Methods and related aspects for tracking biomolecular activities in live cells

Abstract

Provided herein are methods of differentiating cells in a cell population that includes detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce a first detected fluorescent signal data set, in which a first subset of the cells in the cell population each comprise a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and in which the first and second FPs, or the functional portions thereof, differ from one another. The methods also include identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set. Related methods, compositions, kits, systems, and computer readable media are also provided.

Description

Atty Dkt. No.0184.0318-PCT (P18296-02) METHODS AND RELATED ASPECTS FOR TRACKING BIOMOLECULAR ACTIVITIES IN LIVE CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No.63 / 679,700, filed August 6, 2024, the disclosure of which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grants GM136711 and CA098252 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] To understand physiological and pathological processes as well as the responses to pharmacological agents at the cellular level, it is often necessary to track the spatiotemporal dynamics of molecular activities. Biomolecular activities can be monitored by genetically encoded fluorescent biosensors, which contain fluorescent proteins (FPs) that change emission properties or subcellular localizations reflecting the activation status of specific molecules or physicochemical environment of the cell. Compared to other detection methods that require cell lysis or fixation, such as mass spectrometry and antibody-based approaches, biosensors offer several advantages. First, they enable continuous monitoring of various activities in live cells and can be targeted to different subcellular compartments or organelles to report the specific activities at these locations. Second, they can reveal cell-to-cell variability that is not apparent from ensemble measurements. Third, some biochemical events such as the activation of G-proteins involve subtle conformational changes that are hard to detect with other approaches but can be readily monitored using biosensors. Other biosensors can track the physical environments of the cell such as the pH and membrane potential, which are not accessible by mass spectrometry or antibody-based assays. Despite the power andAtty Dkt. No.0184.0318-PCT (P18296-02) promise of biosensors, a major drawback of biosensors in general is their limited multiplexing capability due to the broad emission spectra of FPs and the availability of spectral space.

[0004] Accordingly, there exists an unmet need for additional methods to monitor multiple intracellular activities over long durations for various applications. SUMMARY OF THE INVENTION

[0005] The present disclosure provides methods and related aspects for tracking large numbers of biomolecular activities in live cells over extended periods of time, among many other applications. Some embodiments provide tandem fluorescent protein (FP) barcoding schemes in which barcodes are generated by fusing FPs of different colors into a single polypeptide chain, and the ratio between the emitted signal from different FPs is used as the basis for barcode identification. Since the intensity of FPs can be more precisely determined than their localization, these tandem FP barcoding schemes significantly improve robustness compared to other approaches and facilitate full automation of image analysis. In some embodiments, for example, the enhanced tolerance for image resolution provided by the methods and related aspects disclosed herein enable seamless adaptation to high-throughput formats, where lower magnification images are frequently employed. In some embodiments, the fluorescent barcoding proteins disclosed herein enable expression in hard-to-transfect 2D cell lines as well as in 3D organoids for the long-term measurement of processes that occur over days or weeks instead of just hours. In some embodiments, the methods, systems, and related aspects of the present disclosure are implemented using electronic neural networks, deep learning classifiers, and / or other machine learning techniques. These and other attributes will be apparent upon a complete review of the present disclosure, including the accompanying figures.

[0006] According to various embodiments, a method of differentiating cells in a cell population is presented. The method includes detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a firstAtty Dkt. No.0184.0318-PCT (P18296-02) subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another. The method also includes identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set, thereby differentiating the cells in the cell population.

[0007] Various optional features of the above embodiments include the following. The method includes identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signals emitted by the first FP, or the functional portion thereof, and detected fluorescent signals emitted by the second FP, or the functional portion thereof. The first fluorescent barcoding protein comprises a single polypeptide chain. The first subset of cells in the cell population is a selected cell type. The first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof. The method includes detecting fluorescent signals emitted by multiple sets of fluorescent barcoding proteins in the cell population to produce multiple sets of detected fluorescent signals, wherein at least two subsets of cells in the cell population comprise different fluorescent barcoding proteins and identifying the different fluorescent barcoding proteins in the at least two subsets of cells in the cell population using the multiple sets of detected fluorescent signals. The method includes identifying the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population by determining ratios of detected fluorescent signals emitted by the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population.

[0008] Various additional optional features of the above embodiments include the following. At least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein, and wherein the method comprises detecting fluorescent signalsAtty Dkt. No.0184.0318-PCT (P18296-02) emitted by the first and second fluorescent barcoding proteins to produce the first detected fluorescent signal data set and at least a second detected fluorescent signal data set, and identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population using the first and second sets of detected fluorescent signals. The first and second subsets of cells in the cell population are different selected cell types. The first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. The second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof. The method includes identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population by determining a ratio of detected fluorescent signals emitted by the first fluorescent barcoding protein and a ratio of detected fluorescent signals emitted by the second fluorescent barcoding proteins.

[0009] Various additional optional features of the above embodiments include the following. An organoid comprises the cell population. A two-dimensional (2D) array comprises the cell population. The detecting step comprises imaging and / or identifying one or more cells in the cell population that comprise the fluorescent barcoding proteins at one or more time points during a selected duration. The method further includes detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population. The fluorescent calibration standard biomolecules comprise FPs. At least one synthetic polynucleotide encodes one or more of the fluorescent barcoding proteins and wherein the method comprises expressing the fluorescent barcoding proteins from the synthetic polynucleotide prior to and / or concurrent with the detecting step. An expression cassette comprises the synthetic polynucleotide. A plasmid comprises the expression cassette. The method further includes introducing the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step. The method includes introducing the synthetic polynucleotide into aAtty Dkt. No.0184.0318-PCT (P18296-02) subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

[0010] According to various embodiments, a composition is presented. The composition comprises a cell population, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another.

[0011] Various optional features of the above embodiments include the following. The first fluorescent barcoding protein comprises a single polypeptide chain. The first subset of cells in the cell population is a selected cell type. The first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof. At least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. The first and second subsets of cells in the cell population are different selected cell types. The first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. The second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof. A kit comprises the composition.

[0012] According to various embodiments, a kit is presented. The kit comprises a first synthetic polynucleotide that encodes at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof.

[0013] Various optional features of the above embodiments include the following. The first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or theAtty Dkt. No.0184.0318-PCT (P18296-02) functional portion thereof. The kit further includes a second synthetic polynucleotide that encodes at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. The first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. The second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0014] According to various embodiments, a system for differentiating cells in a cell population is presented. The system comprises: a processor; and a memory communicatively coupled to the processor, the memory storing instructions which, when executed on the processor, perform operations comprising: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set. In some embodiments, the instructions which, when executed on the processor, perform operations comprising: identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signals emitted by the first FP, or the functional portion thereof, and detected fluorescent signals emitted by the second FP, or the functional portion thereof.

[0015] According to various embodiments, a computer readable media is presented. The computer readable media comprises non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in theAtty Dkt. No.0184.0318-PCT (P18296-02) cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set.

[0016] According to various embodiments, a first fluorescent barcoding protein is presented. The first fluorescent barcoding protein comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another.

[0017] Various optional features of the above embodiments include the following. The first fluorescent barcoding protein comprises a single polypeptide chain. The first fluorescent barcoding protein comprises more than one copy of the first FP, or a functional portion thereof, and / or more than one copy of the second FP, or a functional portion thereof. A cell comprising the first fluorescent barcoding protein. A composition comprising the first fluorescent barcoding protein. The composition further comprising a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. A first subset of cells in a cell population each comprise the first fluorescent barcoding protein and wherein a second subset of cells in the cell population each comprise the second fluorescent barcoding protein. The first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. The second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0018] According to various embodiments, a synthetic polynucleotide is presented. The synthetic polynucleotide that encodes a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functionalAtty Dkt. No.0184.0318-PCT (P18296-02) portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another.

[0019] Various optional features of the above embodiments include the following. An expression cassette that comprises the synthetic polynucleotide. A plasmid that comprises the expression cassette. A kit that comprises the expression cassette. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS.1A-1E. Scheme of tandem FP barcodes according to one embodiment. (A) Prior barcodes based FP of different colors targeted to different subcellular locations. (B) The new tandem FP barcodes are generated by fusing FP of different colors at various copy numbers. (C) An example of barcodes created from BFP and RFP in 1:1, 2:1, and 1:2 ratios. The intensity of BFP is proportional to that of RFP in individual cells (D), with the BFP / RFP ratio determined by the copy numbers (E).

[0021] FIG.2 is a flow chart that schematically shows exemplary method steps of differentiating cells in a cell population according to some aspects disclosed herein.

[0022] FIG.3 is a schematic diagram of an exemplary system suitable for use with certain aspects disclosed herein.

[0023] FIGS.4A-4D. Tandem barcodes based on BFP and mCardinal according to one embodiment. (A) Excitation and emission spectrums of BFP (EBFP2) and mCardinal, as well as the excitation wavelengths (vertical lines) and the corresponding emission ranges (boxes). (B) Barcodes made from three different copy numbers of BFP and mCardinal. (C-D). Signals of the 5 channels normalized to that of 659-668 nm channels for individual cells (left), as well as the plot of BFP / RFP vs. RFP intensity (right), under two different imaging settings shown on the left.

[0024] FIGS.5A-5F. Tandem FP barcodes based on BFP and three red FPs according to one embodiment. (A) Excitation and emission spectrums of BFP (EBFP2) and three red FPs, as well as the excitation wavelengths (vertical lines) and the corresponding emission ranges (boxes). (B) Barcodes made from one copy of BFP fused to one copy of a red FP. Signal intensity at the five emission ranges normalized to the red channel with the highest signal for individual cells is shown onAtty Dkt. No.0184.0318-PCT (P18296-02) the bottom. (C) BFP / RFP ratio of the three barcodes in (B). (D-F) Signals of the 5 channels normalized to that of the red channel with the highest signal for individual cells (left), as well as the plot of BFP / RFP vs. RFP intensity (right) of individual cells expressing barcodes made of BFP fused to a red RFP in three different copy numbers (2:1, 1:1, and 1:2).

[0025] FIGS.6A-6G. Tandem FP barcodes. (A) sctFP barcodes are generated by fusing FP of different colors at various copy numbers. (B) Barcodes made from five different copy number ratios of BFP and mCardinal. (C) Plot of BFP vs. mCar signal intensity (SI) of individual cells expressing the five sctFP barcodes. (D) Plot of BFP / mCardinal signal intensity ratio (SIR) vs. mCardinal signal intensity of individual cells expressing the five barcodes. (E) The BFP / mCardinal SIR (mean ± SD of n=29, 35, 24, 27, and 28 cells) for the five barcodes. The SD / mean (σ / μ) of BFP / mCar SIR for each barcode is shown on the top. (F) Hypothetical distribution of the SIR in cells expressing two sctFP barcodes, modeled as normal distributions with means ± SD (standard deviation) of μ₁ ± σ₁ and μ₂ ± σ₂, respectively. The vertical greyscale line indicates a threshold that includes 95% of barcode 1 while excluding all but 1% of barcode 2. (G) The maximum allowable σ / μ that satisfies the condition in (F), plotted as a function of the μ₂ / μ₁ ratio.

[0026] FIGS.7A-7F. Expanding sctFP barcodes. (A) FPs and their sources. (B) Emission spectra of the FPs shown in (A), along with the acquisition ranges used for channels. The spectra are generated using the spectra viewer from FPbase

[0025] . Spectra of 10 (mGarnet2) and 11 (mCarmine) are not shown due to overlapping with 12 (iRFP670). (C-D) Emission profiles represented by the SIR of the indicated channels (traces, left) in individual HeLa cells expressing two BFP-RFP sctFP barcodes that showed consistent (C) and variable (D) BFP / RFP SIR (dots, right). (E- F) SIR (E) and σ / μ (F) of various sctFP constructs generated from combinations of FPs in (A). Prokaryotic FPs are shown in ligther greyscale to the right.

[0027] FIGS.8A-8G. Tandem FP barcodes based on BFP and three red FPs. (A) Excitation and emission spectra of BFP (EBFP2) and three red FPs (RFPs), as well as the excitation wavelengths (vertical lines) and the emission ranges (boxes) for image acquisition. The spectra are generated using the spectra viewer fromAtty Dkt. No.0184.0318-PCT (P18296-02) FPbase. (B) Emission profile of sctFP barcodes made from three BFP-RFP pairs at 1:1 ratio. (C) SIR (BFP / peak RFP) the three sctFP barcodes in (B) expressed in three different cell lines. (D-G) Emission profile and SIR of indicated channels for individual HeLa cells expressing sctFP barcodes made from BFP-mCardinal (D), BFP-TagRFP (E), and BFP-mCherry (F), and mCherry-mCardinal at 1:2, 1:1, and 2:1 ratios. Bar graphs on the right show the mean ± SD as well as SD / mean (σ / μ) of SIR of n=35 cells.

[0028] FIGS.9A-9H. FPs that require exogenous cofactors show variable fluorescence signal intensity. (A) Images of HeLa cells expressing sctFP barcodes made from BFP-iRFP at 1:1 ratio. (B) Plots of FP signal intensity (SI) (see Fig.7A for channel nomenclature) of individual cells expressing the three BFP-iRFP sctFP barcodes. (C) Immunoblots for BFP (anti-GFP), FLAG, and GAPDH of various BFP- iRFP and the iRFP constructs. (D-G) Plots of FP signal intensity (SI) (see Fig.7A for channel nomenclature) of individual cells expressing sctFP barcodes containing TagRFP-iRFP702 (D), monomeric iRFPs (E), the EV-linker (F), and smURFP (G). (H) Bar graphs summarizing the SIR (mean ± SD of n=35 cells) and SD / mean (σ / μ) for the various constructs. The dotted line indicates the 15% σ / μ threshold.

[0029] FIGS.10A-10F. sctFP barcodes facilitate automated image analysis. (A) Schematic of multiplexed biosensor imaging using barcoded cells. Each cell is transfected or transduced with a specific biosensor and a unique barcode. Cells are then mixed for simultaneous imaging, and biosensor activity is extracted from cells based on their corresponding barcodes. (B-C) Comparison of location-based barcodes (B) and sctFP barcodes (C). (D) Schematic of pixelwise analysis enabled by sctFP barcodes, where each pixel is assigned a barcode based on its emission profile. Cell masks are then generated by grouping neighboring pixels sharing the same barcode. (E-F) An example of barcode determination using pixelwise analysis (see text for details).

[0030] FIGS.11A-11E. Multiplexed tracking of biosensor activities using sctFP barcodes. (A) sctFP barcodes made from BFP and three RFPs (TagRFP, mCherry, mCardinal) at different ratios were used to label cells expressing 8 CFP- YFP FRET biosensors for the RTK signaling network and CFP (control). (B) TargetsAtty Dkt. No.0184.0318-PCT (P18296-02) of the RTK signaling network reported by the biosensors (shown in lighter greyscale). (C) Workflow for analyzing multiplexed images. For each pixel, the identity of the RFP is determined from the spectral profile to create cell masks for individual RFPs (Step 1). Next, the BFP / RFP ratio is used to create masks for each barcode (Step 2). Finally, the masks are applied to the CFP and YFP images to calculate the YFP / CFP ratio for each barcode. (D) YFP / CFP ratios for 8 biosensors and CFP (control) in HeLa cells stimulated with 100 ng / ml EGF at 6 min. (E) The YFP / CFP ratios from (C) normalized to prestimulation levels.

[0031] FIGS.12A-12D. Multiplexed tracking of various types of biosensors. (A) Summary of different types of biosensors and the barcode FPs that are spectrally compatible with each. Shown are the excitation wavelengths and the emission wavelength ranges used for acquiring signals from both biosensors and barcodes. (B) Excitation (Ex) and emission (Em) spectra of FPs used in the biosensors and their compatible barcodes. Vertical black lines indicate the excitation wavelengths, and boxes denote the emission ranges used for signal acquisition. (C) sctFP barcodes for ExRai and CFP-YFP FRET biosensors. (D) Simultaneous tracking of ExRai and CFP-YFP FRET biosensors in mixed barcoded cells treated with known activators. The responses (mean ± SEM of n=20 cells) are normalized to prestimulus levels. DEFINITIONS

[0032] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0033] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0034] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, 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 this disclosure pertains. In describing and claiming the methods, systems, and computer readable media, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.

[0035] Biomolecule: As used herein, “biomolecule” refers to an organic molecule produced by a living organism. Exemplary biomolecules, include without limitation macromolecules, such as nucleic acids, proteins, peptides, oligomers, carbohydrates, and lipids.

[0036] Classifier: As used herein, “classifier” generally refers to algorithm computer code that receives, as input, test data and produces, as output, a classification of the input data as belonging to one or another class.

[0037] Data set: As used herein, “data set” refers to a group or collection of information, values, or data points related to or associated with one or more objects, records, and / or variables. In some embodiments, a given data set is organized as, or included as part of, a matrix or tabular data structure. In some embodiments, a data set is encoded as a feature vector corresponding to a given object, record, and / or variable, such as a given test or reference subject. For example, a medical data set for a given subject can include one or more observed values of one or more variables associated with that subject.

[0038] Electronic neural network: As used herein, “electronic neural network” refers to a machine learning algorithm or model that includes layers of at least partially interconnected artificial neurons (e.g., perceptrons or nodes) organized as input and output layers with one or more intervening hidden layers that together form a network that is or can be trained to classify data, such as test subject medical data sets (e.g., medical images or the like).

[0039] Encode: As used herein, “encode” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. In other aspects, a DNA molecule canAtty Dkt. No.0184.0318-PCT (P18296-02) encode an RNA molecule (e.g., by the process of transcription that uses a DNA- dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term “encode” also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.

[0040] Expression: As used herein, the term “expression”, when used in reference to a nucleic acid herein, refers to one or more of the following events: (1) production of an RNA transcript of a DNA template (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and / or 3′ end formation); (3) translation of an RNA into a polypeptide; and / or (4) post- translational modification of a polypeptide.

[0041] Expression Cassette: As used herein, the term "expression cassette" or "expression vector" as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences. Expression cassettes typically include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers. "Operably linked', refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes include plasmids, recombinant viruses, any form of a recombinant vector, and the like.

[0042] Functional Portion Thereof: As used herein, the phrase “functional portion thereof” in the context of a fluorescent protein (e.g., FP biosensors, fluorescent calibration standard biomolecules, fluorescent barcoding proteins, etc.) refers to a modified or unmodified portion or fragment, or a sequence variant (e.g., amino acid sequence variant or encoding polynucleotide sequence variant) of the fluorescent protein that retains its ability to emit or otherwise produce a fluorescentAtty Dkt. No.0184.0318-PCT (P18296-02) signal. In some embodiments, a “functional portion thereof” of a fluorescent protein has at least 80%, 85%, 90%, 95%, 99% identity with a wild-type fluorescent protein amino acid sequence. In some embodiments, a “functional portion thereof” of a polynucleotide encoding a fluorescent protein has at least 80%, 85%, 90%, 95%, 99% identity with a wild-type fluorescent protein gene. In some embodiments, a “functional portion” of a fluorescent protein or polynucleotide encoding a fluorescent protein can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of a corresponding full-length fluorescent protein or polynucleotide encoding a fluorescent protein.

[0043] Host cell: As used herein, the phrase “host cell” or “cell” in the context of the methods and other aspects of the present disclosure refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. For example, host cells may be used to produce FP biosensors, fluorescent calibration standard biomolecules, fluorescent barcoding proteins, or a functional portion thereof, (e.g., wild-type, recombinant, or modified protein molecules) referenced herein by standard production techniques. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells (e.g., induced pluripotent stem (iPS) cells, mesenchymal stem cells, etc.), or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells:Atty Dkt. No.0184.0318-PCT (P18296-02) Chinese Hamster Ovary or CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa cells, HepG2 cells, W138 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, BHK21 cells, Jurkat cells, Daudi cells, A431 (epidermal) cells, CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, SP2 / 0 cells, NS-0 cells, MMT 060562 cells, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and a cell line derived from an aforementioned cell.

[0044] Machine Learning Algorithm: As used herein, "machine learning algorithm" generally refers to an algorithm, executed by computer, that automates analytical model building, e.g., for clustering, classification or pattern recognition. Machine learning algorithms may be supervised or unsupervised. Learning algorithms include, for example, artificial neural networks (e.g., back propagation networks), discriminant analyses (e.g., Bayesian classifier or Fisher’s analysis), multiple-instance learning (MIL), support vector machines, decision trees (e.g., recursive partitioning processes such as CART -classification and regression trees, or random forests), linear classifiers (e.g., multiple linear regression (MLR), partial least squares (PLS) regression, and principal components regression), hierarchical clustering, and cluster analysis. A dataset on which a machine learning algorithm learns can be referred to as "training data." A model produced using a machine learning algorithm is generally referred to herein as a “machine learning model.”

[0045] Nucleic Acid: As used herein, “nucleic acid” refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids can also include nucleotide analogs (e.g., bromodeoxyuridine (BrdU)), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, cfDNA, ctDNA, or any combination thereof.

[0046] Protein: As used herein, “protein” is used interchangeably with “polypeptide” and refers to polymers of amino acids of any length. These terms alsoAtty Dkt. No.0184.0318-PCT (P18296-02) include proteins that are post-translationally modified through reactions that include, but are not limited to, glycosylation, acetylation, phosphorylation, glycation or protein processing. Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence and a protein can be obtained with the same properties. The term “polypeptide” typically refers to a sequence with more than 10 amino acids and the term “peptide” means sequences with up to 10 amino acids in length. However, the terms may be used interchangeably.

[0047] Recombinant: As used herein, the term “recombinant” or “modified” is intended to refer to polypeptides (e.g., a fluorescent protein, or a functional portion thereof) that are designed, engineered, prepared, expressed, created or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant, combinatorial polypeptide library or polypeptides prepared, expressed, created or isolated by any other means that involves splicing selected sequence elements to one another. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source. In some embodiments, one or more such selected sequence elements results from the combination of multiple (e.g., two or more) known sequence elements that are not naturally present in the same polypeptide. In some embodiments, the term "recombinant" refers to (i) nucleic acid molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) nucleic acid molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0048] Vector: As used herein, the term “vector” in the context of fluorescent proteins (e.g., FP biosensors, fluorescent calibration standard biomolecules, fluorescent barcoding proteins, etc.) and polynucleotides encoding such fluorescent proteins refers to a biological carrier or delivery entity used to introduce the proteins or polynucleotides into target organoids or cells. In some embodiments, a “vector” is a nucleic acid that can be used to introduce a heterologous or synthetic polynucleotide into a cell. One type of vector is a “plasmid”, which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., lentiviruses, replication defective retroviruses, adenoviruses and adeno-associated viruses), in which additional DNA or RNA segments can be introduced into the viral genome. Other exemplary vectors in the context of polynucleotides encoding fluorescent proteins, include cells, plasmids, exosome vesicles, cosmids, liposomes, expression cassettes, lipid nanocrystals, lipid nanoparticles, and artificial chromosomes, among others. DETAILED DESCRIPTION

[0049] Genetically encoded fluorescent biosensors are powerful tools for tracking signaling dynamics in live cells, but their multiplexing capacity is severely limited by available spectral space. We overcame this problem by developing a “biosensor barcoding” method for highly multiplexed tracking of signaling activities in live cells. In this method, cells expressing different biosensors are labeled with a pair of barcoding proteins, which are blue or red fluorescent proteins (FPs) targeted to different subcellular locations. The spectra of barcoding proteins can be separated from those of commonly used biosensors based on GFP or the Förster resonance energy transfer (FRET) between CFP and YFP. Barcoded cells expressing different biosensors are mixed for simultaneous imaging, and the identity of each biosensor in each cell is established by analyzing the barcode through machine learning models. Our technique enables massively paralleled tracking of biomolecular activities with fluorescent biosensors in real time.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0050] In the original barcoding scheme, distinguishing the barcodes depends on the distinct patterns of FPs targeted to the nucleus, plasma membrane, nuclear membrane, and cytoplasm (FIG.1A). While the scheme works well for high- resolution confocal images, it can be challenging to apply it to images with lower magnification, such as those used in high-throughput screening. This is because it can be difficult to accurately identify the subcellular localization of the barcoding proteins in low-resolution images. Distinguishing these subcellular structures can also be challenging for cells with scant cytoplasm (e.g. leukocytes) or with a tendency to cluster. These problems are often aggravated by the uneven expression of the barcoding proteins.

[0051] In the present disclosure this challenge is overcome by using a different barcoding scheme that does not rely on subcellular localization. In some aspects, for example, the present disclosure provides a barcoding scheme in which fluorescent proteins of different colors are fused into a single polypeptide chain to form a “tandem fluorescent protein barcode” (FIG.1B). This approach allows for the use of the relative intensity between fluorescent proteins, rather than their subcellular locations or spectral unmixing, as the basis for barcode identification. Because fluorescence intensity can be more accurately determined than subcellular localization, this approach is more robust to image quality and can be easily automated. The improved tolerance for low imaging resolution enables the extension of multiplexed biosensor imaging to high-throughput formats, among other attributes.

[0052] In some embodiments, the tandem FP barcodes are generated by fusing FPs of different colors as a single polypeptide chain. Instead of subcellular localizations, the relative intensity between different FPs, determined by their copy numbers, is used as the basis for distinguishing different barcodes (FIG.1B). FIG. 1C shows an example of three barcodes made from fusing blue (BFP) and red (RFP) in copy numbers 1:1, 2:1, and 1:2. For individual cells, the BFP and RFP signals fall on the three lines with slopes proportional to the copy number ratios (FIG.1D). The ratio of BFP to RFP signals for the same barcode is independent of the expression level but is proportional to the copy number ratio (FIG.1E).Atty Dkt. No.0184.0318-PCT (P18296-02)

[0053] In some embodiments, the present disclosure provides lentiviral or other vectors that encode barcodes, biosensors, and calibration standards to enable expression in hard-to-transfect 2D cell lines as well as 3D organoids. By drug selection of lentivirus transduced cells followed by sorting, the present disclosure provides barcoded cell lines that stably express biosensors or calibration standards with homogeneous fluorescence. These cells can be readily mixed for simultaneous tracking of various biomolecular activities. Stable expression also enables long-term measurement of processes that occur over days or weeks instead of hours. As described further herein, 3D organoids preserve the 3D cell connections and are typically better models for the study of tissue morphogenesis, tumor heterogeneity, drug screening, and tumor / microenvironment interactions than 2D cell lines.

[0054] The present disclosure also relates to an automated image analysis pipeline for analyzing barcoded cells as well as algorithms to calibrate biosensor signals using the standards disclosed herein. In some embodiments, the methods and compositions of the present disclosure are incorporated into kits containing specific biosensor libraries for investigating signaling pathways. In some embodiments, for example, a kit containing plasmids for a receptor tyrosine kinase (RTK) signaling network biosensor and barcodes, calibration standards, and analysis software (e.g., a deep learning model in some embodiments) is provided for investigators and pharmaceutical companies to conduct assays for various anti- cancer drugs. These and other attributes will be apparent upon a complete review of the present disclosure, including the accompanying figures.

[0055] To illustrate, in some embodiments, the present disclosure provides a method of differentiating cells in a cell population (FIG.2). For example, method 200 includes detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set (step 202). At least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof. The first and second FPs, or the functional portions thereof, also differ from one another. In someAtty Dkt. No.0184.0318-PCT (P18296-02) embodiments, the first fluorescent barcoding protein comprises a single polypeptide chain. In some embodiments, the first subset of cells in the cell population is a selected cell type (e.g., a cancer cell type, an immune cell type, and / or the like). In some embodiments, the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, (e.g., two, three, four, five, six, seven, eight, nine, ten, or more copies) and / or more than one copy of the second FP, or the functional portion thereof (e.g., two, three, four, five, six, seven, eight, nine, ten, or more copies).As shown, method 200 also includes identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set to thereby differentiate the cells in the cell population (step 204). Typically, detecting step (step 202) comprises imaging and / or identifying one or more cells in the cell population that comprise the fluorescent barcoding proteins at one or more time points during a selected duration. In some embodiments, method 200 includes identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signals emitted by the first FP and detected fluorescent signals emitted by the second FP. In some embodiments, method 200 also includes detecting fluorescent signals emitted by fluorescent calibration standard biomolecules in the cell population to produce one or more detected calibration standard signal data sets. In some embodiments, a detected calibration standard signal data set is used to normalize the detected fluorescent signal data sets disclosed herein. In some embodiments, the fluorescent calibration standard biomolecules comprise FPs.

[0056] In some embodiments, method 200 includes detecting fluorescent signals emitted by multiple sets of fluorescent barcoding proteins in the cell population to produce multiple sets of detected fluorescent signals. In these embodiments, at least two subsets of cells in the cell population comprise different fluorescent barcoding proteins. In these embodiments, method 200 also includes identifying the different fluorescent barcoding proteins in the at least two subsets of cells in the cell population using the multiple sets of detected fluorescent signals. In these embodiments, method 200 typically includes identifying the different fluorescentAtty Dkt. No.0184.0318-PCT (P18296-02) barcoding proteins in the at least two subsets of the cells in the cell population by determining ratios of detected fluorescent signals emitted by the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population.

[0057] In some embodiments, at least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. In these embodiments, method 200 typically includes detecting fluorescent signals emitted by the first and second fluorescent barcoding proteins to produce the first detected fluorescent signal data set and at least a second detected fluorescent signal data set. In these embodiments, method 200 also typically includes identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population using the first and second sets of detected fluorescent signals. In these embodiments, the first and second subsets of cells in the cell population are different selected cell types. In some of these embodiments, the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. In some of these embodiments, the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof. In some of these embodiments, method 200 includes identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population by determining a ratio of detected fluorescent signals emitted by the first fluorescent barcoding protein and a ratio of detected fluorescent signals emitted by the second fluorescent barcoding proteins.

[0058] In some embodiments, a synthetic polynucleotide (e.g., an expression cassette) encodes the fluorescent barcoding proteins and / or the fluorescent calibration standard biomolecules. In these embodiments, method 200 typically includes expressing the fluorescent barcoding proteins and / or the fluorescent calibration standard biomolecules from the synthetic polynucleotide prior to and / or concurrent with the detecting step. In some embodiments, a first synthetic polynucleotide encodes the fluorescent barcoding proteins and a second syntheticAtty Dkt. No.0184.0318-PCT (P18296-02) polynucleotide encodes the fluorescent calibration standard biomolecules. In some embodiments, a plasmid comprises the expression cassette. Typically, method 200 includes introducing (e.g., using a lentiviral vector or another type of vector) the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step.

[0059] In some embodiments, the present disclosure provides a composition that includes a cell population in which at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and in which the first and second FPs, or the functional portions thereof, differ from one another. In some embodiments, the first fluorescent barcoding protein comprises a single polypeptide chain. In some embodiments, the first subset of cells in the cell population is a selected cell type (e.g., a cancer cell type or the like). In some embodiments, the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, (e.g., two, three, four, five, six, seven, eight, nine, ten, or more copies) and / or more than one copy of the second FP, or the functional portion thereof (e.g., two, three, four, five, six, seven, eight, nine, ten, or more copies). In some embodiments, at least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. In some embodiments, the first and second subsets of cells in the cell population are different selected cell types. In some embodiments, the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. In some embodiments, the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof. In some embodiments, the compositions of the present disclosure are included as components of kits.

[0060] In some embodiments, the present disclosure provides a kit that includes a first synthetic polynucleotide that encodes at least a first fluorescent barcodingAtty Dkt. No.0184.0318-PCT (P18296-02) protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof. In some embodiments, the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof. In some embodiments, the kit further includes a second synthetic polynucleotide that encodes at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. In some embodiments, the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another. In some embodiments, the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0061] The present disclosure also provides various fluorescent barcoding proteins. In some embodiments, for example, the present disclosure provides a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another. In some embodiments, the first fluorescent barcoding protein comprises a single polypeptide chain. In some embodiments, the first fluorescent barcoding protein comprises more than one copy of the first FP, or a functional portion thereof, and / or more than one copy of the second FP, or a functional portion thereof. In some embodiments, a cell includes the first fluorescent barcoding protein. In some embodiments, a composition includes the first fluorescent barcoding protein. In some of these embodiments, the composition also includes a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein. In some of these embodiments, a first subset of cells in a cell population each comprise the first fluorescent barcoding protein and wherein a second subset of cells in the cell population each comprise the second fluorescent barcoding protein. In some of these embodiments, the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, orAtty Dkt. No.0184.0318-PCT (P18296-02) the functional portions thereof, that differ from one another. In some of these embodiments, the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0062] In some embodiments, the present disclosure also provides a synthetic polynucleotide that encodes a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another. In some embodiments, an expression cassette that comprises the synthetic polynucleotide. In some embodiments, a plasmid that comprises the expression cassette. In some embodiments, a kit includes the expression cassette and / or the plasmid.

[0063] The present disclosure also provides various systems and computer program products or machine readable media. In some aspects, for example, the methods described herein are optionally performed or facilitated at least in part using systems, distributed computing hardware and applications (e.g., cloud computing services), electronic communication networks, communication interfaces, computer program products, machine readable media, electronic storage media, software (e.g., machine-executable code or logic instructions) and / or the like. To illustrate, FIG.3 provides a schematic diagram of an exemplary system suitable for use with implementing at least aspects of the methods disclosed in this application. As shown, system 300 includes at least one controller or computer, e.g., server 302 (e.g., a search engine server), which includes processor 304 and memory, storage device, or memory component 306, and one or more other communication devices 314, 316, (e.g., client-side computer terminals, telephones, tablets, laptops, other mobile devices, etc. (e.g., for receiving data sets or results, etc.) in communication with the remote server 302, through electronic communication network 312, such as the Internet or other internetwork. Communication devices 314, 316 typically include an electronic display (e.g., an internet enabled computer or the like) in communication with, e.g., server 302 computer over network 312 in which the electronic display comprises a user interface (e.g., a graphical user interface (GUI), aAtty Dkt. No.0184.0318-PCT (P18296-02) web-based user interface, and / or the like) for displaying results upon implementing the methods described herein. In certain aspects, communication networks also encompass the physical transfer of data from one location to another, for example, using a hard drive, thumb drive, or other data storage mechanism. System 300 also includes program product 308 (e.g., for calibrating fluorescent signals, for differentiating cells in a cell population, etc. as described herein) stored on a computer or machine readable medium, such as, for example, one or more of various types of memory, such as memory 306 of server 302, that is readable by the server 302, to facilitate, for example, a guided search application or other executable by one or more other communication devices, such as 314 (schematically shown as a desktop or personal computer). In some aspects, system 300 optionally also includes at least one database server, such as, for example, server 310 associated with an online website having data stored thereon (e.g., entries corresponding to detected fluorescent signal data set and / or a calibration standard signal data set, etc.) searchable either directly or through search engine server 302. System 300 optionally also includes one or more other servers positioned remotely from server 302, each of which are optionally associated with one or more database servers 310 located remotely or located local to each of the other servers. The other servers can beneficially provide service to geographically remote users and enhance geographically distributed operations.

[0064] As understood by those of ordinary skill in the art, memory 306 of the server 302 optionally includes volatile and / or nonvolatile memory including, for example, RAM, ROM, and magnetic or optical disks, among others. It is also understood by those of ordinary skill in the art that although illustrated as a single server, the illustrated configuration of server 302 is given only by way of example and that other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 302 shown schematically in FIG.3, represents a server or server cluster or server farm and is not limited to any individual physical server. The server site may be deployed as a server farm or server cluster managed by a server hosting provider. The number of servers and their architecture and configuration may be increased based on usage, demand andAtty Dkt. No.0184.0318-PCT (P18296-02) capacity requirements for the system 300. As also understood by those of ordinary skill in the art, other user communication devices 314, 316 in these aspects, for example, can be a laptop, desktop, tablet, personal digital assistant (PDA), cell phone, server, or other types of computers. As known and understood by those of ordinary skill in the art, network 312 can include an internet, intranet, a telecommunication network, an extranet, or world wide web of a plurality of computers / servers in communication with one or more other computers through a communication network, and / or portions of a local or other area network.

[0065] As further understood by those of ordinary skill in the art, exemplary program product or machine readable medium 308 is optionally in the form of microcode, programs, cloud computing format, routines, and / or symbolic languages that provide one or more sets of ordered operations that control the functioning of the hardware and direct its operation. Program product 308, according to an exemplary aspect, also need not reside in its entirety in volatile memory, but can be selectively loaded, as necessary, according to various methodologies as known and understood by those of ordinary skill in the art.

[0066] As further understood by those of ordinary skill in the art, the term "computer-readable medium" or “machine-readable medium” refers to any medium that participates in providing instructions to a processor for execution. To illustrate, the term "computer-readable medium" or “machine-readable medium” encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 308 implementing the functionality or processes of various aspects of the present disclosure, for example, for reading by a computer. A "computer-readable medium" or “machine-readable medium” may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks. Volatile media includes dynamic memory, such as the main memory of a given system. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared dataAtty Dkt. No.0184.0318-PCT (P18296-02) communications, among others. Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.

[0067] Program product 308 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 308, or portions thereof, are to be run, it is optionally loaded from their distribution medium, their intermediate storage medium, or the like into the execution memory of one or more computers, configuring the computer(s) to act in accordance with the functionality or method of various aspects disclosed herein. All such operations are well known to those of ordinary skill in the art of, for example, computer systems.

[0068] In some aspects, program product 308 includes non-transitory computer- executable instructions which, when executed by electronic processor 304, perform at least: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, in which at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and in which the first and second FPs, or the functional portions thereof, differ from one another, and identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set.

[0069] In some embodiments, detecting fluorescent signals emitted by fluorescent barcoding proteins in cell populations are measured using device 318. As shown, device 318 includes excitation laser 320 and detector 324. Detector 324 receives detectable signals from, e.g., fluorescent calibration standard biomolecules and / or fluorescent barcoding proteins. In some embodiments, device 318 includes sample container receiving area 326 on which sample container 322 is positioned. SampleAtty Dkt. No.0184.0318-PCT (P18296-02) container 322 includes cells (e.g., 2D cell arrays, 3D organoids, etc.) that include fluorescent protein (FP) biosensors, fluorescent calibration standard biomolecules, and fluorescent barcoding proteins and / or the like. EXAMPLES

[0100] EXAMPLE 1: Barcoding Platform Based on Tandem Fluorescent Proteins

[0101] RESULTS

[0102] In this example, we created tandem FP barcodes by fusing BFP and mCardinal (FIG.4A) at 2:1, 1:1, and 1:2 ratios (FIG.4B). We imaged HeLa cells transfected with these barcodes using a Zeiss 780 confocal microscope equipped with a spectral detector. BFP emission was collected between 410-432 nm under 405 nm excitation, whereas mCardinal emission was collected at four ranges between 579 and 700 nm under two different excitation wavelengths, 561 nm and 633 nm (FIG.4A). For each cell, the signal of each of the 5 channels was normalized to that of the red channel with the highest signal. As shown in FIGS.4C and 4D, the profiles for cells expressing different barcodes were separated into three non- overlapping groups. Between the groups, the relative BFP / RFP ratio of the three barcodes closely matches the expected ratios based on the copy number of BFP and mCardinal in the three barcodes, i.e.1:2:4. The BFP / RFP ratio was independent of the expression level except in very low expressors, in which the FP signal was close to the background noise (FIGS.4C, 4D, right).

[0103] To expand the number of barcodes, we paired BFP with three different red FPs: TagRFP, mCherry, and mCardinal (FIG.5A). The spectral profiles obtained from individual cells expressing the three barcodes showed distinct patterns that are highly conserved between individual cells (FIG.5B). The BFP / RFP ratios remain consistent when barcodes are expressed in different cell lines (FIG.5C). We next generated tandem FP barcodes made from BFP and the three red FPs at 2:1, 1:1, and 1:2 ratios. As expected, barcodes based on the same red FP displayed the same spectral profiles in the emission ranges of the red FPs (FIGS.5D-5F, left). Moreover, the BFP / RFP ratios of cells expressing different barcodes are proportionalAtty Dkt. No.0184.0318-PCT (P18296-02) to the copy number ratios. Despite slight variability in the ratio in individual cells, there is no overlap between groups of cells expressing different barcodes (FIGS.5D- 5F, right). These results suggest that tandem FP barcodes made from different copy numbers and colors can be robustly identified using the spectral profile and BFP / RFP ratio.

[0104] DISCUSSION

[0105] In this example, we developed a method to enable robust tracking of multiple molecular activities in live cells using genetically encoded fluorescent biosensors. In contrast to the original biosensor barcoding method that relies on discerning the subcellular location of barcoding protein, the new tandem FP barcoding scheme uses intensity of FPs as the basis of barcoding. Since the intensity of an FP can be more accurately determined than its subcellular patterns, the tandem FP barcoding scheme has greatly improved robustness compared to the original scheme and will facilitate full automation of image analysis. The greater tolerance for image resolution allows for ready adaptation to high throughput format in which lower magnification images are often used.

[0106] We envision our technique to find broad applications including but not limited to the following: 1) Study the dynamics and structure of signaling networks: By simultaneously tracking signaling activities in response to pharmacological or genetic perturbations, the regulatory structure of the network can be delineated. The dynamic information can be used to build quantitative models for generating testable predictions. Understanding the regulatory structure of signaling networks has important clinical implications, e.g. for developing strategies to overcome cancer resistance to targeted therapy.2) Study the interactions between different cell populations: By barcoding heterogeneous populations of cells, their interactions can be revealed, e.g. those between tumor cells and other cells in the microenvironment such as fibroblasts and immune cells, or the effects of oncogenic mutations on neighboring cells.3) Identify cellular pathways targeted by pharmacological agents: By continuously monitoring different molecular activities, biosensor barcoding can reveal the pathways that respond to treatments with chemical agents, obtain theAtty Dkt. No.0184.0318-PCT (P18296-02) dynamics and dose curves of different responding molecules, and identify off-target effects.

[0107] EXAMPLE 2: Cell barcoding with tandem fluorescent proteins enables high-throughput signaling dynamics analysis

[0108] Background

[0109] Cell barcodes play a vital role in a variety of experimental applications focused on monitoring individual cells. These unique identifiers allow researchers to map clonal relationships within cell populations and track individual cells over time. For example, barcodes enable the tracing of cell lineages and simultaneous evaluation of multiple genetic modifications to accelerate the discovery of functional gene targets. For single-cell analysis, barcoding enables the differentiation and tracking of different cells, providing comprehensive insights into cellular heterogeneity and the dynamics of complex biological systems.

[0110] Cell barcoding is typically achieved using either nucleotide-based or fluorescent barcodes. Nucleotide-based barcodes take advantage of the large number of combinations that can be generated from relatively short nucleotide sequences. However, nucleotide-based barcodes are in general destructive when identifying cells. Cell lysis and sequencing, amplification, or hybridization assays are required, thus are suitable for applications where the complexity and depth of information outweigh the need for dynamic and longitudinal observation. In contrast, fluorescent barcodes utilize the spectral and spatial patterns of fluorophores to identify different cell populations. Barcodes based on fluorescent proteins (FPs) permit longitudinal monitoring of dynamic cell behaviors and phenotypes.

[0111] A recent application of FP-based barcodes is multiplex imaging of genetically encoded fluorescent biosensors. In these applications, cells expressing different biosensors are tagged with barcodes created from combinations of FPs that are spectrally distinct from the biosensors. To mitigate the reduction of available spectral space due to biosensor fluorescence, barcoding FPs are directed to different subcellular locations. However, this localization-based barcoding approach presents several challenges. Firstly, identifying the barcodes often relies on high- resolution confocal imaging, making it difficult to apply to lower magnification imagesAtty Dkt. No.0184.0318-PCT (P18296-02) used in high-throughput screening. Additionally, distinguishing subcellular structures becomes challenging for cells with minimal cytoplasm (e.g., leukocytes) or those prone to clustering. These difficulties are often compounded by uneven expression levels of barcoding proteins, posing further obstacles to automating image analysis.

[0112] In this example, we explored a new strategy for generating a large number of FP-based barcodes with robust color patterns for labeling various cell populations, which can be combined effectively with fluorescent biosensors. This approach, termed single-chain tandem FP (sctFP) barcodes, involves linking different FPs within a single polypeptide chain (Fig.6A). We demonstrate that sctFP barcodes can be reliably distinguished by analyzing the fluorescence signal intensity at different wavelengths. This method also supports a simple pixelwise image analysis protocol that is robust and does not require prior segmentation of individual cells, thus enabling automated image processing. Unlike previous barcoding strategies limited to certain types of biosensors, the defined emission ratios of sctFP barcodes accommodate a broader range of biosensors.

[0113] Results

[0114] A new barcoding platform based on single-chain tandem fluorescent proteins

[0115] To test the sctFP barcoding strategy, we fused BFP (EBFP2) with the red FP mCardinal at 1:3, 1:2, 1:1, and 2:1, and 3:1 ratios (Fig.6B). We imaged HeLa cells transfected with these barcodes and plotted the fluorescence signal intensity of BFP and mCardinal of individual cells (Fig.6C). As expected, cells expressing the same barcodes fell on straight lines with slopes reflecting the copy number ratios of BFP and mCardinal (Fig.6C). Importantly, the BFP / mCardinal signal intensity ratio (SIR) for individual cells were separated into five non-overlapping groups regardless of the expression level (Fig.6D). Between the groups, the relative SIR of the five barcodes closely matched the expected ratios based on the copy number of BFP and mCardinal (Fig.6E). These results establish the proof-of-principle that the SIR between FPs can be reliably used to distinguish different sctFP barcodes.

[0116] To quantitatively understand how SIR variations affect the ability to distinguish between sctFP barcodes, we considered a hypothetical scenarioAtty Dkt. No.0184.0318-PCT (P18296-02) involving sctFPs composed of two FPs, FP1 and FP2, at two different copy number ratios. Assuming a normal distribution of the SIR for the two sctFPs with mean ± SD (standard deviation) = μ1 ± σ1 and μ2 ± σ2, respectively (^^^^2>^^^^1), we estimated the SD required to achieve a minimum 95% identification rate of the two sctFPs in a 1:1 mixture with no more than a 1% error rate (Fig.6F). In this scenario, the threshold for distinguishing barcode 1 (Fig.6F, vertical line) need to be at least 2σ1 from μ1 and at least 2.33σ2 from μ2, i.e.

[0117] Similarly, the threshold also needs to be at least 2.33σ1 from μ1 and at least 2σ2 from μ2, i.e.

[0118] Since the same pair of FPs is used for sctFP construction, it is reasonable to assume that the SD scales with the mean, i.e. σ1 / μ1=σ2 / μ2. This is supported by observing the σ / μ (SD / mean) of the SIR for the five BFP-mCardinal sctFP barcodes (Fig.6F, top). Denoting the σ / μ ratio as ^^^^, we have σ1 = μ1 ^^^^ and σ2 = μ2 ^^^^. Substituting and rearranging (1) and (2) give where ^^^^=^^^^2 / ^^^^1 is the ratio between the mean SIR of the two sctFP constructs. Note that inequality (2a) is more stringent than (2b) as ^^^^>1, and gives the upper limit for σ / μ that enables reliable separation of two sctFP constructs with known FP1 / FP2 ratios. For example, distinguishing 2:1 and 1:1 barcodes (corresponding to μ2 / μ1 = 2) requires σ / μ to be smaller than 15%, whereas distinguish 3:1 and 2:1 barcodes (corresponding to μ2 / μ1 = 1.5) requires σ / μ to be smaller than 9% (Fig.6G). Note that the σ / μ for the BFP-mCardinal constructs was below 9% (Fig.6E), thus enabling the separation of all five sctFP constructs in Fig.6B.

[0119] Expanding sctFP barcodes

[0120] We next sought to expand the sctFP barcode repertoire by incorporating additional FP combinations. This would enable a broader range of barcodes suitedAtty Dkt. No.0184.0318-PCT (P18296-02) for diverse applications with varying constraints on barcode color selection. Based on the above analysis, we screened FP pairs to identify those with an SIR σ / μ below 15%, which would allow the generation of at least three distinct sctFPs (at 1:1, 1:2, and 2:1 ratios).

[0121] Using a selected panel of FPs, we generated sctFPs composed of one copy each of two different FPs (Fig.7A). We measured their fluorescence signals across multiple wavelengths (Fig.7B). The resulting spectral profiles were generally consistent with predictions based on FPbase data (Fig.7C,D). We calculated SIRs using the channels with peak intensities for the two component FPs (Fig.7E) and determined their corresponding σ / μ values (Fig.7F). Surprisingly, not all sctFPs exhibited consistent SIRs across the cell population, with some FP pairs showing σ / μ values exceeding the 15% threshold (Fig.7D,E).

[0122] We selected three FP pairs with σ / μ values below 15%, including BFP- TagRFP, BFP-mCherry, and BFP-mCardinal, for further evaluation of their robustness in mixed populations. These FP pairs were previously used to barcode cells expressing CFP-, GFP-, and YFP-based biosensors due to their spectral separation from the biosensor emission ranges (Fig.8A). Spectral profiles from individual cells expressing each of the three barcodes showed distinct, highly conserved patterns across the population, enabling reliable FP identification (Fig. 8B). Importantly, their SIR remained consistent when expressed in different cell lines (Fig.8C).

[0123] We next imaged cells expressing sctFP barcodes generated by fusing BFP with TagRFP, mCherry, or mCardinal at 1:1, 1:2, and 2:1 ratios. As expected, barcodes incorporating the same red FP exhibited identical spectral profiles within the red emission range (Fig.8D-F, left). Moreover, the BFP / RFP SIR scaled proportionally with the copy number ratios (Fig.8D-F, middle, right). Consistent with their low σ / μ values, the distributions for each barcode were clearly separated with no overlap between groups (Fig.8D-F, middle). Together, these results demonstrate that FP pairs meeting the 15% σ / μ threshold can reliably generate three distinguishable sctFP barcodes based on their spectral profiles and SIRs.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0124] In addition to BFP-red FP combinations, we tested barcodes composed solely of red FPs by fusing mCardinal and mCherry at 1:1, 1:2, and 2:1 ratios. Due to the overlapping spectrum of mCardinal and mCherry, the SIR did not scale proportionally with their copy number ratios. Nevertheless, these barcodes displayed distinct spectral profiles and SIRs, enabling robust classification (Fig.8G).

[0125] FPs that require exogenous cofactors show variable fluorescence intensity ratios

[0126] Our analysis revealed high SIR variability when one of the FPs belonged to the iRFP family, which consists of near-infrared (NIR) FPs derived from prokaryotes (Fig.7D-F). To investigate the basis of this variability, we examined images of cells expressing BFP-iRFP constructs. Interestingly, across the population, iRFP signals appeared more uniform than BFP signals (Fig.9A). When we plotted iRFP versus BFP fluorescence, we observed a proportional relationship at low expression levels, but iRFP signal plateaued at higher BFP levels (Fig.9B). For comparison, BFP and mCardinal signals fell on a straight line across the entire range (Fig.6C).

[0127] To rule out protein degradation, we performed immunoblotting. Since BFP is derived from GFP, we used an anti-GFP antibody to detect BFP, and added a FLAG tag to iRFP for detection with an anti-FLAG antibody. Immunoblotting analysis confirmed that both FPs were expressed primarily as full-length fusion proteins (Fig. 9C). The variability was not specific to the BFP-iRFP combination, as constructs using TagRFP-iRFP702 also showed high SIR variability across the population (Fig. 9D).

[0128] To rule out the possibility that iRFP dimerization caused spectral interference, we generated sctFPs using monomeric iRFP variants—including emiRFP703, emiRFP670, and miRFP680—fused to BFP or mCardinal. None of these constructs showed consistent SIRs (Fig.9E). We further tested whether intramolecular FP interactions contributed to the variability by inserting an EV linker, a long, flexible peptide designed to minimize baseline interactions between FPs in biosensor constructs. However, both BFP–EV–iRFP702 and TagRFP–EV–iRFP702 still displayed high SIR variability (Fig.9F).Atty Dkt. No.0184.0318-PCT (P18296-02)

[0129] These findings suggest that iRFP fluorescence intensity does not scale linearly with protein abundance. A likely explanation is that, unlike cnidarian FPs, iRFPs require incorporation of biliverdin as a chromophore. Biliverdin incorporation rate may vary between cells, particularly at high expression levels where it becomes limiting. Consistent with this idea, previous studies have shown that exogenously added biliverdin enhances iRFP fluorescence.

[0130] Together, our results indicate that cnidarian-derived FPs—such as BFP and YFP (jellyfish), mCherry (coral), and TagRFP (anemone)—exhibit robust and reproducible SIRs because they do not rely on exogenous chromophores. In contrast, FPs that require external factors like biliverdin show greater variability. To further test this hypothesis, we evaluated BFP–smURFP constructs, as smURFP is a cyanobacteria-derived FP that also requires biliverdin. As expected, BFP–smURFP barcodes exhibited high SIR variability across the population (Fig.9G). The SIR and σ / μ for various constructs are summarized in Fig.9H. It should be noted that while BFP–iRFP constructs at varying ratios cannot be reliably distinguished based on SIR, they remain distinguishable from BFP-only or iRFP-only barcodes.

[0131] sctFP barcodes enable high-throughput image analysis of biosensor activities

[0132] We applied sctFPs to enable multiplexed tracking of signaling activities in live cells using genetically encoded fluorescent biosensors. In this strategy, different populations of cells are separately transfected with a distinct biosensor paired with a unique sctFP barcode. The cells are then pooled and imaged together under the same perturbation conditions. Signals from cells expressing the same biosensor are grouped by barcode to reconstruct the corresponding signaling dynamics over time (Fig.10A).

[0133] A previous implementation of this concept used location-based barcodes, in which pairs of fluorescent proteins were targeted to distinct subcellular compartments (Fig.10B). While effective with high-resolution confocal imaging, accurately identifying the subcellular localization of barcode proteins becomes challenging in lower-resolution images typically used for high-throughput screening, particularly in cells with scant cytoplasm. These challenges are often exacerbated byAtty Dkt. No.0184.0318-PCT (P18296-02) uneven expression of the barcoding proteins. Additionally, directing FPs to different subcellular compartments may differentially affect cell physiology. As discussed below, inconsistent expression levels between the two FPs further limit the applicability of this approach to biosensors outside the CFP, GFP, and YFP emission range. We reasoned that the simpler sctFP design could overcome these limitations by enabling robust barcode identification from lower-resolution images, increasing compatibility with high-throughput imaging, and expanding the usable biosensor spectral range (Fig.10C).

[0134] A key advantage of sctFP barcodes is their simplified image analysis. For location-based barcodes, identification of the subcellular localization of FPs starts with segmentation of individual cells. Theoretically the process can be accomplished using machine learning models trained on different barcode images. However, due to the heterogeneity of the spatial patterns of barcodes, model training would require a large number of images encompassing the full range of cell density, fluorescence intensity, and spatial patterns, and new models will likely be needed for each cell type due to differences in cell morphologies or imaging parameters. A significant fraction of cells (sometimes over 50%) are also eliminated during analysis due to ambiguous location patterns, as high-magnification imaging is limited by a thin focal plane (< 500 nm) and many cells in the field of view can be out of the focal plane. Therefore, image segmentation has been the rate limiting step in the analysis of biosensor barcoding images.

[0135] sctFP barcodes can circumvent the image segmentation problem: since the barcode can be uniquely determined by the SIR of different FPs, spatial information is not necessary for barcode identification. As a result, sctFP barcodes can be imaged using low magnification objectives, which allow relatively thick focal plane. Then each pixel can be assigned a barcode based on its spectral profile. Masks created for each barcode can then be used to define individual cell areas (Fig.9D). A key benefit of pixelwise analysis is that it eliminates the need to define individual cell boundaries, which are often irregular or poorly defined, particularly for dim cells.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0136] We tested this approach using an image of mixed cells expressing BFP- RFP (mCardinal) sctFP barcodes at 1:1, 1:2, and 2:1 ratios. By analyzing signals across different channels, we computed deviations from a reference RFP intensity profile for pixels along a horizontal line intersecting multiple cells (Fig.10E, upper plot). This deviation plot effectively identified cellular pixels, including those with low signals that only became apparent upon contrast adjustment (Fig.10E, ovals). We also plotted the BFP / RFP intensity ratio along the same line (Fig.10E, black plot). Due to chromatic aberration, caused by wavelength-dependent differences in refraction, the BFP / RFP SIR was skewed near cell edges, increasing pixel-to-pixel variance (Fig.10E, lower plot). By excluding pixels with high SIR variance, we accurately assigned barcodes based on BFP / RFP SIR (Fig.10E, darker and lighter rectangles). Applying this analysis across the full image field yielded regions corresponding to distinct barcodes, which aligned well with cellular boundaries (Fig. 10F).

[0137] We next used sctFP barcodes to track the responses of RTK network biosensors to epidermal growth factor (EGF) stimulation. We barcoded cells expressing eight FRET-based biosensors and a CFP control using sctFPs composed of BFP paired with TagRFP, mCherry, or mCardinal at 1:1, 1:2, and 2:1 ratios (Fig. 11A). The biosensors detect the activity of RTK (EGFR) and its downstream signaling proteins (Fig.11B). Images were acquired across BFP and four red FP channels for barcode identification, and CFP and YFP channels for biosensor readout, following EGF stimulation. Image analysis proceeded as follows: (1) red FP profiles were used to identify TagRFP, mCherry, and mCardinal (Fig.11C, step 1); (2) the BFP / peak red FP ratio was used to assign barcode identities (Fig.11C, step 2); and (3) YFP / CFP ratios, used as a proxy for biosensor FRET level, were calculated pixelwise and averaged across pixels sharing the same barcode (Fig. 11C, step 3). We plotted both raw YFP / CFP values (Fig.11D) and values normalized to prestimulus levels (Fig.11E). The biosensor responses were consistent with previous reports of EGF-stimulated signaling, while the CFP control showed no response, as expected (Fig.11D,E). Importantly, the entire analysis was fullyAtty Dkt. No.0184.0318-PCT (P18296-02) automated. These results demonstrate that sctFP barcoding facilitates high- throughput image analysis by obviating the need for cell segmentation.

[0138] sctFP barcodes enable multiplexed tracking of different types of biosensors

[0139] We tested whether sctFP barcodes could be robustly implemented in multiplexed dynamic measurement using biosensors with different spectral properties. Previous barcodes based on BFP and RFPs are spectrally separable from biosensors traditionally made from CFP, GFP, or YFP (Fig.10B). In principle, biosensors and barcodes only need to be distinguishable within the same cells. However, due to the uncoordinated expression level of different FPs in earlier barcode designs, a complete separation between biosensor and barcode spectrums at the population level is essential. This is because accommodating biosensors that spectrally overlap with BFP or RFP may require using single-FP barcodes, which are difficult to distinguish from two-FP barcodes with weak expression of one FP. Although CFP-, GFP-, and YFP-based biosensors remain widely used, alternative fluorescent proteins with enhanced dynamic range and sensitivity are increasingly incorporated into biosensor development. We reasoned that the robust SIR offered by sctFP barcodes could overcome the limitations of previous designs, thereby enabling broader compatibility with a wider range of biosensor types.

[0140] We considered the following types of biosensors that involve FPs beyond the CFP-GFP-YFP emission range: 1) GFP-RFP FRET-based biosensors, such as those using the mClover-mRuby3 pair, which offer greater spectral separation, reduced autofluorescence, and lower phototoxicity.2) cpGFP-RFP biosensors, such as the ATP biosensor iATPsnFR, which consist of a circularly permuted GFP (cpGFP) whose fluorescence changes upon target binding, fused to a red FP (e.g., mCherry) used for normalization.3) Excitation-ratiometric (ExRai) biosensors, which typically exhibit high dynamic ranges and use ratiometric measurements based on excitation rather than emission. These biosensors are spectrally separable from only a subset of FPs used in the BFP and RFP based barcodes (Fig.12A,B). For example, ExRai biosensors require an excitation range that overlaps with BFP and are only compatible with RFP based barcodes. The robust SIR of sctFP barcodesAtty Dkt. No.0184.0318-PCT (P18296-02) would enable the simultaneous use of BFP-RFP and RFP-only barcodes in the population mix.

[0141] As a proof of principle, we tested simultaneous tracking of ExRai and FRET biosensors. We used RFP-only barcodes for cells expressing four ExRai biosensors that detect the activity of PKC (CKAR), cAMP (AKAR), AKT (AKTAR2), and AMPK (AMPKAR), and BFP-RFP barcodes for cells expressing two CFP-YFP FRET biosensors that detect the activity of FAK (CytoFAK) and ERK (EKAR-1, derived from EKAR) (Fig.12C). Cells were mixed and simultaneously imaged during stimulation with four known activators: 2-deoxy-D-glucose (2-DG) for AMPK, EGF for AKT and FAK, forskolin for cAMP, and phorbol 12,13-dibutyrate (PDBu) for PKC. We were able to reliably distinguish the barcodes and quantify the responses of the corresponding biosensors (Fig.12D). All biosensors responded to their expected stimuli, with magnitudes and kinetics comparable to those observed in pure populations of cells expressing individual biosensors. In addition to the expected responses, we also observed unexpected responses, such as activation of ExRai- AKAR2 by PDBu. The significance of these responses is currently under investigation. Together, these results demonstrate that sctFP barcodes enable simultaneous tracking of biosensors with distinct spectral properties.

[0142] Discussion

[0143] In this example, we developed a new method for barcoding cells by combining FPs with distinct spectral properties in varying ratios into a single polypeptide chain. Our findings demonstrate that these sctFP barcodes can be reliably differentiated through the relative intensity of the component FPs. Interestingly, this strategy proved effective for cnidarian FPs, but not for prokaryotic FPs, which exhibited variability in fluorescence signals likely due to their reliance on exogenous cofactors. We also showed that the analysis of sctFP barcodes can be streamlined through a pixelwise analysis approach. Additionally, we successfully applied this method in multiplexed imaging of various types of biosensors designed to detect different signaling activities.

[0144] Genetically encoded fluorescent biosensors serve as powerful tools for monitoring signaling dynamics in live cells. While these biosensors have primarilyAtty Dkt. No.0184.0318-PCT (P18296-02) been employed to investigate individual molecular activities, there is a growing interest in multiplexing biosensors to gain insights into the interactions among various signaling pathways. Most existing strategies focus on distinguishing biosensors expressed in the same cells based on differences in their spectral, spatial, or dynamic properties. More recently, an alternative approach utilizing FP- based barcodes has emerged to label cells expressing different biosensors for simultaneous imaging. This barcoding strategy eliminates interference between different biosensors within the same cells, with any potential adverse effects confined to the cells expressing a specific biosensor. Additionally, the approach is easily scalable to accommodate high multiplexity and can readily integrate existing biosensors. In prior implementations of biosensor barcodes, various FPs targeted to distinct subcellular locations were combined to enhance multiplexity. While this method has proven effective, the use of FPs localized to different regions within the cell can produce variable effects and necessitates high-resolution imaging to accurately identify their subcellular locations. Furthermore, imbalances in expression levels of barcoding FPs may hamper barcode identification in subsets of cells.

[0145] Our sctFP barcodes effectively address these challenges. They provide defined and reproducible fluorescence signatures, allowing for the use of relative intensity between FPs as the basis for barcoding. Because the intensity of an FP can be measured more accurately than its subcellular localization, sctFP barcodes offer significantly enhanced robustness compared to location-based sensors. Furthermore, these barcodes can be decoded directly from pixel-level spectral data, eliminating the need for time-consuming cell segmentation and enabling fast, fully automated image analysis. This greater tolerance for variations in image resolution and the streamlined image analysis process facilitate adaptation to high-throughput formats, where lower magnification images are often employed. Additionally, the simplified design of sctFP barcodes enhances compatibility with a wider range of biosensors and may synergize effectively with synthetic biology tools. Although not demonstrated in the current study, sctFP barcodes could also be applicable in bacterial cells, which do not possess the subcellular compartments required for location-based barcoding.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0146] While the primary motivation for this work is the multiplexing of genetically encoded fluorescent biosensors, fluorescent cell barcodes have also been developed for various other applications. For instance, Brainbow employs the Cre- Lox recombination system to randomly express different ratios of FPs in individual neurons, facilitating the tracing of neural connections and the study of brain architecture. However, the random nature of Brainbow limits their utility in labeling defined cell populations, which is essential for biosensor barcoding or genetic screening. Pairing a small set of FPs can generate a large number of barcodes that is compatible with genetic screening and lineage tracing, and fluorescent tagging of endogenous proteins has been employed to visualize their spatiotemporal dynamics on a large scale under various perturbation conditions, but these strategies present challenges when integrating additional fluorescent readouts, such as biosensors. Barcodes based on sequential multicolor immunofluorescence of epitope combinations have been utilized for protein engineering, but they require additional steps involving cell fixation for immunofluorescence following live-cell imaging. As a result, this method cannot sustain longitudinal monitoring. Our approach provides a simple and robust alternative to existing barcoding strategies, particularly for applications that demand precise detection of fluorescence readouts, such as biosensor use.

[0147] In summary, the sctFP barcodes represent a simple and versatile tool for studying signaling dynamics and beyond.

[0148] Methods

[0149] Cells

[0150] HeLa, HEK293T, SiHa and Panc 10.05 cells, purchased from ATCC, were grown at 37°C, 5% CO2 in DMEM high glucose medium (Gibco, #11965092) supplemented with 10% FBS (Corning Cellgro, 35-010-CV), 1 mM sodium pyruvate (Gibco, #11360070), and 1X nonessential amino acids (Gibco, #11140076).

[0151] Chemical reagents

[0152] Stocks of 200 μM phorbol-12,13-dibutyrate (PDBu, EMD Millipore, #524390),10mM Forskolin (Torcis Bioscience, #1099) were prepared by dissolving the chemicals in DMSO. Stocks were diluted to the indicated final concentrations inAtty Dkt. No.0184.0318-PCT (P18296-02) the culture medium. The EGF stock solution was prepared by dissolving EGF (Sigma-Aldrich, E9644) in 10 mM acetic acid to a final concentration of 1 mg / ml. All drug stocks were stored at -20°C.2-Deoxyglucose (2-DG, MilliporeSigma, #D8375) was dissolved in culture medium to 100 mM and used immediately.

[0153] Plasmids

[0154] sctFP barcodes

[0155] To construct the entry vector backbone containing a multiple cloning site (MCS) for tandem barcode gene cloning, a gBlocks gene fragment (5’- ggatccGGCTCTTCCactagtGGAAGCggtaccTGATCTTCTG gagctcTGGGAGGaagcttGTGAGGCAGCAGCGgaattcTGGGGTCCTCCcatatgTGAG GGTCTTCCgcggccgcAAC-3’) was synthesized by Integrated DNA Technologies and first amplified by PCR. The purified product was then re-amplified using primers containing attB sequences to add attB1 site to 5’ and attB2 site to 3’ ends. This second PCR product was purified and recombined with pDONR221 (Invitrogen, #12536017) in a Gateway BP reaction using BP Clonase II enzyme mix (Invitrogen, #11789020), resulting in the entry vector pENTR221-sctFP-MCS.

[0156] pENTR221-sctFP-MCS serves as a versatile cloning vector for inserting up to four fluorescent proteins (FPs) in tandem. The designated restriction sites for FP insertion are: FP1 – BamHI and SpeI; FP2 – KpnI and SacI; FP3 – HindIII and EcoRI; FP4 – NdeI and NotI. A Kozak sequence and an ATG start codon must be included at the 5’ end of FP1 after the BamHI restriction site. To maintain the correct reading frame, a G nucleotide should be added to the 3’ end of FP2 and to both the 5’ and 3’ ends of FP3 if it is included. Internal stop codons are present for FP1, FP2, and FP3 when in-frame, while FP4 must include a stop codon at its 3’ end.

[0157] Single FP constructs were obtained from Addgene, amplified to add appropriate restriction sites, and then cloned into pENTR221-sctFP-MCS. These various entry vectors were then recombined with the lentiviral destination vector pLex307 (Addgene, #41392) using Gateway LR Clonase II (Invitrogen, #11791020). The resulting expression plasmids were used for both transient transfection and lentivirus production.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0158] To construct the pcDNA3.1-BFP–EV–iRFP702 and pcDNA3.1-TagRFP– EV–iRFP702 plasmids, we first generated the pcDNA3.1-PicchuEV-1 construct by inserting the PicchuEV sequence

[0052] into pcDNA3.1(+) using the HindIII and BamHI restriction sites. BFP or TagRFP was then inserted by replacing the smaller fragment between HindIII and KpnI, while iRFP702 was introduced by replacing the smaller fragment between BspEI and BamHI.

[0159] Biosensors

[0160] Plasmids for the following biosensors were purchased from Addgene: CytoFAK (Addgene #78300), LynFAK (Addgene #78299),

[0041] , RhoA2G (Addgene #40176)

[0053] , Src (Addgene #78302)

[0054] , Syk (Addgene #125729)

[0055] , EKAR (Addgene #18679)

[0042] , ExRai-CKAR (Addgene #118409), ExRai-AKAR2 (Addgene #161753), ExRai-AKTAR2 (Addgene #184047), and ExRai-AMPKAR (Addgene #192446). PicchuEV and EV-ROCK were generously provided by Dr. Michiyuki Matsuda’s laboratory.

[0161] Cell transfection and lentiviral transduction

[0162] Cells were transiently transfected using the GenJet™ In Vitro DNA Transfection Reagent (Ver. II) following the manufacturer's protocol (SignaGen Laboratories, #SL100489). Briefly, for one 3.5 cm culture dish, 4x105 cells were seeded the day before transfection. On the day of transfection, the culture medium was replaced with 1 mL fresh complete medium without antibiotics. The GenJet-DNA complex was prepared by diluting (1) 1 μg of DNA in 50 μL serum-free DMEM, and (2) 3 μL GenJet reagent in 50 μL serum-free DMEM in two separate tubes, and then mix the two together by adding GenJet into DNA. The complex was allowed to form at room temperature for 15 min, and then added dropwise onto the cells. Cell expression was visible 24 to 48 h post transfection.

[0163] For lentiviral transduction, 4x105 cells were seeded in one 3.5 cm culture dish the day before transduction. The culture medium was then replaced with a complete medium containing lentiviruses and polybrene (final concentration 8 μg / mL). Cells were incubated with the viruses at 37°C, 5% CO2 overnight and medium replaced the next day. Cell expression was visible 48 h post transduction.

[0164] Lentivirus productionAtty Dkt. No.0184.0318-PCT (P18296-02)

[0165] Lentiviruses were collected from HEK293T cells transfected with a transfer plasmid, a packaging plasmid psPAX2 (Addgene #12260), and an envelope plasmid pMD2.G (Addgene #12259) at the mass ratio of 4:3:1. The culture medium was replaced 5 h after transfection and the conditioned medium was collected 24 h later. More fresh medium was added to the transfected cells and the conditioned medium was collected again after another 24 h. The two batches of conditioned medium containing lentiviruses were pooled together and concentrated 10 times with the Lenti-X Concentrator following the manufacturer’s protocol (Takara Bio, #631231). The concentrated lentiviruses were aliquoted, flash frozen in liquid nitrogen, and stored at -80°C.

[0166] Microscopy

[0167] Cells were transfected with plasmids encoding barcode constructs and different biosensor constructs in separate wells. Prior to imaging, cells expressing distinct barcode and biosensor combinations were harvested, pooled, and re-plated onto imaging dishes. Confocal microscopy was performed using a Zeiss LSM 780 or 880 equipped with a spectral detector (for barcode emission profiling) and a motorized stage (for automated acquisition of multiple fields of view).

[0168] For the sctFP barcodes, spectral imaging was performed on a Zeiss LSM 780 or 880 confocal microscope equipped with a spectral detector. Each fluorescent protein was excited using its optimal laser line (405, 488, 561, or 633 nm), based on its excitation spectrum. For each sctFP, emission signals were collected across 4–5 detection channels, with wavelength ranges selected according to the emission profile of the corresponding FP (see Fig.7B).

[0169] For CFP–YFP FRET biosensor imaging, cells were excited with a 458 nm laser. Emission signals were collected using two detection windows: 457–500 nm for CFP and 507–544 nm for YFP. FRET activity was quantified as the YFP / CFP emission ratio.

[0170] For ExRai biosensor imaging, cells were sequentially excited with 488 nm and 405 nm lasers, and emission signals from both excitations were collected in the 507–544 nm range. ExRai activity was represented by the ratio of emission intensities upon 488 nm versus 405 nm excitation.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0171] Biosensor responses were quantified by calculating pixel-by-pixel intensity differences between time-lapse frames separated by defined intervals. To reduce noise, the 2–3 frames immediately prior to stimulation were averaged to establish a baseline. The percentage change in intensity was then calculated by normalizing each difference to the baseline average.

[0172] Image analysis

[0173] Images were processed and analyzed with NIH ImageJ and Fiji [56,57]. The signal intensity ratio (SIR) of individual cells was calculated in Microsoft Excel from the signal intensity (SI) of different channels. Microsoft Excel was also used for statistical analysis and to generate graphs.

[0174] Pixelwise analysis of sctFP barcodes comprises the following steps: 1) The SI of all channels (typically a BFP channel plus 4 RFP channels) was measured within a 10×10 pixel area and normalized to the SI of the peak RFP channel.2) The normalized SI values of the RFP channels were compared to reference RFP spectra to calculate the deviation.3) The variance of the SIR of BFP / RFP across 5 adjacent pixels was calculated.4) A pixels was assigned a barcode when it met the following conditions: A) low deviation from the RFP reference spectrum; B) low variance in BFP / RFP across neighboring pixels; C) a BFP / RFP within 10% of the reference ratio, based on the empirical distribution of BFP / RFP values. Thresholds for acceptable RFP deviation and BFP / RFP variance were empirically determined.

[0175] Immunoblotting

[0176] The following primary antibodies were used: anti-GFP (Cell Signaling Technology, #2956, 1:1000), anti-GAPDH (Cell Signaling Technology, #2118, 1:2000), and anti-FLAG (Millipore, F7425) for detecting FLAG-tagged sctFPs. The secondary antibody used was Donkey anti-Rabbit Alexa Fluor™ 647 (Thermo Fisher Scientific, A-31573, 1:5000).

[0177] Immunoblotting was done as described in our previous publication

[0058] . Briefly, cells were harvested and lysed in 1X RIPA buffer (Cell Signaling, #9806) containing 1X protease inhibitor cocktail (Roche, #11873580001) and 1X phosphatase inhibitor (Sigma, #P5726). Cell lysates were collected after centrifugation. After mixing with sample buffer and boiling at 95℃ for 5 min, proteinsAtty Dkt. No.0184.0318-PCT (P18296-02) were separated by SDS-PAGE using 4–20% Criterion™ TGX™ Precast Midi Protein Gels (Bio-Rad, #5671094), and then transferred to the low fluorescence background PVDF membrane (Millipore, #IPFL00005) in an ice bath at 80V for 1h. Membranes were incubated with primary antibodies overnight followed by incubation with secondary antibodies in 5% BSA in TBST at room temperature for 1h. Images were taken by a Pharos Molecular Imager (BioRad).

[0178] Some further aspects are also defined in the following clauses:

[0179] Clause 1: A method of differentiating cells in a cell population, the method comprising: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set, thereby differentiating the cells in the cell population.

[0180] Clause 2: The method of Clause 1, comprising identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signals emitted by the first FP, or the functional portion thereof, and detected fluorescent signals emitted by the second FP, or the functional portion thereof.

[0181] Clause 3: The method of Clause 1 or Clause 2, wherein the first fluorescent barcoding protein comprises a single polypeptide chain.

[0182] Clause 4: The method of any of Clauses 1-3, wherein the first subset of cells in the cell population is a selected cell type.

[0183] Clause 5: The method of any of Clauses 1-4, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0184] Clause 6: The method of any of Clauses 1-5, comprising detecting fluorescent signals emitted by multiple sets of fluorescent barcoding proteins in the cell population to produce multiple sets of detected fluorescent signals, wherein at least two subsets of cells in the cell population comprise different fluorescent barcoding proteins, and identifying the different fluorescent barcoding proteins in the at least two subsets of cells in the cell population using the multiple sets of detected fluorescent signals.

[0185] Clause 7: The method of any of Clauses 1-6, comprising identifying the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population by determining ratios of detected fluorescent signals emitted by the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population.

[0186] Clause 8: The method of any of Clauses 1-7, wherein at least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein, and wherein the method comprises detecting fluorescent signals emitted by the first and second fluorescent barcoding proteins to produce the first detected fluorescent signal data set and at least a second detected fluorescent signal data set, and identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population using the first and second sets of detected fluorescent signals.

[0187] Clause 9: The method of any of Clauses 1-8, wherein the first and second subsets of cells in the cell population are different selected cell types.

[0188] Clause 10: The method of any of Clauses 1-9, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

[0189] Clause 11: The method of any of Clauses 1-10, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0190] Clause 12: The method of any of Clauses 1-11, comprising identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population by determining a ratio of detected fluorescent signals emitted by the first fluorescent barcoding protein and a ratio of detected fluorescent signals emitted by the second fluorescent barcoding proteins.

[0191] Clause 13: The method of any of Clauses 1-12, wherein an organoid comprises the cell population.

[0192] Clause 14: The method of any of Clauses 1-13, wherein a two-dimensional (2D) array comprises the cell population.

[0193] Clause 15: The method of any of Clauses 1-14, wherein the detecting step comprises imaging and / or identifying one or more cells in the cell population that comprise the fluorescent barcoding proteins at one or more time points during a selected duration.

[0194] Clause 16: The method of any of Clauses 1-15, further comprising detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population.

[0195] Clause 17: The method of any of Clauses 1-16, wherein the fluorescent calibration standard biomolecules comprise FPs.

[0196] Clause 18: The method of any of Clauses 1-17, wherein at least one synthetic polynucleotide encodes one or more of the fluorescent barcoding proteins and wherein the method comprises expressing the fluorescent barcoding proteins from the synthetic polynucleotide prior to and / or concurrent with the detecting step.

[0197] Clause 19: The method of any of Clauses 1-18, wherein an expression cassette comprises the synthetic polynucleotide.

[0198] Clause 20: The method of any of Clauses 1-19, wherein a plasmid comprises the expression cassette.

[0199] Clause 21: The method of any of Clauses 1-20, further comprising introducing the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0200] Clause 22: The method of any of Clauses 1-21, comprising introducing the synthetic polynucleotide into a subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

[0201] Clause 23: A composition, comprising a cell population, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another.

[0202] Clause 24: The composition of Clause 23, wherein the first fluorescent barcoding protein comprises a single polypeptide chain.

[0203] Clause 25: The composition of Clause 23 or Clause 24, wherein the first subset of cells in the cell population is a selected cell type.

[0204] Clause 26: The composition of any of Clauses 23-25, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof.

[0205] Clause 27: The composition of any of Clauses 23-26, wherein at least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein.

[0206] Clause 28: The composition of any of Clauses 23-27, wherein the first and second subsets of cells in the cell population are different selected cell types.

[0207] Clause 29: The composition of any of Clauses 23-28, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

[0208] Clause 30: The composition of any of Clauses 23-29, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0209] Clause 31: A kit comprising the composition of any of Clauses 23-30.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0210] Clause 32: A kit, comprising a first synthetic polynucleotide that encodes at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof.

[0211] Clause 33: The kit of Clause 32, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof.

[0212] Clause 34: The kit of Clause 32 or Clause 33, further comprising a second synthetic polynucleotide that encodes at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein.

[0213] Clause 35: The kit of any of Clauses 32-34, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

[0214] Clause 36: The kit of any of Clauses 32-35, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0215] Clause 37: A system for differentiating cells in a cell population, comprising: a processor; and a memory communicatively coupled to the processor, the memory storing instructions which, when executed on the processor, perform operations comprising: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0216] Clause 38: The system of Clause 37, wherein the instructions which, when executed on the processor, further perform operations comprising: identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signals emitted by the first FP, or the functional portion thereof, and detected fluorescent signals emitted by the second FP, or the functional portion thereof.

[0217] Clause 39: A computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set.

[0218] Clause 40: A first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another.

[0219] Clause 41: The fluorescent barcoding protein of Clause 40, wherein the first fluorescent barcoding protein comprises a single polypeptide chain.

[0220] Clause 42: The fluorescent barcoding protein of Clause 40 or Clause 41, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or a functional portion thereof, and / or more than one copy of the second FP, or a functional portion thereof.

[0221] Clause 43: A cell comprising the first fluorescent barcoding protein of any of Clauses 40-42.

[0222] Clause 44: A composition comprising the first fluorescent barcoding protein of any of Clauses 40-43.Atty Dkt. No.0184.0318-PCT (P18296-02)

[0223] Clause 45: The composition of any of Clauses 40-44, further comprising a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein.

[0224] Clause 46: The composition of any of Clauses 40-45, wherein a first subset of cells in a cell population each comprise the first fluorescent barcoding protein and wherein a second subset of cells in the cell population each comprise the second fluorescent barcoding protein.

[0225] Clause 47: The composition of any of Clauses 40-46, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

[0226] Clause 48: The composition of any of Clauses 40-47, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

[0227] Clause 49: A synthetic polynucleotide that encodes a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another.

[0228] Clause 50: An expression cassette that comprises the synthetic polynucleotide of Clause 49.

[0229] Clause 51: A plasmid that comprises the expression cassette of Clause 50.

[0230] Clause 52: A kit that comprises the expression cassette of Clause 51.

[0231] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0232] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims)Atty Dkt. No.0184.0318-PCT (P18296-02) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0233] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Atty Dkt. No.0184.0318-PCT (P18296-02) Claims:

1. A method of differentiating cells in a cell population, the method comprising: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and, identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set, thereby differentiating the cells in the cell population.

2. The method of claim 1, comprising identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signals emitted by the first FP, or the functional portion thereof, and detected fluorescent signals emitted by the second FP, or the functional portion thereof.

3. The method of claim 1, wherein the first fluorescent barcoding protein comprises a single polypeptide chain.

4. The method of claim 1, wherein the first subset of cells in the cell population is a selected cell type.Atty Dkt. No.0184.0318-PCT (P18296-02) 5. The method of claim 1, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof.

6. The method of claim 1, comprising detecting fluorescent signals emitted by multiple sets of fluorescent barcoding proteins in the cell population to produce multiple sets of detected fluorescent signals, wherein at least two subsets of cells in the cell population comprise different fluorescent barcoding proteins, and identifying the different fluorescent barcoding proteins in the at least two subsets of cells in the cell population using the multiple sets of detected fluorescent signals.

7. The method of claim 6, comprising identifying the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population by determining ratios of detected fluorescent signals emitted by the different fluorescent barcoding proteins in the at least two subsets of the cells in the cell population.

8. The method of claim 1, wherein at least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein, and wherein the method comprises detecting fluorescent signals emitted by the first and second fluorescent barcoding proteins to produce the first detected fluorescent signal data set and at least a second detected fluorescent signal data set, and identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population using the first and second sets of detected fluorescent signals.

9. The method of claim 8, wherein the first and second subsets of cells in the cell population are different selected cell types.

10. The method of claim 8, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.Atty Dkt. No.0184.0318-PCT (P18296-02) 11. The method of claim 8, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

12. The method of claim 8, comprising identifying the first and second fluorescent barcoding proteins in the first and second subsets of the cells in the cell population by determining a ratio of detected fluorescent signals emitted by the first fluorescent barcoding protein and a ratio of detected fluorescent signals emitted by the second fluorescent barcoding proteins.

13. The method of claim 1, wherein an organoid comprises the cell population.

14. The method of claim 1, wherein a two-dimensional (2D) array comprises the cell population.

15. The method of claim 1, wherein the detecting step comprises imaging and / or identifying one or more cells in the cell population that comprise the fluorescent barcoding proteins at one or more time points during a selected duration.

16. The method of claim 1, further comprising detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population.

17. The method of claim 16, wherein the fluorescent calibration standard biomolecules comprise FPs.

18. The method of claim 1, wherein at least one synthetic polynucleotide encodes one or more of the fluorescent barcoding proteins and wherein the methodAtty Dkt. No.0184.0318-PCT (P18296-02) comprises expressing the fluorescent barcoding proteins from the synthetic polynucleotide prior to and / or concurrent with the detecting step.

19. The method of claim 18, wherein an expression cassette comprises the synthetic polynucleotide.

20. The method of claim 19, wherein a plasmid comprises the expression cassette.

21. The method of claim 18, further comprising introducing the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step.

22. The method of claim 21, comprising introducing the synthetic polynucleotide into a subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

23. A composition, comprising a cell population, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another.

24. The composition of claim 23, wherein the first fluorescent barcoding protein comprises a single polypeptide chain.

25. The composition of claim 23, wherein the first subset of cells in the cell population is a selected cell type.Atty Dkt. No.0184.0318-PCT (P18296-02) 26. The composition of claim 23, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof.

27. The composition of claim 23, wherein at least a second subset of the cells in the cell population each comprise at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein.

28. The composition of claim 27, wherein the first and second subsets of cells in the cell population are different selected cell types.

29. The composition of claim 27, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

30. The composition of claim 27, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

31. A kit comprising the composition of claim 23.

32. A kit, comprising a first synthetic polynucleotide that encodes at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof.

33. The kit of claim 32, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or the functional portion thereof, and / or more than one copy of the second FP, or the functional portion thereof.Atty Dkt. No.0184.0318-PCT (P18296-02) 34. The kit of claim 32, further comprising a second synthetic polynucleotide that encodes at least a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein.

35. The kit of claim 34, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

36. The kit of claim 34, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

37. A system for differentiating cells in a cell population, comprising: a processor; and a memory communicatively coupled to the processor, the memory storing instructions which, when executed on the processor, perform operations comprising: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and, identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set.

38. The system of claim 37, wherein the instructions which, when executed on the processor, perform operations comprising: identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population by determining a ratio between detected fluorescent signalsAtty Dkt. No.0184.0318-PCT (P18296-02) emitted by the first FP, or the functional portion thereof, and detected fluorescent signals emitted by the second FP, or the functional portion thereof,.

39. A computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: detecting fluorescent signals emitted by at least one set of fluorescent barcoding proteins in the cell population to produce at least a first detected fluorescent signal data set, wherein at least a first subset of the cells in the cell population each comprise at least a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, and wherein the first and second FPs, or the functional portions thereof, differ from one another; and, identifying the first fluorescent barcoding protein in the first subset of the cells in the cell population using the first detected fluorescent signal data set.

40. A first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another.

41. The fluorescent barcoding protein of claim 40, wherein the first fluorescent barcoding protein comprises a single polypeptide chain.

42. The fluorescent barcoding protein of claim 40, wherein the first fluorescent barcoding protein comprises more than one copy of the first FP, or a functional portion thereof, and / or more than one copy of the second FP, or a functional portion thereof.

43. A cell comprising the first fluorescent barcoding protein of claim 40.Atty Dkt. No.0184.0318-PCT (P18296-02) 44. A composition comprising the first fluorescent barcoding protein of claim 40.

45. The composition of claim 44, further comprising a second fluorescent barcoding protein that differs from the first fluorescent barcoding protein.

46. The composition of claim 45, wherein a first subset of cells in a cell population each comprise the first fluorescent barcoding protein and wherein a second subset of cells in the cell population each comprise the second fluorescent barcoding protein.

47. The composition of claim 45, wherein the first and second fluorescent barcoding proteins each comprise copy number combinations of the first and second FPs, or the functional portions thereof, that differ from one another.

48. The composition of claim 45, wherein the second fluorescent barcoding protein comprises at least one copy of a third FP, or a functional portion thereof, that differs from the first and second FPs, or the functional portions thereof.

49. A synthetic polynucleotide that encodes a first fluorescent barcoding protein that comprises at least one copy of a first fluorescent protein (FP), or a functional portion thereof, and at least one copy of a second FP, or a functional portion thereof, wherein the first and second FPs, or the functional portions thereof, differ from one another.

50. An expression cassette that comprises the synthetic polynucleotide of claim 49.

51. A plasmid that comprises the expression cassette of claim 50.Atty Dkt. No.0184.0318-PCT (P18296-02) 52. A kit that comprises the expression cassette of claim 51.

Images