Methods and related aspects of biosensors

Abstract

Provided herein are methods of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors that include detecting fluorescent signals emitted by one or more FP biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof, and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set. Related methods, compositions, kits, systems, and computer readable media are also provided.

Description

Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) METHODS AND RELATED ASPECTS OF BIOSENSORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No.63 / 514,728, filed July 20, 2023, the disclosure of which is incorporated herein by reference. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant GM136711 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. A large number of biosensors are based on a single GFP or the Förster or fluorescence resonanceAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) energy transfer (FRET) between CFP and YFP. Despite the power and 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. We previously overcame the limitation by developing the "biosensor barcoding" to overcome the limited multiplexing capability of fluorescent biosensors (FIG.3). This involves labeling cells expressing different biosensors with barcodes consisting of a pair of barcoding proteins - blue or red fluorescent proteins that are targeted to distinct subcellular locations. The emission spectra of these barcoding proteins are distinguishable from those of the commonly used CFP, GFP, and YFP in biosensors, allowing for the simultaneous imaging and identification of the biosensor-expressing cells.

[0004] Another limitation of biosensors is that direct comparison of biosensor signals from different imaging experiments cannot be reliably achieved. For example, for FRET-based biosensors, the YFP / CFP ratio is often used as an indicator for the FRET efficiency, which cannot be directly measured, whereas for single FP biosensors, the fluorescence intensity in either subcellular compartments or the entire cell is used as the readout. However, the fluorescence ratio or intensity depends on factors including laser intensity, which may fluctuate over time, or camera detector and other imaging settings. Thus, due to the lack of a reliable method to calibrate the signals from different imaging experiments, most biosensor experiments are conducted in a single imaging session with limited duration (up to hours).

[0005] Accordingly, there exists an unmet need for an effective method to monitor multiple intracellular activities over long durations for various applications. SUMMARY OF THE INVENTION

[0006] 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 calibration standards for FRET-based biosensors that can be used to calibrate the biosensor signals from images taken at different time points. In some embodiments, the present disclosureAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) provides fluorescent calibration standard proteins involving dual intein systems of use in calibrating detectable signal emitted from single fluorescent protein biosensors. Related algorithms for calibrating biosensor signals using these standards are also provided. In some embodiments, the barcodes, biosensors, and calibration standards 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.

[0007] According to various embodiments, a method of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors is presented. The method includes detecting fluorescent signals emitted by one or more FP biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof, and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set, thereby calibrating the fluorescent signals emitted by the FP biosensors.

[0008] According to various embodiments, a method of differentiating cells in a cell population is presented. The method includes detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from theAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) different targeting sites in the subset of the cells, thereby differentiating the cells in the cell population. In some embodiments, an organoid comprises the cell population. In some embodiments, a two-dimensional (2D) array comprises the cell population. In some embodiments, different subsets of cells in the cell population comprise different pairs of the fluorescent barcoding proteins, wherein a first pair of fluorescent barcoding proteins comprises a combination of fluorescent labels and / or targeting sites that differs a second pair of fluorescent barcoding proteins. In some embodiments, 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. In some embodiments, the method further comprises detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors in the cell population. In some embodiments, the method further comprises detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population.

[0009] According to various embodiments, the present disclosure provides a composition, comprising a cell population, wherein at least a first set of cells in the cell population comprises one or more fluorescent protein (FP) biosensors and wherein at least a second set of cells in the cell population comprises one or more fluorescent calibration standard biomolecules, wherein: at least a first set of the FP biosensors comprises fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety; and at least a first set of the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety, and wherein one or more subsets of cells in the second set of cells comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations; and / or, at least a second set of the FP biosensors that each comprise a first set of one or more FPs, or functional portions thereof, wherein the FP biosensors are fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked to the FP biosensors via one or moreAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) inteins, and at least a second set of the fluorescent calibration standard biomolecules that each comprise a second set of one or more FPs, or functional portions thereof, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the fluorescent calibration standard biomolecules are fused to polypeptide constructs that each comprise the third set of one or more FPs, or functional portions thereof, operably linked to the fluorescent calibration standard biomolecules via the one or more inteins. In some embodiments, a kit comprises the composition.

[0010] According to various embodiments, the present disclosure provides a kit that comprises a first synthetic polynucleotide that encodes at least a first set of one or more fluorescent protein (FP) biosensors, or functional portions thereof, and a second synthetic polynucleotide that encodes at least a first set of one or more fluorescent calibration standard biomolecules, or functional portions thereof. In some embodiments, the method further includes a third synthetic polynucleotide that encodes at least a first set of one or more fluorescent barcoding proteins, or functional portions thereof.

[0011] According to various embodiments, a system for calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors, 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 one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set. In some embodiments, the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by a set of fluorescent barcodingAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0012] According to various embodiments, 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 a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells. In some embodiments, the instructions which, when executed on the processor, further perform operations comprise: detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in the cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

[0013] According to various embodiments, a computer readable media comprising non-transitory computer executable instructions which, when executed by at least one electronic processor, perform at least: detecting fluorescent signalsAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set. In some embodiments, the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0014] According to various embodiments, 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 a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells. In some embodiments, the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules inAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) the cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

[0015] Various optional features of the above embodiments include the following. The detecting step comprises imaging and / or identifying one or more cells in the cell population that comprise the FP biosensors at one or more time points during a selected duration. The first and second sets of FPs are identical to one another. At least one synthetic polynucleotide encodes the FP biosensors and / or the fluorescent calibration standard biomolecules and wherein the method comprises expressing the FP biosensors and / or the fluorescent calibration standard biomolecules from the synthetic polynucleotide prior to and / or concurrent with the detecting step. A first synthetic polynucleotide encodes the FP biosensors and wherein a second synthetic polynucleotide encodes the fluorescent calibration standard biomolecules. An expression cassette comprises the synthetic polynucleotide. A plasmid comprises the expression cassette. The method further comprises introducing the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step. The method comprises introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector. At least one subset of the cells in the cell population comprises one or more fluorescent barcoding proteins. The subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells.

[0016] Various additional optional features of the above embodiments include the following. The first and second fluorescent labels comprise FPs or functional portions thereof. at least one synthetic polynucleotide encodes the fluorescent barcodingAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) 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 comprises introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step. The method comprises introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector. The fluorescent calibration standard biomolecules comprise FPs. The FP biosensors comprise fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety. The fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety. One or more subsets of cells in the cell population comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations. Various additional optional features of the above embodiments include the following. The FP biosensors each comprise only a single FP or a functional portion thereof. The FP biosensors and the fluorescent calibration standard biomolecules are separately fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked via one or more inteins, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the inteins undergo self-cleavage to release the FP biosensors and the fluorescent calibration standard biomolecules from the third set of FPs prior to and / or concurrent with the detecting step. The third set of FPs is further operably linked to a cellular targeting moiety. The fluorescent calibration standard biomolecules are further operably linked to the cellular targeting moiety. The cellular targeting moiety comprises a nuclear export signal (NES). At least one synthetic polynucleotide encodes the polypeptide constructs and wherein the method comprises expressing the polypeptide constructs from the synthetic polynucleotide prior to and / orAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) concurrent with the detecting step. An expression cassette comprises the synthetic polynucleotide. A plasmid comprises the expression cassette. The method further comprises introducing the synthetic polynucleotide into at least a subset of the cells in the cell population prior to the detecting step. The method comprises introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG.1A is a flow chart that schematically shows exemplary method steps of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors according to some aspects disclosed herein.

[0018] FIG.1B is a flow chart that schematically shows exemplary method steps of differentiating cells in a cell population according to some aspects disclosed herein.

[0019] FIG.2 is a schematic diagram of an exemplary system suitable for use with certain aspects disclosed herein.

[0020] FIGS.3A-3F. Biosensor barcoding method. (A) Excitation and emission spectra of barcoding proteins (BFP, mCherry, mCardinal, and iRFP702) and biosensors (CFP, GFP, YFP). Excitation laser wavelengths are indicated by lines (405 nm and 633 nm for BFP and red FPs used in barcodes, respectively; 458 nm for biosensors) and the corresponding ranges of emission detection are indicated by boxes. (B) Targeting sites for barcoding proteins. (C) Representative pseudo-colored images of cells expressing different barcodes (cover image of Cell 184). (D) A pair of barcoding proteins (different colors, different targeting sites) are co-expressed with a fluorescent biosensor in the cell. (E) Cells are mixed and imaged for the barcodes using spectral detectors, and for the biosensors using CFP / YFP channels. The identity of the biosensor can be inferred after spectral unmixing of the barcodes. (F) Activities corresponding to the same barcodes are averaged to obtain the temporal profile of each biosensor.

[0021] FIGS.4A-4C. Calibration for FRET biosensors. (A) Schematic of FRET calibration standards (FRET-OFF, FRET-ON) and biosensors. (B) Expression ofAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) calibration standards and biosensors in barcoded cells. (C) Normalization of YFP / CFP of biosensors using the calibration standards.

[0022] FIGS.5A-5F. Example of FRET biosensor calibration. (A) Schematic of FRET calibration standards (FRET-OFF, FRET-ON) and biosensors (CytoFAK, EKAR) as well as the FRET donor-acceptor pair used in each construct. (B) The level of laser power and gain settings for CFP and YFP. (C) CFP and YFP levels (mean ± SEM) of FRET-OFF under different imaging settings. (D) YFP / CFP ratio of individual cells expressing the FRET constructs plotted against the YFP signal. (E) YFP / CFP (mean ± SEM) across cells expressing the FRET constructs under different imaging settings. (F) YFP / CFP normalized to the calibration standards (mean ± SEM) of the FRET constructs, with FRET-OFF and FRET-ON set to 0 and 1, respectively, under different imaging settings.

[0023] FIGS.6A-6D. Calibration for single FP biosensors. (A) Schematic of the constructs for biosensors and calibration standard, which undergoes autocatalytic cleavage to release the biosensor and cytoplasm-targeted NES-CFP. (B) GFP and CFP images of translocation and intensity based biosensors and NES-CFP. (C) Expression of GFP-DI-CFP and biosensor-DI-CFP in barcoded cells.(D) Normalization of GFP / CFP of biosensors using the calibration standard. Note that GFP is imaged in the YFP channel to simplify the imaging protocol when mixed with FRET-based biosensors.

[0024] FIGS.7A-7C. Automated barcode reading using deep learning models. (A) Schematic of the barcode analysis by deep learning models. (B) The confusion matrix and identification rate (%) for barcoded HeLa cells identified by model 2 (top) and model 3 (bottom) under indicated certainty thresholds. The 4 numbers denote the location of mCherry, mCardinal, iRFP702, and BFP (in that order), with 1, 2, 3, and 4 denoting the nucleus, plasma membrane, nuclear membrane, and cytoplasm, respectively. (C) Confusion matrix (%) of MCF7 (top) and U2OS (bottom) cells barcoded with nuclear or cytoplasmic BFP plus mCherry targeted to one of the four subcellular locations using the deep learning models trained on HeLa cells (original model, left) and new models trained on MCF7 or U2OS cell images (right).Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0025] FIGS.8A-8C. Deep learning models for barcode reading in organoids. (A) Representative images of mouse intestinal organoids expressing RFP (top) or BFP (middle) and PDAC organoids expressing RFP (bottom) targeted to the nucleus, plasma membrane, nuclear membrane, or cytosol. Scale bars: 20 μm (unpublished). (B) Performance of deep learning models on images of intestinal organoids expressing barcoding proteins localized to the nucleus, plasma membrane, and cytosol. The accuracy and validation loss of models on training and validation image sets over 100 epochs of training are shown (unpublished) (C) Workflow of barcode prediction in organoids using deep learning models. DEFINITIONS

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Biomolecule: As used herein, “biomolecule” refers to an organic molecule produced by a living organism. Exemplary biomolecules, include without limitationAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) macromolecules, such as nucleic acids, proteins, peptides, oligomers, carbohydrates, and lipids.

[0030] 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.

[0031] 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.

[0032] 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).

[0033] 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 can 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.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0034] 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.

[0035] 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.

[0036] 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 fluorescent 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.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0037] 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: 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.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0038] Locked FRET-OFF Conformation: As used herein, “locked FRET-OFF conformation” in the context of FRET calibration standard biomolecules refers to a biomolecule in which a FRET acceptor moiety and a FRET donor moiety are disposed relative to one another at a substantially fixed distance such that the FRET donor moiety, in its electronic excited state, is substantially unable to transfer energy to the acceptor moiety through nonradiative dipole–dipole coupling.

[0039] Locked FRET-ON Conformation: As used herein, “locked FRET-ON conformation” in the context of FRET calibration standard biomolecules refers to a biomolecule in which a FRET acceptor moiety and a FRET donor moiety are disposed relative to one another at a substantially fixed distance such that the FRET donor moiety, in its electronic excited state, may transfer energy to the acceptor moiety through nonradiative dipole–dipole coupling.

[0040] 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.”

[0041] 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) orAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, cfDNA, ctDNA, or any combination thereof.

[0042] Protein: As used herein, “protein” is used interchangeably with “polypeptide” and refers to polymers of amino acids of any length. These terms also 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.

[0043] 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 moleculesAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) 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.

[0044] 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

[0045] A large number of diseases, such as cancer, result from defects in signaling pathways. In various types of cancer, mutations in one or several proteins along biochemical pathways governing cell growth and replication can mutate, causing rapid cell overgrowth and tumorigenesis. By understanding the specific signaling pathways involved in a disease, scientists and physicians can develop targeted treatments that modify the activity of these pathways to correct the underlying cellular problems and ameliorate the symptoms of the disease.

[0046] To assist with the analysis of signaling pathways, among other attributes, the present disclosure solves various major limitations that hamper the wider application of, for example, FRET-based biosensors in studying such signaling pathways in cells. In one exemplary aspect, the present disclosure providesAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) solutions to the poor multiplexing capability of FRET-based biosensors, due to the overlapping spectra of fluorescent proteins (FPs) and limited availability of spectral space. In another exemplary aspect, the present disclosure provides solutions to the problem that the comparison of measurements across experiments is unreliable in the absence of calibration standards for fluorescence signals.

[0047] More specifically, in some embodiments, the present disclosure provides calibration standards for FRET-based biosensors. An overview of some of these embodiments is schematically depicted in FIGS.4A-4C. In some embodiments, for example, these standards are cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) constructs that are locked in either FRET-on or FRET-off conformations. In some embodiments, a barcoding system, as described further herein, can be used in conjunction with introducing the calibration standards in a subset of cells so that they can be imaged together with the biosensors in populations of mixed barcoded cells. As described further with respect to FIGS.5A- 5F, the YFP / CFP ratio for FRET biosensors normalized to these calibration standards remains constant under different imaging settings, demonstrating that the standards can be used to calibrate the biosensor signals from images taken at different time points. In addition, for single FP biosensors, calibration can be achieved by normalizing, for example, to a cytoplasmic CFP that is automatically cleaved using a dual intein system. This embodiment is described further, for example, in connection with FIGS.6A-6D.

[0048] 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 in relation to FIGS.8A-8C, 3D organoids preserve the 3D cell connections and are typically better models for the study of tissue morphogenesis,Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) tumor heterogeneity, drug screening, and tumor / microenvironment interactions than 2D cell lines.

[0049] 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. Some of these aspects are further described in connection, for example, with FIGS.7A-7C. 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.

[0050] To illustrate, in some embodiments, the present disclosure provides a method of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors (FIG.1A). For example, method 100 includes detecting fluorescent signals emitted by one or more FP biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof (step 102). Method 100 also includes normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set (step 104). In some embodiments, the fluorescent calibration standard biomolecules comprise FPs. Typically, detecting step (step 102) comprises imaging and / or identifying one or more cells in the cell population that comprise the FP biosensors at one or more time points during a selected duration. In some embodiments, the first and second sets of FPs are identical to one another.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0051] In some embodiments, a synthetic polynucleotide (e.g., an expression cassette) encodes the FP biosensors and / or the fluorescent calibration standard biomolecules. In these embodiments, method 100 typically includes expressing the FP biosensors 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 FP biosensors and a second synthetic polynucleotide encodes the fluorescent calibration standard biomolecules. In some embodiments, a plasmid comprises the expression cassette. Typically, method 100 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.

[0052] In some embodiments, a subset of the cells in the cell population comprises one or more fluorescent barcoding proteins. In some embodiments, the subset of the cells each comprise a pair of the fluorescent barcoding proteins in which a first member of the pair comprises a first fluorescent label, a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and the first and second members are located at different targeting sites in a given cell of the subset of the cells. In some embodiments, the first and second fluorescent labels comprise FPs or functional portions thereof. In some embodiments, a synthetic polynucleotide (e.g., an expression cassette) encodes the fluorescent barcoding proteins and method 100 comprises expressing the fluorescent barcoding proteins from the synthetic polynucleotide prior to and / or concurrent with the detecting step. Typically, a plasmid comprises the expression cassette. In some of these embodiments, method 100 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.

[0053] In some embodiments, the FP biosensors comprise fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety. In some embodiments, the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and atAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) least one FRET donor moiety. In some embodiments, one or more subsets of cells in the cell population comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations.

[0054] In some embodiments, the FP biosensors each comprise only a single FP or a functional portion thereof. In some embodiments, the FP biosensors and the fluorescent calibration standard biomolecules are separately fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked via one or more inteins, in which the first and second sets of FPs are identical to one another, the third set of FPs is different from the first and second sets of FPs, and the inteins undergo self-cleavage to release the FP biosensors and the fluorescent calibration standard biomolecules from the third set of FPs prior to and / or concurrent with the detecting step. In some embodiments, the third set of FPs is further operably linked to a cellular targeting moiety. In some embodiments, the fluorescent calibration standard biomolecules are further operably linked to the cellular targeting moiety. In some embodiments, the cellular targeting moiety comprises a nuclear export signal (NES) or another type of targeting moiety. In some of these embodiments, a synthetic polynucleotide (e.g., an expression cassette) encodes the polypeptide constructs and method 100 comprises expressing the polypeptide constructs from the synthetic polynucleotide prior to and / or concurrent with the detecting step. Typically, a plasmid comprises the expression cassette. In some of these embodiments, method 100 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.

[0055] To further illustrate, in some embodiments, the present disclosure provides a method of differentiating cells in a cell population. As shown in FIG.1B, for example, method 106 includes detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescentAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells (step 108). Method 106 also includes identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells (step 110). In some embodiments, an organoid comprises the cell population. In some embodiments, a two-dimensional (2D) array comprises the cell population. In some embodiments, different subsets of cells in the cell population comprise different pairs of the fluorescent barcoding proteins, wherein a first pair of fluorescent barcoding proteins comprises a combination of fluorescent labels and / or targeting sites that differs a second pair of fluorescent barcoding proteins. In some embodiments, 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. In some embodiments, method 106 further includes detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors in the cell population. In some embodiments, method 106 further includes detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population.

[0056] the present disclosure provides a composition, comprising a cell population, wherein at least a first set of cells in the cell population comprises one or more fluorescent protein (FP) biosensors and wherein at least a second set of cells in the cell population comprises one or more fluorescent calibration standard biomolecules, wherein: at least a first set of the FP biosensors comprises fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety; and at least a first set of the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety, and wherein one or more subsets of cells in the second set of cells comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations; and / or, at least a second set of the FP biosensors that each comprise a first set of one or more FPs, or functionalAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) portions thereof, wherein the FP biosensors are fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked to the FP biosensors via one or more inteins, and at least a second set of the fluorescent calibration standard biomolecules that each comprise a second set of one or more FPs, or functional portions thereof, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the fluorescent calibration standard biomolecules are fused to polypeptide constructs that each comprise the third set of one or more FPs, or functional portions thereof, operably linked to the fluorescent calibration standard biomolecules via the one or more inteins. In some embodiments, a kit comprises the composition.

[0057] In some embodiments, the present disclosure provides a kit that includes a first synthetic polynucleotide that encodes at least a first set of one or more fluorescent protein (FP) biosensors, or functional portions thereof, and a second synthetic polynucleotide that encodes at least a first set of one or more fluorescent calibration standard biomolecules, or functional portions thereof. In some embodiments, the kit further includes a third synthetic polynucleotide that encodes at least a first set of one or more fluorescent barcoding proteins, or functional portions thereof.

[0058] 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.2 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 200 includes at least one controller or computer, e.g., server 202 (e.g., a search engine server), which includes processor 204 and memory, storage device, or memory component 206, and one or more other communication devicesAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) 214, 216, (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 202, through electronic communication network 212, such as the Internet or other internetwork. Communication devices 214, 216 typically include an electronic display (e.g., an internet enabled computer or the like) in communication with, e.g., server 202 computer over network 212 in which the electronic display comprises a user interface (e.g., a graphical user interface (GUI), a 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 200 also includes program product 208 (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 206 of server 202, that is readable by the server 202, to facilitate, for example, a guided search application or other executable by one or more other communication devices, such as 214 (schematically shown as a desktop or personal computer). In some aspects, system 200 optionally also includes at least one database server, such as, for example, server 210 associated with an online website having data stored thereon (e.g., entries corresponding to a calibration standard signal data set, etc.) searchable either directly or through search engine server 202. System 200 optionally also includes one or more other servers positioned remotely from server 202, each of which are optionally associated with one or more database servers 210 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.

[0059] As understood by those of ordinary skill in the art, memory 206 of the server 202 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 202 is given only by way of exampleAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) and that other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 202 shown schematically in FIG.2, 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 and capacity requirements for the system 200. As also understood by those of ordinary skill in the art, other user communication devices 214, 216 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 212 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.

[0060] As further understood by those of ordinary skill in the art, exemplary program product or machine readable medium 208 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 208, 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.

[0061] 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 208 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 notAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) 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 data 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.

[0062] Program product 208 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 208, 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.

[0063] In some aspects, program product 208 includes non-transitory computer- executable instructions which, when executed by electronic processor 204, perform at least: detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof, and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set. In some aspects, program product 208 includes non-transitory computer-executable instructionsAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) which, when executed by electronic processor 204, perform at least: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0064] In some embodiments, calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors and the like are measured using device 218. As shown, device 218 includes excitation laser 220 and detector 224. Detector 224 receives detectable signals from, e.g., fluorescent protein biosensors, fluorescent calibration standard biomolecules, and fluorescent barcoding proteins. In some embodiments, device 218 includes sample container receiving area 226 on which sample container 222 is positioned. Sample container 222 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 the like. EXAMPLES

[0100] EXAMPLE 1: Method for Highly Multiplexed and Standardized Measurement of Biomolecular Activities in Cells

[0101] METHOD

[0102] 1. Multiplexed biosensor imaging in barcoded cells

[0103] Constructing lentiviral vectors for biosensors: Lentiviral plasmids are constructed using the Gateway Recombination Cloning Technology. Briefly, entry vectors are generated by integrating PCR-generated biosensor sequences into pDONR221 (Invitrogen, #12536017) with Gateway BP Clonase (Invitrogen, #11789100). The final expression vectors were generated by transferring the biosensor sequence from the entry vector to the destination vector pLenti-CMV-Neo,Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) which allows for neomycin selection, with Gateway LR Clonase (Invitrogen, #11791100).

[0104] Lentiviral transduction of 2D cells and organoids. Lentivirus production is carried out using techniques know to those having ordinary skill in the art. Briefly, HEK293T are transfected with pMD2.G (Addgene, #12259), psPAX2 (Addgene, #12260), and pLenti-CMV-Neo vector encoding the biosensors using GenJet In Vitro DNA Transfection Reagent ver. II. Medium containing lentiviral particles is harvested in 2 batches at 24 and 48 hrs after transfection and filtered through a 0.45 μm PVDF filter. Lentiviral transduction of 2D cells are carried out by seeding cells into an 8-well chamber for attachment overnight, followed by incubation with lentiviral particles in a medium containing 8 μg / ml polybrene for 24 hr. Neomycin is added 48-72 hours after transduction for selection. Organoids are passaged in Matrigel and dissociated into single cells using TrypLE solution at 37℃. The cell pellet is then resuspended and incubated in a medium containing lentiviral particles and 5 μg / mL polybrene at 37℃ for 6 h. The cells are pelleted, resuspended with Matrigel, and plated as domes in Sato medium containing 10 μM Y-27632. Neomycin is added for selection when the organoid density reaches 80-90%.

[0105] Multiplexed imaging of biosensors in barcoded cells. Cells separately transduced with biosensors and barcodes composed of a pair of barcoding proteins, which are blue or red FPs targeted to different subcellular locations. With appropriately chosen excitation wavelengths and emission ranges, the spectra of barcoding proteins can be separated from those of the cyan, green, or yellow FPs (450-550 nm emission) used in the majority of biosensor designs (FIGS.3A-3C). For biosensors not in this emission range, many of them can be reconstructed by replacing single fluorophores with GFP or FRET pairs with CFP and YFP. Therefore our strategy is compatible with most biosensors. Barcoded cells expressing different biosensors are mixed together before imaging on a fluorescence microscope equipped with a spectral detector. At the start of the experiment, spectral images of the barcodes are acquired, followed by time-lapse imaging of biosensors (FIG.3D, E). The identity of the biosensor in each cell is inferred from the barcode, which can be identified using machine learning models (see below). The signals from cellsAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) expressing the same biosensors can then be pooled together for further analysis (FIG.3F).

[0106] 2. Calibration of FRET biosensors

[0107] Generation of of calibration standards. We generated two calibration constructs that lock the CFP-YFP pairs in the FRET (FREF-ON) or non-FRET (FRET-OFF) configurations (FIG.4A). The FRET-ON construct was based on the Cyto-FAK biosensor, which responds to FAK activation with loss of FRET. The tyrosine in the FAK substrate peptide was mutated to phenylalanine to render the construct insensitive to FAK and stay at the FRET state. The FRET-OFF construct was based on the H3K9me3(W45A) biosensor, which was used as a non-FRET control, with Histone H3 removed from the construct.

[0108] Calibration of FRET biosensors. Calibration standards are expressed in barcoded cells using lentiviral vectors and mixed with barcoded cells expressing biosensors for simultaneous imaging (FIG.4B). The YFP / CFP ratio from cells expressing various biosensors (identified by their barcodes) is calibrated using the following formula such that FRET-OFF and FRET-ON have a ratio of 0 and 1, respectively (FIG.4C):

[0109] As a demonstration, in the example below, we mixed cells expressing four FRET constructs: FRET-ON, FRET-OFF, Cyto-FAK, and EKAR (FIG.5A) and imaged the cells under two different laser power and three different detector gains (FIG.5B). The fluorescence intensity of CFP and YFP varied widely between different imaging settings (FIG.5C). Under the same imaging setting, YFP / CFP ratios remained relatively unaffected by the expression level (indicated by YFP fluorescence) for individual FRET constructs (FIG.5D). However, the ratios between different imaging settings showed significant variation (FIG.5E). We then calibrated the YPF / CFP ratios using the following formula (1). Remarkably, the YFP / CFP ratios for Cyto-FAK and EKAR became uniform across the imaging settings after calibration (FIG.5F). Note that the EKAR contains different CFP and YFP variantsAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) from the standards. These results suggest that the FRET-OFF and FRET-ON can be used to calibrate CFP-YFP FRET biosensor in different imaging experiments regardless of the CFP and YFP variants used in the biosensor design.

[0110] 3. Calibration of single FP biosensors

[0111] Generation of of biosensor-dual-intein (DI)-CFP and calibration standard constructs. The biosensor-DI-CFP construct is created by fusing any GFP-based biosensor with a CFP-NES flanking two mini-inteins SspDNAE(N159A)and SspDnaB(C1A)

[0016] , which undergoes self-cleavage to release the biosensor and cytoplasm-targeted CFP-NES (i.e. nuclear export signal fused to CFP) (FIG.6A). This system ensures equimolar expression of GFP / biosensor and CFP, allowing for the use of the GFP / CFP ratio as a readout for both translocation and intensity based biosensors (FIG.6B). The calibration standard GFP-DI-CFP is created by fusing GFP-NES with a CFP-NES flanking the two mini-inteins (FIG.6A).

[0112] Calibration of single FP biosensors. The biosensor-DI-CFP and GFP-DI- CFP standard constructs are expressed in barcoded cells using lentiviral vectors and mixed for simultaneous imaging (FIG.6C). To simplify the imaging protocol, GFP and CFP are imaged in the same setting as with the FRET-based biosensors due to the spectral overlapping between GFP and YFP. The YFP / CFP ratio from cells expressing various biosensors (identified by their barcodes) is calibrated using the following formula:

[0113] 4. Image analysis of barcode and biosensors in different cell lines and organoids

[0114] Barcode identification in 2D cells: We have developed deep learning models to analyze the barcode images. Prior to barcode analysis, three deep learning models are trained on cells expressing BFP images (Model 1), red FP images combined with nuclear BFP (Model 2), and red FP images combined with plasma membrane BFP (Model 3). To analyze the barcodes from imaging experiments, the BFP images are first classified by Model 1. For the red barcoding proteins, spectral profiles are used to determine which of the three unmixedAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) channels corresponded to the correct FP. The spectral images of red FPs are unmixed using the reference spectra of mCherry, mCardinal, and iRFP702 in the ZEN software. The unmixed red FP images are then combined with BFP images and classified by Model 2 or Model 3 depending on whether the BFP signal was in the nucleus or membrane. The output is filtered by a certainty threshold and examined for the existence of the barcode. (FIG.7A) Using the workflow, we achieved an overall accuracy of 99% in barcode identification in HeLa cells (FIG.7B). When these models were used to classify images of barcoded MCF7 and U2OS cells, which have very different morphologies, the accuracy of these decreased significantly (Fig.5C, left), but new models can be readily trained using images of these cells expressing known barcodes to achieve >99% accuracy (FIG.7C, right).

[0115] Barcode identification in 3D organoids: Following the approach established in 2D cells, we will train deep learning models for automated identification of barcoded organoids. To create datasets for model training and testing, we will manually segment processed images of organoids expressing mCherry targeted to one of four aforementioned subcellular locations in ImageJ (FIG.8A). The images will be used to train deep convolutional neural network models using Keras with the TensorFlow backend. The models will be trained for ~100 epochs, selected according to validation loss (FIG.8B), and subsequently tested on the testing set. Various threshold levels of the deep learning models will be tested to determine the optimal level that achieves over 99% accuracy and high identification rate. The final model is used for barcode identification in organoids (i.e. Model 1 in FIG.8C).

[0116] To analyze the images of barcoded organoids, the identity of the red FP (mCherry, mCardinal, or iRFP702) is first determined by spectral analysis. The images of BFP and red FPs are then separately classified using a single deep learning model (Model 1) to determine their subcellular localization (FIG.8C). Note that the workflow is simpler than that in 2D cells (FIG.7A) since the classification is facilitated by the presence of dozens of identically barcoded cells in each organoid. In contrast, barcode identification in 2D cells is performed on individual cells that express different barcodes. The wide variation in morphology and brightness in those cells require a more stringent analysis procedure.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0117] Quantification of biosensor activities CFP and YFP Images are processed and analyzed with NIH ImageJ and Fiji. The barcode of each cell, determined as described above, allows for identification of the biosensors and calibration standards expressed in cells. The mean intensity of YFP over the entire cell or organoid is divided by that of CFP. The mean YFP / CFP ratio for cells or organoids expressing biosensors are normalized to the YFP / CFP ratio of FRET-ON and FRET-OFF for FRET-based biosensors according to formula (1), or to the YFP / CFP ratio of GFP- DI-CFP for single FP biosensors according to formula (2). Normalized activities of the same biosensors are then pooled together to calculate the mean and S.D.

[0118] Exemplary Applications

[0119] Our technique enables massively paralleled tracking of biomolecular activities with fluorescent biosensors in real time. There is an ever-growing number of biosensors, with a recent review listing over a thousand biosensor designs for ~200 cellular targets such as proteins, voltage, ions, and metabolites. Therefore, we envision our technique to find broad applications including but not limited to the following:

[0120] 1. Study the dynamics and structure of signaling networks: By simultaneous tracking of 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.

[0121] 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.

[0122] 3. Identify cellular pathways targeted by pharmacological agents: By continuously monitoring different molecular activities, biosensor barcoding can revealAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) the pathways that respond to treatments with chemical agents, obtain the dynamics and dose curves of different responding molecules, and identify off-target effects.

[0123] CONCLUSIONS

[0124] The present disclosure enables the monitoring of numerous biomolecular processes in living cells over extended durations, significantly expanding the range of application of biosensor technology for studying biological and pathological processes, as well as therapeutic development.

[0125] Some further aspects are also defined in the following clauses:

[0126] Clause 1: A method of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors, the method comprising: detecting fluorescent signals emitted by one or more FP biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set, thereby calibrating the fluorescent signals emitted by the FP biosensors.

[0127] Clause 2: The method of Clause 1, wherein the detecting step comprises imaging and / or identifying one or more cells in the cell population that comprise the FP biosensors at one or more time points during a selected duration.

[0128] Clause 3: The method of Clause 1 or Clause 2, wherein the first and second sets of FPs are identical to one another.

[0129] Clause 4: The method of any of Clauses 1-3, wherein at least one synthetic polynucleotide encodes the FP biosensors and / or the fluorescent calibration standard biomolecules and wherein the method comprises expressing the FP biosensors and / or the fluorescent calibration standard biomolecules from the synthetic polynucleotide prior to and / or concurrent with the detecting step.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0130] Clause 5: The method of any of Clauses 1-4, wherein a first synthetic polynucleotide encodes the FP biosensors and wherein a second synthetic polynucleotide encodes the fluorescent calibration standard biomolecules.

[0131] Clause 6: The method of any of Clauses 1-5, wherein an expression cassette comprises the synthetic polynucleotide.

[0132] Clause 7: The method of any of Clauses 1-6, wherein a plasmid comprises the expression cassette.

[0133] Clause 8: The method of any of Clauses 1-7, further comprising introducing the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step.

[0134] Clause 9: The method of any of Clauses 1-8, comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

[0135] Clause 10: The method of any of Clauses 1-9, wherein at least one subset of the cells in the cell population comprises one or more fluorescent barcoding proteins.

[0136] Clause 11: The method of any of Clauses 1-10, wherein the subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells.

[0137] Clause 12: The method of any of Clauses 1-11, wherein the first and second fluorescent labels comprise FPs or functional portions thereof.

[0138] Clause 13: The method of any of Clauses 1-12, wherein at least one synthetic polynucleotide encodes 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.

[0139] Clause 14: The method of any of Clauses 1-13, wherein an expression cassette comprises the synthetic polynucleotide.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02)

[0140] Clause 15: The method of any of Clauses 1-14, wherein a plasmid comprises the expression cassette.

[0141] Clause 16: The method of any of Clauses 1-15, further comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step.

[0142] Clause 17: The method of any of Clauses 1-16, comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

[0143] Clause 18: The method of any of Clauses 1-17, wherein the fluorescent calibration standard biomolecules comprise FPs.

[0144] Clause 19: The method of any of Clauses 1-18, wherein the FP biosensors comprise fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety.

[0145] Clause 20: The method of any of Clauses 1-19, wherein the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety.

[0146] Clause 21: The method of any of Clauses 1-20, wherein one or more subsets of cells in the cell population comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations.

[0147] Clause 22: The method of any of Clauses 1-21, wherein the FP biosensors each comprise only a single FP or a functional portion thereof.

[0148] Clause 23: The method of any of Clauses 1-22, wherein the FP biosensors and the fluorescent calibration standard biomolecules are separately fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked via one or more inteins, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the inteins undergo self- cleavage to release the FP biosensors and the fluorescent calibration standardAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) biomolecules from the third set of FPs prior to and / or concurrent with the detecting step.

[0149] Clause 24: The method of any of Clauses 1-23, wherein the third set of FPs is further operably linked to a cellular targeting moiety.

[0150] Clause 25: The method of any of Clauses 1-24, wherein the fluorescent calibration standard biomolecules are further operably linked to the cellular targeting moiety.

[0151] Clause 26: The method of any of Clauses 1-25, wherein the cellular targeting moiety comprises a nuclear export signal (NES).

[0152] Clause 27: The method of any of Clauses 1-26, wherein at least one synthetic polynucleotide encodes the polypeptide constructs and wherein the method comprises expressing the polypeptide constructs from the synthetic polynucleotide prior to and / or concurrent with the detecting step.

[0153] Clause 28: The method of any of Clauses 1-27, wherein an expression cassette comprises the synthetic polynucleotide.

[0154] Clause 29: The method of any of Clauses 1-28, wherein a plasmid comprises the expression cassette.

[0155] Clause 30: The method of any of Clauses 1-29, further comprising introducing the synthetic polynucleotide into at least a subset of the cells in the cell population prior to the detecting step.

[0156] Clause 31: The method of any of Clauses 1-30, comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

[0157] Clause 32: A method of differentiating cells in a cell population, the method comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and, identifying the fluorescent signals emittedAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) by the first and second fluorescent labels from the different targeting sites in the subset of the cells, thereby differentiating the cells in the cell population.

[0158] Clause 33: The method of Clause 32, wherein an organoid comprises the cell population.

[0159] Clause 34: The method of Clause 32 or Clause 33, wherein a two- dimensional (2D) array comprises the cell population.

[0160] Clause 35: The method of any of Clauses 32-34, wherein different subsets of cells in the cell population comprise different pairs of the fluorescent barcoding proteins, wherein a first pair of fluorescent barcoding proteins comprises a combination of fluorescent labels and / or targeting sites that differs a second pair of fluorescent barcoding proteins.

[0161] Clause 36: The method of any of Clauses 32-35, 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.

[0162] Clause 37: The method of any of Clauses 32-36, further comprising detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors in the cell population.

[0163] Clause 38: The method of any of Clauses 32-37, further comprising detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population.

[0164] Clause 39: A composition, comprising a cell population, wherein at least a first set of cells in the cell population comprises one or more fluorescent protein (FP) biosensors and wherein at least a second set of cells in the cell population comprises one or more fluorescent calibration standard biomolecules, wherein: at least a first set of the FP biosensors comprises fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety; and at least a first set of the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety, and wherein one or more subsets of cells in the second set of cells compriseAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations; and / or, at least a second set of the FP biosensors that each comprise a first set of one or more FPs, or functional portions thereof, wherein the FP biosensors are fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked to the FP biosensors via one or more inteins, and at least a second set of the fluorescent calibration standard biomolecules that each comprise a second set of one or more FPs, or functional portions thereof, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the fluorescent calibration standard biomolecules are fused to polypeptide constructs that each comprise the third set of one or more FPs, or functional portions thereof, operably linked to the fluorescent calibration standard biomolecules via the one or more inteins.

[0165] Clause 40: A kit comprising the composition of Clause 39.

[0166] Clause 41: A kit, comprising: a first synthetic polynucleotide that encodes at least a first set of one or more fluorescent protein (FP) biosensors, or functional portions thereof, and, a second synthetic polynucleotide that encodes at least a first set of one or more fluorescent calibration standard biomolecules, or functional portions thereof.

[0167] Clause 42: The kit of Clause 41, further comprising a third synthetic polynucleotide that encodes at least a first set of one or more fluorescent barcoding proteins, or functional portions thereof.

[0168] Clause 43: A system for calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors, 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 one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one orAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

[0169] Clause 44: The system of Clause 43, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0170] Clause 45: 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 a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and, identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0171] Clause 46: The system of Clause 45, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in the cell population to produce a detected FP biosensor signal data set and a detected calibration standardAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

[0172] Clause 47: 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 one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

[0173] Clause 48: The computer readable media of Clause 47, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0174] Clause 49: 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 a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member ofAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and, identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

[0175] Clause 50: The computer readable media of Clause 49, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in the cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

[0176] 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.

[0177] 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) 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”)Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 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.

[0178] 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.0184.0267-PCT (C17746_P17746-02) Claims:

1. A method of calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors, the method comprising: detecting fluorescent signals emitted by one or more FP biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set, thereby calibrating the fluorescent signals emitted by the FP biosensors.

2. 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 FP biosensors at one or more time points during a selected duration.

3. The method of claim 1, wherein the first and second sets of FPs are identical to one another.

4. The method of claim 1, wherein at least one synthetic polynucleotide encodes the FP biosensors and / or the fluorescent calibration standard biomolecules and wherein the method comprises expressing the FP biosensors and / or the fluorescent calibration standard biomolecules from the synthetic polynucleotide prior to and / or concurrent with the detecting step.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 5. The method of claim 4, wherein a first synthetic polynucleotide encodes the FP biosensors and wherein a second synthetic polynucleotide encodes the fluorescent calibration standard biomolecules.

6. The method of claim 5, wherein an expression cassette comprises the synthetic polynucleotide.

7. The method of claim 6, wherein a plasmid comprises the expression cassette.

8. The method of claim 4, further comprising introducing the synthetic polynucleotide into at least a subset of cells in the cell population prior to the detecting step.

9. The method of claim 8, comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

10. The method of claim 1, wherein at least one subset of the cells in the cell population comprises one or more fluorescent barcoding proteins.

11. The method of claim 10, wherein the subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells.

12. The method of claim 11, wherein the first and second fluorescent labels comprise FPs or functional portions thereof.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 13. The method of claim 11, wherein at least one synthetic polynucleotide encodes 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.

14. The method of claim 13, wherein an expression cassette comprises the synthetic polynucleotide.

15. The method of claim 14, wherein a plasmid comprises the expression cassette.

16. The method of claim 13, further comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step.

17. The method of claim 16, comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

18. The method of claim 1, wherein the fluorescent calibration standard biomolecules comprise FPs.

19. The method of claim 1, wherein the FP biosensors comprise fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety.

20. The method of claim 19, wherein the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 21. The method of claim 20, wherein one or more subsets of cells in the cell population comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations.

22. The method of claim 1, wherein the FP biosensors each comprise only a single FP or a functional portion thereof.

23. The method of claim 22, wherein the FP biosensors and the fluorescent calibration standard biomolecules are separately fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked via one or more inteins, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the inteins undergo self-cleavage to release the FP biosensors and the fluorescent calibration standard biomolecules from the third set of FPs prior to and / or concurrent with the detecting step.

24. The method of claim 23, wherein the third set of FPs is further operably linked to a cellular targeting moiety.

25. The method of claim 23, wherein the fluorescent calibration standard biomolecules are further operably linked to the cellular targeting moiety.

26. The method of claim 25, wherein the cellular targeting moiety comprises a nuclear export signal (NES).

27. The method of claim 23, wherein at least one synthetic polynucleotide encodes the polypeptide constructs and wherein the method comprises expressing the polypeptide constructs from the synthetic polynucleotide prior to and / or concurrent with the detecting step.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 28. The method of claim 27, wherein an expression cassette comprises the synthetic polynucleotide.

29. The method of claim 28, wherein a plasmid comprises the expression cassette.

30. The method of claim 27, further comprising introducing the synthetic polynucleotide into at least a subset of the cells in the cell population prior to the detecting step.

31. The method of claim 30, comprising introducing the synthetic polynucleotide into the subset of the cells in the cell population prior to the detecting step using at least one lentiviral vector.

32. A method of differentiating cells in a cell population, the method comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and, identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells, thereby differentiating the cells in the cell population.

33. The method of claim 32, wherein an organoid comprises the cell population.Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 34. The method of claim 32, wherein a two-dimensional (2D) array comprises the cell population.

35. The method of claim 32, wherein different subsets of cells in the cell population comprise different pairs of the fluorescent barcoding proteins, wherein a first pair of fluorescent barcoding proteins comprises a combination of fluorescent labels and / or targeting sites that differs a second pair of fluorescent barcoding proteins.

36. The method of claim 32, 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.

37. The method of claim 32, further comprising detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors in the cell population.

38. The method of claim 32, further comprising detecting fluorescent signals emitted by one or more fluorescent calibration standard biomolecules in the cell population.

39. A composition, comprising a cell population, wherein at least a first set of cells in the cell population comprises one or more fluorescent protein (FP) biosensors and wherein at least a second set of cells in the cell population comprises one or more fluorescent calibration standard biomolecules, wherein: at least a first set of the FP biosensors comprises fluorescence resonance energy transfer (FRET) FP biosensors that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety; and at least a first set of the fluorescent calibration standard biomolecules comprise FRET calibration standard biomolecules that each comprise at least one FRET acceptor moiety and at least one FRET donor moiety, and wherein one orAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) more subsets of cells in the second set of cells comprise FRET calibration standard biomolecules having locked FRET-ON conformations and / or FRET calibration standard biomolecules having locked FRET-OFF conformations; and / or, at least a second set of the FP biosensors that each comprise a first set of one or more FPs, or functional portions thereof, wherein the FP biosensors are fused to polypeptide constructs that each comprise a third set of one or more FPs, or functional portions thereof, operably linked to the FP biosensors via one or more inteins, and at least a second set of the fluorescent calibration standard biomolecules that each comprise a second set of one or more FPs, or functional portions thereof, wherein the first and second sets of FPs are identical to one another, wherein the third set of FPs is different from the first and second sets of FPs, and wherein the fluorescent calibration standard biomolecules are fused to polypeptide constructs that each comprise the third set of one or more FPs, or functional portions thereof, operably linked to the fluorescent calibration standard biomolecules via the one or more inteins.

40. A kit comprising the composition of claim 39.

41. A kit, comprising: a first synthetic polynucleotide that encodes at least a first set of one or more fluorescent protein (FP) biosensors, or functional portions thereof, and, a second synthetic polynucleotide that encodes at least a first set of one or more fluorescent calibration standard biomolecules, or functional portions thereof.

42. The kit of claim 41, further comprising a third synthetic polynucleotide that encodes at least a first set of one or more fluorescent barcoding proteins, or functional portions thereof.

43. A system for calibrating fluorescent signals emitted by fluorescent protein (FP) biosensors, comprising:Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) 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 one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

44. The system of claim 43, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

45. 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 a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise aAtty Dkt. No.0184.0267-PCT (C17746_P17746-02) pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and, identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

46. The system of claim 45, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in the cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

47. 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 one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in a cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and,Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

48. The computer readable media of claim 47, wherein the instructions which, when executed on the processor, further perform operations comprising: detecting fluorescent signals emitted by a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

49. 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 a set of fluorescent barcoding proteins in the cell population, wherein at least a subset of the cells each comprise a pair of the fluorescent barcoding proteins, wherein a first member of the pair comprises a first fluorescent label, wherein a second member of the pair comprises a second fluorescent label that differs from the first fluorescent label, and wherein the first and second members are located at different targeting sites in a given cell of the subset of the cells; and, identifying the fluorescent signals emitted by the first and second fluorescent labels from the different targeting sites in the subset of the cells.

50. The computer readable media of claim 49, wherein the instructions which, when executed on the processor, further perform operations comprising:Atty Dkt. No.0184.0267-PCT (C17746_P17746-02) detecting fluorescent signals emitted by one or more fluorescent protein (FP) biosensors and one or more fluorescent calibration standard biomolecules in the cell population to produce a detected FP biosensor signal data set and a detected calibration standard signal data set, wherein the FP biosensors comprise at least a first set of one or more FPs, or functional portions thereof, and wherein the calibration standard biomolecules comprise at least a second set of one or more FPs, or functional portions thereof; and, normalizing the detected FP biosensor signal data set to the detected calibration standard signal data set.

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