluorescent moiety or the fluorescent dye that a combination of the emission spectrum of each fluorescent moiety or dye and the emission band of its paired emission filter minimize fluorescence cross talk between each filter.

27. A method for multiplexed cell morphology imaging, comprising: providing a first fluorescently labeled antibody to a first cell and a second fluorescently labeled antibody to a second cell, wherein the first and second fluorescently labeled antibodies are specific to a surface antigen on each cell and the first and second fluorescently labeled antibodies possess different emission spectra; fixing the first cell and the second cell to a well of an imaging plate; imaging the well across multiple filters to identify a position of the first cell and a position of the second cell; removing a signal from the first and second fluorescently labeled antibodies in accordance with any one of claims 16-18; andAtty Dkt: ALTI-739WO painting the first and second cells via the method of any one of claims 1 -26.

28. The method of claim 27, further comprising: providing both the first and second fluorescently labeled antibodies to a third cell; fixing the third cell to the well of the imaging plate; wherein imaging the well across multiple filters further identifies a position of the third; wherein painting the first and second cells via the method of any one of claims 1 - 24 also paints the third cell.

29. The method of claim 27 or 28, wherein the first cell, the second cell, and the third cell are from different sources.

30. A kit comprising the mixture of fluorescent markers listed claim 3.

31. The kit of claim 30, further comprising a stripping agent, optionally selected from the agents according to claim 18.

32. The kit of claim 30 or 31 , further comprising one or more filters selected from the filters listed in claim 2.

Images