Compositions and methods for analyzing cells in mammalian tissues

Abstract

The present application relates to, in certain aspects, methods and compositions for imaging mammalian brain tissue via detection of multiple different epitope tags delivered via a plurality of vectors. Certain aspects provided herein relate to binder sets for the multiple different epitope tags, and resulting compositions.

Description

COMPOSITIONS AND METHODS FOR ANALYZING CELLS IN MAMMALIAN TISSUESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 598,780, filed on November 14, 2023, entitled “Compositions and Methods for Analyzing Cells in Mammalian Tissues,” which is herein incorporated by reference in its entirety for all purposes.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (314342000140SEQLIST.xml; Size: 65,783 bytes; and Date of Creation: November 5, 2024) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure in some aspects relate to compositions and methods for analyzing cells in a mammalian neural tissue, such as for detecting connectivity between cells in a brain tissue sample.BACKGROUND

[0004] US Patent Application No. 2019 / 0071,666 (Zador et al) discloses “a composition comprising a plurality of labeled neurons, each of which is labeled by an expression construct that encodes a unique barcoded nucleic acid”.

[0005] US Patent Application No. 2020 / 0299340 (Brown et al.,) discloses “a fusion protein comprising a scaffold protein and a series of two or more epitopes, where the distinct epitopes are recognized by distinct antibodies, and where the series of epitopes forms a detectable protein tag.”

[0006] Shen et al., Nature Communications (2020) 11 :4632 | https: / / doi.org / 10.1038 / s41467- 020-18422-8 states “we devise a light microscopy approach for connectivity analysis of defined cell types called spectral connectomics. We combine multicolor labeling (Brainbow) of neurons with multi-round immunostaining Expansion Microscopy (miriEx) to simultaneously interrogate morphology, molecular markers, and connectivity in the same brain section.”

[0007] An et al., Society for Neuroscience 2022 Poster discloses development progress towards “a scalable set of protein epitopes that can be safely expressed in combinations in neurons, so that each cell gets a unique combination of epitopes, and can be distinguished during serial staining, imaging, and washing steps.”BRIEF SUMMARY

[0008] The present disclosure is based, at least in part, on discoveries and findings regarding methods for imaging brain circuit connectivity mapping in mammals that reduce from high error rates and large volume acquisition requirements of many previous methods.

[0009] In some aspects and embodiments of the disclosure a composition, comprising a plurality of different fusion proteins is provided, wherein each fusion protein in the plurality of fusion proteins independently comprises: (a) a fluorescent protein comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 40-43; and (b)one or more peptide epitopes comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31; wherein the composition in total comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all of the peptide epitopes of SEQ ID NO: 1-31.

[0010] In certain aspects and embodiments of the disclosure, provided is a composition comprising a plurality of antibodies, wherein the plurality of antibodies comprises antibodies that in total selectively bind to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more peptide epitopes comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-31.

[0011] In some aspects a composition is provided wherein the composition includes a plurality of viral particles, wherein the plurality of viral particles comprise plurality of nucleic acids encoding different fusion proteins as disclosed elsewhere herein.

[0012] In one aspect, a method for detecting connectivity between cells in a brain tissue sample is provided wherein the method comprises: (a) expressing the protein composition and / or the nucleic acid composition of any embodiment in a brain tissue sample; (b) contacting the brain tissue sample with:• (i) the antibody composition of any embodiment herein under conditions to promote binding of the antibodies to the peptide epitopes to form detectable antibody-epitope complexes; and• (ii) antibodies selective for synaptic markers under conditions to promote binding of the antibodies to the synaptic markers to form detectable antibody-synaptic marker complexes;(c) obtaining images of the detectable antibody-epitope complexes and the detectable synaptic markers in the brain sample; and (d) analyzing the images to identify connectivity between cells in the brain tissue sample.

[0013] In some aspects, provided herein is a method for analyzing a mammalian tissue, comprising: (a) contacting the mammalian tissue with a plurality of different vectors for expressing multiple different epitope tags in cells of the mammalian tissue, wherein each different vector encodes a different epitope tag, and wherein two or more cells in the mammalian tissue each expresses a different combination of different epitope tags; (b) contacting the mammalian tissue with a first plurality of binders recognizing a first subset of the multiple different epitope tags; (c) detecting first signals associated with the first plurality of binders in the mammalian tissue; (d) contacting the mammalian tissue with a second plurality of binders recognizing a second subset of the multiple different epitope tags which is different from the first subset; (e) detecting second signals associated with the second plurality of binders in the mammalian tissue; and (f) generating a codeword for each of the two or more cells in the mammalian tissue, wherein the codeword comprises signal codes corresponding to the presence or absence of the first signals and signal codes corresponding to the presence or absence of the second signals, and the codeword for a particular cell corresponding to the combination of different epitope tags expressed in the cell.

[0014] In some embodiments, the mammalian tissue is a neural tissue, and the each of two or more cells is independently selected from the group consisting of a neuron, an oligodendrocyte, an astrocyte, an ependymal cell, a microglia, a Schwann cell, and a satellite cell. In some embodiments, the mammalian tissue is a brain tissue or a spinal cord tissue, and the two or more cells are neurons. In some embodiments, i)the mammalian tissue is a cell culture comprising cultured neurons, optionally wherein the cell culture is a patient derived cell culture; ii) the mammalian tissue is a cultured tissue, optionally wherein the cultured tissue is a cultured brain tissue; or iii) the mammalian tissue is in a live mammalian individual and the plurality of different vectors are introduced into the individual to contact with the mammalian tissue. In some embodiments, the plurality of different vectors are viral vectors.

[0015] In some embodiments, the plurality of different vectors are AAV vectors. In some embodiments, the plurality of different vectors comprises stoichiometric ratios of the different vectors or non-stoichiometric ratios of the different vectors. In some embodiments, the plurality of different vectors comprises more than 10 different vectors. In some embodiments, the plurality of different vectors comprises about 30 different vectors. In some embodiments, the plurality of different vectors comprises about 100 different vectors.

[0016] In some embodiments, the multiple different epitope tags comprise more than 10 different epitope tags. In some embodiments, the multiple different epitope tags comprise about 30 different epitope tags. In some embodiments, the multiple different epitope tags compriseabout 50 different epitope tags. In some embodiments, the multiple different epitope tags comprise peptide tags. In some embodiments, the multiple different epitope tags are each between about 6 and about 30 amino acid residues in length. In some embodiments, each different vector encodes a different fusion protein comprising the epitope tag linked to a scaffold protein.

[0017] In some embodiments, the scaffold protein is common among the different fusion proteins encoded by the plurality of different vectors, or wherein the scaffold protein is different among the different fusion proteins encoded by two or more different vectors of the plurality of different vectors. In some embodiments, the scaffold protein is a fluorescent protein, optionally wherein the scaffold protein is an eGFP, a mNeonGreen, mGreenLantem, or a momomeric GFP.

[0018] In some embodiments, each different vector encodes a different fusion protein comprising the epitope tag linked to a localization domain, optionally wherein the localization domain is common among the different fusion proteins encoded by the plurality of different vectors, or optionally wherein the localization domain is different among the different fusion proteins encoded by two or more different vectors of the plurality of different vectors. In some embodiments, each different vector comprises a promoter operably linked to a sequence encoding the epitope tag and / or scaffold protein. In some embodiments, the promoter is a CAG promoter or a Sindbis virus subgenomic promoter.

[0019] In some embodiments, the mammalian tissue is the brain of an mammalian individual, wherein: each different epitope tag is expressed randomly in about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the neurons labeled by the different epitope tags, or each different epitope tag is expressed randomly in over 50% of the neurons labeled by the different epitope tags, optionally wherein each different epitope tag is expressed randomly in about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the neurons labeled by the different epitope tags. In some embodiments, at least or about 10, at least or about 100, at least or about 103, at least or about 104, at least or about 105, at least or about 106, at least or about 107, at least or about 108, at least or about 109, at least or about 1010, or at least or about 1011 neurons in the mammalian tissue each expresses a unique combination of different epitope tags.

[0020] In some embodiments, a method of any of the above embodiments is provided herein, comprising: contacting the mammalian tissue with a third plurality of binders recognizing a third subset of the multiple different epitope tags; detecting third signals associated with the third plurality of binders in the mammalian tissue, wherein the codeword further comprises signalcodes corresponding to the presence or absence of the third signals. In some embodiments, the method of the above is provided herein, comprising: contacting the mammalian tissue with a fourth plurality of binders recognizing a fourth subset of the multiple different epitope tags; detecting fourth signals associated with the fourth plurality of binders in the mammalian tissue, wherein the codeword further comprises signal codes corresponding to the presence or absence of the fourth signals. In some embodiments, the method of the above is provided herein, comprising: contacting the mammalian tissue with a fifth plurality of binders recognizing a fifth subset of the multiple different epitope tags; detecting fifth signals associated with the fifth plurality of binders in the mammalian tissue, wherein the codeword further comprises signal codes corresponding to the presence or absence of the fifth signals.

[0021] In some embodiments, each plurality of binders comprises binders recognizing two, three, four, five, or more different epitope tags. In some embodiments, in each cycle of binder recognition and signal detection, a signal associated with each different epitope tag detected at a particular neuron, or the absence of the signal at the neuron, is recorded as a signal code at a bit in the codeword for the neuron. In some embodiments, the method comprises two, three, four, five, or more cycles of binder recognition and signal detection, and in each cycle the plurality of binders comprises two, three, four, five, or more different binders each recognizing a different epitope tag of the multiple different epitope tags. In some embodiments, the method comprises five cycles of binder recognition and signal detection, and in each cycle the plurality of binders comprises three different binders each recognizing a different epitope tag of the multiple different epitope tags. In some embodiments, in a particular cycle of binder recognition and signal detection, each different binder is detected in a different channel of fluorescent microscopy. In some embodiments, the codeword for the neuron is between 2 and 100 bits, optionally wherein the codeword for the neuron is 15 bits, 30 bits, 50 bits, or 100 bits. In some embodiments, prior to a particular cycle of binder recognition and signal detection, the method comprises a step of removing the plurality of binders of a previous cycle from the mammalian tissue, and / or extinguishing signals associated with the plurality of binders of the previous cycle. In some embodiments, each plurality of binders comprises primary antibodies or epitope-binding fragments thereof that bind to the epitope tags, optionally wherein the primary antibodies or epitope-binding fragments thereof are detectably labeled. In some embodiments, each plurality of binders further comprises secondary antibodies or epitope-binding fragments thereof that bind to the primary antibodies or epitope-binding fragments thereof, optionally wherein the secondary antibodies or epitope-binding fragments thereof are detectably labeled. In some embodiments, each of the primary antibodies or epitope-binding fragments thereof or the secondary antibodiesor epitope-binding fragments thereof is conjugated to a nucleic acid tag, optionally wherein the nucleic acid tag comprises one or more barcode sequences.

[0022] In some embodiments, the mammalian tissue is a brain tissue and the method comprises detecting a pre-synaptic marker, a post-synaptic marker, and / or a neurotransmitter marker in the brain tissue. In some embodiments, the pre-synaptic marker is selected from the group consisting of piccolo, bassoon, CASK, one or more SNARE types, SNAP25, VAMP, and syntaxin. In some embodiments, the post-synaptic marker is selected from the group consisting of Homer, post-synaptic density-95 (PSD95), neuroligin, SAP 102, SAPAP, SHANK, and calcium -dep endent protein kinase II. In some embodiments, the neurotransmitter marker is selected from the group consisting of a marker for glutamatergic transmission, a marker for GABAergic transmission, a marker for dopaminergic transmission, a marker for cholinergic transmission, and a marker for serotonergic transmission. In some embodiments, the neurotransmitter marker is selected from the group consisting of VGAT, GABRA1, gephyrin, NMDA-1, and vGluTl. In some embodiments, the mammalian tissue is expanded or not expanded. In some embodiments, the mammalian tissue is a brain tissue and the method comprises generating a plurality of different codewords at cellular structures in the mammalian tissue. In some embodiments, a method provided herein comprises identifying two or more cellular structures having the same codeword as belonging to the same neuron. In some embodiments, the method comprises identifying two or more cellular structures each having a different codeword as belonging to different neurons. In some embodiments, the cellular structures are selected from the group consisting of a nucleus or a portion thereof, a cell body or a portion thereof, an axon or a portion thereof, and a dendrite or a portion thereof. In some embodiments, the method comprises embedding the brain tissue in a swellable polymer matrix and expanding the swellable polymer matrix and the brain tissue embedded therein.

[0023] In some aspects, provided herein is a composition comprising a plurality of different vectors at stoichiometric ratios, wherein each different vector encodes a fusion protein comprising a different epitope tag linked to a scaffold protein, and wherein the plurality of different vectors are configured to express the fusion proteins in cells of a mammalian tissue. In some embodiments, the plurality of different vectors are viral vectors. In some embodiments, the plurality of different vectors are AAV vectors or sindbis virus vectors. In some embodiments, the composition comprises more than 10 different vectors each expressing a fusion protein comprising a different epitope tag, and two or more or all of the different epitope tags are linked to the same scaffold protein.

[0024] In some embodiments, the composition comprises about 30 different vectors each expressing a fusion protein comprising a different epitope tag linked to a common scaffold protein. In some embodiments, the different epitope tags are peptide tags between about 6 and about 30 amino acid residues in length. In some embodiments, the scaffold protein is a fluorescent protein. In some aspects, provided herein are a plurality of different fusion proteins each comprising a different peptide tag linked to a common scaffold protein, wherein the different peptide tags are between about 6 and about 30 amino acid residues in length and the common scaffold protein is a fluorescent protein. In some embodiments, the plurality of different fusion proteins comprises more than 10 different fusion proteins each comprising a different peptide tag. In some embodiments, the plurality of different fusion proteins comprises about 30 different fusion proteins each comprising a different peptide tag.

[0025] In some aspects, provided herein is a mammalian tissue comprising a plurality of different vectors in contact with cells of the mammalian tissue, wherein each different vector encodes a fusion protein comprising a different epitope tag linked to a scaffold protein, and wherein the plurality of different vectors are configured to express the fusion proteins in cells of the mammalian tissue. In some embodiments, the mammalian tissue comprises more than 10 different vectors each encoding a different peptide tag. In some embodiments, the mammalian tissue comprises about 30 different vectors each encoding a different peptide tag. In some embodiments, provided herein is a mammalian tissue comprising a plurality of different fusion proteins expressed in cells of the mammalian tissue, wherein each different fusion protein comprises a different peptide tag linked to a scaffold protein, and wherein two or more cells in the mammalian tissue each expresses a different combination of peptide tags selected from the plurality of different fusion proteins. In some embodiments, the mammalian tissue is a brain tissue, and: each different peptide tag of the plurality of different fusion proteins is expressed randomly in about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the neurons labeled by the different peptide tags, or each different peptide tag of the plurality of different fusion proteins is expressed randomly in over 50% of the neurons labeled by the different peptide tags, optionally wherein each different peptide tag is expressed randomly in about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the neurons labeled by the different peptide tags. In some embodiments, the mammalian tissue comprises more than 10 different fusion proteins each comprising a different peptide tag linked to a common scaffold protein expressed in cells of the mammalian tissue. In some embodiments, themammalian tissue comprises about 30 different fusion proteins each comprising a different peptide tag linked to a common scaffold protein expressed in cells of the mammalian tissue.

[0026] In some aspects, provided herein is a set of binders, comprising: i) a first plurality of binders recognizing a first subset of multiple different epitope tags, wherein each binder in the first plurality is configured to be detected in a different channel of fluorescent microscopy, and ii) a second plurality of binders recognizing a second subset of the multiple different epitope tags, wherein the second subset is different from the first subset, and wherein each binder in the second plurality is configured to be detected in a different channel of fluorescent microscopy, wherein the multiple different epitope tags are peptide tags between about 6 and about 30 amino acid residues in length. In some embodiments, the set of binders further comprises: iii) a third plurality of binders recognizing a third subset of the multiple different epitope tags, wherein each binder in the third plurality is configured to be detected in a different channel of fluorescent microscopy, iv) a fourth plurality of binders recognizing a fourth subset of the multiple different epitope tags, wherein the second subset is different from the first subset, and wherein each binder in the fourth plurality is configured to be detected in a different channel of fluorescent microscopy, and v) a fifth plurality of binders recognizing a fifth subset of multiple different epitope tags, wherein each binder in the fifth plurality is configured to be detected in a different channel of fluorescent microscopy. In some embodiments, each subset comprises two, three, four, five, or more different epitope tags and is nonoverlapping with another subset. In some embodiments, each binder is an antibody or epitope binding fragment thereof.

[0027] In some aspects, provided herein is a mammalian tissue comprising a plurality of about 30 different fusion proteins expressed in cells of the mammalian tissue, wherein each of the plurality of fusion proteins comprises a different epitope tag linked to a common scaffold protein, wherein two or more cells in the mammalian tissue each expresses a different combination of epitope tags selected from the plurality of different fusion proteins, wherein the mammalian tissue is a brain tissue and each different epitope tag of the plurality of different fusion proteins is expressed randomly in about 50% of the neurons labeled by the different epitope tags, and wherein the mammalian tissue is in contact with a plurality of binders recognizing a subset of the different epitope tags, wherein each binder in the plurality is configured to be detected in a different channel of fluorescent microscopy.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows an exemplary workflow of the invention.

[0029] FIG. 2 shows an example workflow for protein barcode synthesis and delivery, and protein barcode detection using antibodies.

[0030] FIG. 3 shows a workflow wherein samples are embedded in a swellable hydrogel and expanded to improve resolution.

[0031] FIGs. 4A-4B show probability distributions for pooled short epitope segments in AAVs. FIG. 4A shows a comparison of the probability of each epitope in the plasmid pools along with the final probability of each epitope in the final AAV pool. FIG. 4B shows the probability of each epitope when each epitope is packaged separately into AAVs and pooled afterwards at putatively equal titers. Both probability distributions were acceptably balanced (e.g., no epitope was completely dominant or completely absent) and putative barcoding diversity remained high regardless of pooling and synthesis strategy.

[0032] FIG. 5 shows quantification of eGFP+ and epitope-labeled cells in a sample using Tyl, in a pool of 24 total epitopes with approximately balanced distribution. Approximately 500 eGFP+ cell bodies were counted, 42% of which were positive for Tyl.

[0033] FIG. 6 shows representative image samples taken from an assay in samples with predicted low MOIs, showing that epitope distribution in the sample broadly reflects expectations based on NGS results.

[0034] FIG. 7 shows an assay measuring the presence or absence of individual barcode bits in tertiary axons of the superior colliculus of mice injected in the motor cortex with the barcoding pools.

[0035] FIG. 8A shows a brightfield image showing the anatomical location of the superior colliculus and region of interest. FIG. 8B shows eGFP, a pan-barcode tracer, in many axons. FIG. 8C shows a subsampled stain of Suntag, an epitope tag, brightly visible in a subset of axons.

[0036] FIGs. 9A-9E show labeling of neurons via an injection in the motor cortex. FIG. 9A shows a schematic of the experiment (top), with an injection site of the motor cortex and a readout assessed from the superior colliculus. The motor cortex is imaged at a single barcode bit (positive control, bottom), showing signal. FIG. 9B shows imaging of the signal in the superior colliculus (top) and examples of individual putative synapses, labeled with arrowheads, that are filled with barcode protein. FIG. 9C shows a validation assay that was performed independently for each of the 15 barcode bits. FIG. 9D shows the validation of four additional epitope tags in mouse brain. FIG. 9E shows frequency distribution of the 19 epitope tags in a distal region ~4mm from the injection site (n = 242 axons).

[0037] FIG. 10A shows an example of a single channel and cycle of imaging for a single barcode “bit”, the ALFA tag. FIG. 10B shows a merge of 15 channels collected across 5 imaging cycles. FIG. 10C shows a set of all imaging channels split out over each iterative imaging cycle.

[0038] FIG. 11A shows a single imaging channel in the region of interest, with the cell body denoted. FIG. 11B shows the region of interest across 15 imaging cycles, with annotation (1 = stain present, 0 = stain absent).

[0039] FIG. 12A shows all somas at imaged volume of barcode injection site (motor cortex), registered and segmented. FIG. 12B shows bit barcode vector (raw data) corresponding to each cell in the volume. FIG. 12C shows a codebook of barcodes in volume following annotation.

[0040] FIGs. 13A-13C show identification of synapses. FIG. 13A shows positive identification of synapses shown by colocalization of bassoon and shank2 with a dendritic spine labeled with an epitope tag. FIG. 13B shows examples of individual synapses resolved in MAGNIFY. FIG. 13C show barcoding in distal synapses ~3mm from injection site, putatively in the superior colliculus, as identified by synaptophysin.

[0041] FIG. 14 shows the generation of a library in which, in a first iteration, oligo pools incorporating 3 epitopes per construct and a unique DNA barcode per oligo were used. Oligo pools were cloned as a library into pSin-GFP, a constitutive sindbis virus plasmid, to generate a library of a putative 2,300 short epitopes.

[0042] FIG. 15 shows assaying of the diversity of the pool via NGS analysis of DNA barcodes to profile the frequency distributions. A reasonably even distribution (i.e., with no barcodes completely dominating sequencing results) was found.

[0043] FIG. 16 shows GFP in green and 2 epitopes stained in red and far red. Epitopes show a distribution across all labeled cells, indicating unique combinations can be generated.

[0044] FIG. 17 shows eGFP containing 29 short epitope sequences in a Flp-dependent system driven by a Tet response element.

[0045] FIGs. 18A-18B show reading out four bits in a gel-stabilized slice of tissue. FIG. 18A shows a 4-color image of epitopes overlaid. Different colors broadly represent different combinations of epitopes or differentials in brightness. FIG. 18B shows individual channels for the epitope tags VSV, Tag 100, NWS, and OLLAS (clockwise from top left).

[0046] FIG. 19 shows the frequency distribution of epitopes in cells in two biological conditions. Demonstration of pooled labeling and the presence of up to 26 epitope bits in the same sample.

[0047] FIG. 20 shows detection of 20 peptide tags in a single non-expanded tissue sample of the motor cortex.

[0048] FIG. 21 shows epitope distribution in a site 4mm distal from the injection site.

[0049] FIGs. 22A-22B show characterization of the sensitivity of the Cre-dependent systems to recombinases, while demonstrating stable barcode lengths across multiple density labels.

[0050] FIGs. 23A-23B show epitope expression and frequency distribution from a sindbis virus using a GFP scaffold and a 6-bit shuffled assembly at the C terminal.

[0051] FIGs. 24A-24B show epitope expression and frequency distribution from an AAV virus using an mNeonGreen scaffold and a 3 -bit shuffled assembly at the C terminal. This reduces the number of infections required to generate long barcodes, and increases the overall length of barcodes.

[0052] FIGs. 25A-25B show detection of cytosolic and mitochondrially-targeted barcodes in the same sample. FIG. 25A shows a merged image. FIG. 25B shows the split channel images. This demonstrates effective localization of a COX8 motif in neurons that is distinguishable from cytosolically expressed versions of the epitopes in cell bodies and local regions, and effectively doubles the bit information per round of imaging.

[0053] FIGs. 26A-26B show detection of synaptically-targeted scaffold in primary motor cortex (MOp; FIG. 26A) and superior colliculus (FIG. 26B). This demonstrates that a synaptically targeted epitope trafficks and is detectable at pre-synapses >4mm away from the target region, potentially reducing the resolution requirements required for synaptic assignment and increasing trafficking.DETAILED DESCRIPTION

[0054] All references cited are herein incorporated by reference in their entirety. All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0055] Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319-322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0056] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0057] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0058] Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted.

[0059] As used herein, an ‘antibody” may comprise a full length antibody or an antigen binding fragment thereof. Fragments with antigen-binding activity include, but are not limited to, Fab', F(ab')2, Fab, Fv and rlgG, single chain Fv fragments (scFv), bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies, and single domain molecules such as VH and VL that are capable of specifically binding to an antigen.

[0060] All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

[0061] Current methods are also limited in their ability to acquire biomolecular information together with cellular connectivity in the same sample. Approaches based on genetically labeling (barcoding) single neurons have the potential to substantially reduce error rates and reduce acquisition volumes, since cell segments can be associated via barcodes without directly tracingintervening morphology. A further potential advantage of barcoding is that barcodes can be efficiently detected optically, and thus can be combined with efficient methods for optical biomolecular measurements. However, a primary issue with barcoding is the inability of genetically expressed barcode molecules (e.g. uniquely identifying sets of RNA molecules, fluorescent proteins, epitopes) to traffic to the entirety of the neuron, thus complicating accurately establishing connected cells and cell extensions.

[0062] In some aspects, disclosed herein is a method for analyzing a mammalian tissue, comprising: (a) contacting the mammalian tissue with a plurality of different vectors for expressing multiple different epitope tags in cells of the mammalian tissue, wherein each different vector can encode a different epitope tag, and wherein two or more cells in the mammalian tissue can each express a different combination of different epitope tags; (b) contacting the mammalian tissue with a first plurality of binders recognizing a first subset of the multiple different epitope tags; (c) detecting first signals associated with the first plurality of binders in the mammalian tissue; (d) contacting the mammalian tissue with a second plurality of binders recognizing a second subset of the multiple different epitope tags which can be different from the first subset; (e) detecting second signals associated with the second plurality of binders in the mammalian tissue; and (f) generating a codeword for each of the two or more cells in the mammalian tissue, wherein the codeword can comprise signal codes corresponding to the presence or absence of the first signals and signal codes corresponding to the presence or absence of the second signals, and the codeword for a particular cell corresponding to the combination of different epitope tags expressed in the cell.

[0063] In some embodiments, the mammalian tissue can be a neural tissue, and the each of two or more cells can be independently selected from the group consisting of a neuron, an oligodendrocyte, an astrocyte, an ependymal cell, a microglia, a Schwann cell, and a satellite cell. In some embodiments, the mammalian tissue can be a brain tissue or a spinal cord tissue, and the two or more cells can be neurons. In any of the embodiments herein, i) the mammalian tissue can be a cell culture comprising cultured neurons, optionally wherein the cell culture can be a patient-derived cell culture; ii) the mammalian tissue can be a cultured tissue, optionally wherein the cultured tissue can be a cultured brain tissue; or iii) the mammalian tissue can be in a live mammalian individual and the plurality of different vectors can be introduced into the individual to contact with the mammalian tissue.

[0064] In any of the embodiments herein, the plurality of different vectors can be viral vectors. In any of the embodiments herein, the plurality of different vectors can be AAV vectors. In any of the embodiments herein, the plurality of different vectors can comprise stoichiometricratios of the different vectors or non-stoichiometric ratios of the different vectors. In any of the embodiments herein, the plurality of different vectors can comprise more than 10 different vectors. In any of the embodiments herein, the plurality of different vectors can comprise about 30 different vectors. In any of the embodiments herein, the plurality of different vectors can comprise about 100 different vectors.

[0065] In any of the embodiments herein, the multiple different epitope tags can comprise more than 10 different epitope tags. In any of the embodiments herein, the multiple different epitope tags can comprise about 30 different epitope tags. In any of the embodiments herein, the multiple different epitope tags can comprise about 50 different epitope tags. In any of the embodiments herein, the multiple different epitope tags can comprise peptide tags. In any of the embodiments herein, the multiple different epitope tags can each be between about 6 and about 30 amino acid residues in length. In any of the embodiments herein, each different vector can encode a different fusion protein comprising the epitope tag linked to a scaffold protein. In some embodiments, the scaffold protein can be common among the different fusion proteins encoded by the plurality of different vectors, or the scaffold protein can be different among the different fusion proteins encoded by two or more different vectors of the plurality of different vectors. In any of the embodiments herein, the scaffold protein can be a fluorescent protein, optionally wherein the scaffold protein can be an eGFP, a mNeonGreen, mGreenLantem, or a momomeric GFP.

[0066] In any of the embodiments herein, the each different vector can encode a different fusion protein comprising the epitope tag linked to a localization domain, optionally wherein the localization domain can be common among the different fusion proteins encoded by the plurality of different vectors, or optionally wherein the localization domain can be different among the different fusion proteins encoded by two or more different vectors of the plurality of different vectors. In any of the embodiments herein, each different vector can comprise a promoter operably linked to a sequence encoding the epitope tag and / or scaffold protein. In some embodiments, the promoter can be a CAG promoter or a Sindbis virus subgenomic promoter.

[0067] In any of the embodiments herein, the mammalian tissue can be the brain of an mammalian individual, wherein: each different epitope tag can be expressed randomly in about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the neurons labeled by the different epitope tags, or each different epitope tag can be expressed randomly in over 50% of the neurons labeled by the different epitope tags, optionally wherein each different epitope tag is expressed randomly in about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 99% of the neurons labeled by the different epitope tags. In any of the embodiments herein, at least or about 10, at least or about 100, at least or about 103, at least or about 104, at least or about 105, at least or about 106, at least or about 107, at least or about 108, at least or about 109, at least or about IO10, or at least or about 1011neurons in the mammalian tissue can each express a unique combination of different epitope tags.

[0068] In any of the embodiments herein, the method can comprise: contacting the mammalian tissue with a third plurality of binders recognizing a third subset of the multiple different epitope tags; detecting third signals associated with the third plurality of binders in the mammalian tissue, wherein the codeword can further comprise signal codes corresponding to the presence or absence of the third signals. In some embodiments, the method can comprise: contacting the mammalian tissue with a fourth plurality of binders recognizing a fourth subset of the multiple different epitope tags; detecting fourth signals associated with the fourth plurality of binders in the mammalian tissue, wherein the codeword can further comprise signal codes corresponding to the presence or absence of the fourth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with a fifth plurality of binders recognizing a fifth subset of the multiple different epitope tags; detecting fifth signals associated with the fifth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the fifth signals. In any of the embodiments herein, each plurality of binders can comprise binders recognizing two, three, four, five, or more different epitope tags. In some embodiments, the method can comprise: contacting the mammalian tissue with a sixth plurality of binders recognizing a sixth subset of the multiple different epitope tags; detecting sixth signals associated with the sixth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the sixth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with a seventh plurality of binders recognizing a seventh subset of the multiple different epitope tags; detecting seventh signals associated with the seventh plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the seventh signals. In some embodiments, the method can comprise: contacting the mammalian tissue with a eighth plurality of binders recognizing a eighth subset of the multiple different epitope tags; detecting eighth signals associated with the eighth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the eighth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with a nineth plurality of binders recognizing a nineth subset of the multiple different epitope tags; detectingnineth signals associated with the nineth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the nineth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with a tenth plurality of binders recognizing a tenth subset of the multiple different epitope tags; detecting tenth signals associated with the tenth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the tenth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with up to a fifteenth plurality of binders recognizing up to a fifteenth subset of the multiple different epitope tags; detecting up to fifteenth signals associated with the up to fifteenth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the fifteenth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with up to a twentieth plurality of binders recognizing up to a twentieth subset of the multiple different epitope tags; detecting up to twentieth signals associated with the up to twentieth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the twentieth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with up to a twenty-fifth plurality of binders recognizing up to a twenty-fifth subset of the multiple different epitope tags; detecting up to twenty-fifth signals associated with the up to twenty-fifth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the twenty -fifth signals. In some embodiments, the method can comprise: contacting the mammalian tissue with up to a fiftieth plurality of binders recognizing up to a fiftieth subset of the multiple different epitope tags; detecting up to fiftieth signals associated with the up to fiftieth plurality of binders in the mammalian tissue, wherein the codeword further can comprise signal codes corresponding to the presence or absence of the fiftieth signals.

[0069] In some embodiments, in each cycle of binder recognition and signal detection, a signal associated with each different epitope tag detected at a particular neuron, or the absence of the signal at the neuron, can be recorded as a signal code at a bit in the codeword for the neuron. In some embodiments, the method can comprise two, three, four, five, or more cycles of binder recognition and signal detection, and in each cycle the plurality of binders can comprise two, three, four, five, or more different binders each recognizing a different epitope tag of the multiple different epitope tags. In some embodiments, the method can comprise five cycles of binder recognition and signal detection, and in each cycle the plurality of binders can comprise three different binders each recognizing a different epitope tag of the multiple different epitopetags. In some embodiments, the method can comprise two, three, four, five, six, seven, eight, nine, ten, up to fifteen, up to twenty, up to twenty-five, up to fifty, or more cycles of binder recognition and signal detection, and in each cycle the plurality of binders can comprise two, three, four, five, six, seven, eight, nine, ten, up to fifteen, up to twenty, up to twenty-five, up to fifty, or more different binders each recognizing a different epitope tag of the multiple different epitope tags. In some embodiments, the method can comprise five cycles of binder recognition and signal detection, and in each cycle the plurality of binders can comprise three different binders each recognizing a different epitope tag of the multiple different epitope tags. In any of the embodiments herein, in a particular cycle of binder recognition and signal detection, each different binder can be detected in a different channel of fluorescent microscopy.

[0070] In any of the embodiments herein, the codeword for the neuron can be between 2 and 100 bits, optionally wherein the codeword for the neuron can be 15 bits, 30 bits, 50 bits, or 100 bits. In any of the embodiments herein, prior to a particular cycle of binder recognition and signal detection, the method can comprise a step of removing the plurality of binders of a previous cycle from the mammalian tissue, and / or extinguishing signals associated with the plurality of binders of the previous cycle.

[0071] In any of the embodiments herein, each plurality of binders can comprise primary antibodies or epitope-binding fragments thereof that bind to the epitope tags, optionally wherein the primary antibodies or epitope-binding fragments thereof can be detectably labeled. In some embodiments, each plurality of binders can further comprise secondary antibodies or epitopebinding fragments thereof that bind to the primary antibodies or epitope-binding fragments thereof, optionally wherein the secondary antibodies or epitope-binding fragments thereof can be detectably labeled. In any of the embodiments herein, each of the primary antibodies or epitopebinding fragments thereof or the secondary antibodies or epitope-binding fragments thereof can be conjugated to a nucleic acid tag, optionally wherein the nucleic acid tag can comprise one or more barcode sequences.

[0072] In any of the embodiments herein, the mammalian tissue can be a brain tissue and the method can comprise detecting a pre-synaptic marker, a post-synaptic marker, and / or a neurotransmitter marker in the mammalian tissue. In some embodiments, the pre-synaptic marker can be selected from the group consisting of piccolo, bassoon, CASK, one or more SNARE types, SNAP25, VAMP, and syntaxin. In any of the embodiments herein, the post- synaptic marker can be selected from the group consisting of Homer, post-synaptic density-95 (PSD95), neuroligin, SAP 102, SAPAP, SHANK, and calcium-dependent protein kinase II. In any of the embodiments herein, the neurotransmitter marker can be selected from the groupconsisting of a marker for glutamatergic transmission, a marker for GABAergic transmission, a marker for dopaminergic transmission, a marker for cholinergic transmission, and a marker for serotonergic transmission. In any of the embodiments herein, the neurotransmitter marker can be selected from the group consisting of VGAT, GABRA1, gephyrin, NMDA-1, and vGluTl.

[0073] In any of the embodiments herein, the mammalian tissue can be expanded or not expanded. In any of the embodiments herein, the mammalian tissue can be a brain tissue and the method can comprise generating a plurality of different codewords at cellular structures in the mammalian tissue. In some embodiments, the method can comprise identifying two or more cellular structures having the same codeword as belonging to the same neuron. In some embodiments, the method can identify two or more cellular structures each having a different codeword as belonging to different neurons. In any of the embodiments herein, the cellular structures can be selected from the group consisting of a nucleus or a portion thereof, a cell body or a portion thereof, an axon or a portion thereof, and a dendrite or a portion thereof. In any of the embodiments herein, the method can comprise embedding the brain tissue in a swellable polymer matrix and expanding the swellable polymer matrix and the brain tissue embedded therein.

[0074] In some aspects, disclosed herein is a composition comprising a plurality of different vectors at stoichiometric ratios, wherein each different vector can encode a fusion protein comprising a different epitope tag linked to a scaffold protein, and wherein the plurality of different vectors can be configured to express the fusion proteins in cells of a mammalian tissue. In some embodiments, the plurality of different vectors can be viral vectors. In some embodiments, the plurality of different vectors can be AAV vectors or sindbis virus vectors. In any of the embodiments herein, the composition can comprise more than 10 different vectors each expressing a fusion protein comprising a different epitope tag, and two or more or all of the different epitope tags can be linked to the same scaffold protein. In some embodiments, the composition can comprise about 30 different vectors each expressing a fusion protein comprising a different epitope tag linked to a common scaffold protein. In any of the embodiments herein, the different epitope tags can be peptide tags between about 6 and about 30 amino acid residues in length. In any of the embodiments herein, the scaffold protein can be a fluorescent protein.

[0075] In some aspects, disclosed herein is a plurality of different fusion proteins each comprising a different peptide tag linked to a common scaffold protein, wherein the different peptide tags can be between about 6 and about 30 amino acid residues in length and the common scaffold protein can be a fluorescent protein. In some embodiments, the plurality of different fusion proteins can comprise more than 10 different fusion proteins each comprising a differentpeptide tag. In some embodiments, the plurality of different fusion proteins can comprise about 30 different fusion proteins each comprising a different peptide tag.

[0076] In some aspects, disclosed herein is a mammalian tissue comprising a plurality of different vectors in contact with cells of the mammalian tissue, wherein each different vector can encode a fusion protein comprising a different epitope tag linked to a scaffold protein, and wherein the plurality of different vectors can be configured to express the fusion proteins in cells of the mammalian tissue. In some embodiments, the mammalian tissue can comprise more than 10 different vectors each encoding a different peptide tag. In some embodiments, the mammalian tissue can comprise about 30 different vectors each encoding a different peptide tag.

[0077] In some aspects, disclosed herein is a mammalian tissue comprising a plurality of different fusion proteins expressed in cells of the mammalian tissue, wherein each different fusion protein can comprise a different peptide tag linked to a scaffold protein, and wherein two or more cells in the mammalian tissue can each express a different combination of peptide tags selected from the plurality of different fusion proteins. In some embodiments, the mammalian tissue can be a brain tissue, and wherein: each different peptide tag of the plurality of different fusion proteins is expressed randomly in about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the neurons labeled by the different peptide tags, or each different peptide tag of the plurality of different fusion proteins is expressed randomly in over 50% of the neurons labeled by the different peptide tags, optionally wherein each different peptide tag can be expressed randomly in about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the neurons labeled by the different peptide tags. In any of the embodiments herein, the mammalian tissue can comprise more than 10 different fusion proteins each comprising a different peptide tag linked to a common scaffold protein expressed in cells of the mammalian tissue. In some embodiments, the mammalian tissue can comprise about 30 different fusion proteins each comprising a different peptide tag linked to a common scaffold protein expressed in cells of the mammalian tissue.

[0078] In some aspects, disclosed herein is a set of binders, comprising: i) a first plurality of binders recognizing a first subset of multiple different epitope tags, wherein each binder in the first plurality can be configured to be detected in a different channel of fluorescent microscopy, and ii) a second plurality of binders recognizing a second subset of the multiple different epitope tags, wherein the second subset can be different from the first subset, and wherein each binder in the second plurality can be configured to be detected in a different channel of fluorescent microscopy, wherein the multiple different epitope tags can be peptide tags between about 6 andabout 30 amino acid residues in length. The set of binders can further comprise: iii) a third plurality of binders recognizing a third subset of the multiple different epitope tags, wherein each binder in the third plurality can be configured to be detected in a different channel of fluorescent microscopy, iv) a fourth plurality of binders recognizing a fourth subset of the multiple different epitope tags, wherein the second subset can be different from the first subset, and wherein each binder in the fourth plurality can be configured to be detected in a different channel of fluorescent microscopy, and v) a fifth plurality of binders recognizing a fifth subset of multiple different epitope tags, wherein each binder in the fifth plurality can be configured to be detected in a different channel of fluorescent microscopy. In any of the embodiments herein, each subset can comprise two, three, four, five, or more different epitope tags and can be nonoverlapping with another subset. In any of the embodiments herein, each binder can be an antibody or epitope binding fragment thereof.

[0079] In some aspects, disclosed herein is a mammalian tissue comprising a plurality of about 30 different fusion proteins expressed in cells of the mammalian tissue, wherein each of the plurality of fusion proteins can comprise a different epitope tag linked to a common scaffold protein, wherein two or more cells in the mammalian tissue each can express a different combination of epitope tags selected from the plurality of different fusion proteins, wherein the mammalian tissue can be a brain tissue and each different epitope tag of the plurality of different fusion proteins can be expressed randomly in about 50% of the neurons labeled by the different epitope tags, and wherein the mammalian tissue can be in contact with a plurality of binders recognizing a subset of the different epitope tags, wherein each binder in the plurality can be configured to be detected in a different channel of fluorescent microscopy.

[0080] In one aspect, the disclosure provides compositions, comprising a plurality of different fusion proteins, wherein each fusion protein in the plurality of fusion proteins independently comprises:(a) a fluorescent protein comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 40-43; and(b) one or more peptide epitopes comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31; wherein the composition in total comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, or all of the peptide epitopes of SEQ ID NO: 1-31.

[0081] As used herein, a “plurality” means at least two.

[0082] The sequences of SEQ ID NO: 1-31 are provided in Table 1 below, and the sequences of fluorescent proteins SEQ ID NO:40-43 are provided in Table 2. The inventors have discovered that the compositions of the disclosure are superior to previously available markers for labeling cells in a brain tissue sample to permit mapping connectivity between cells in the brain tissue sample. Specifically, the inventors have surprisingly discovered that the recited fluorescent protein component of the fusion proteins are far superior than previously used detectable protein fusion in filling cells in the brain tissue sample, including in axons and dendrites many millimeters from the cell body. The inventors have also identified the peptide epitope targets of SEQ ID NO: 1-31, and antibodies detecting them, as ideally suited for detection in cells in brain tissue samples relative to a starting set of approximately 250 peptide epitopeantibody pairs.

[0083] For mapping brain circuit connectivity (“connectomics”), detection of marker combinations (such as the compositions of the disclosure), also referred to herein as “barcoding”, the barcodes need to fill the cell, including in axons and dendrites many millimeters from the cell body. The inventors have demonstrated that the compositions and methods of the disclosure can be used to examine expression in distant areas of the brain given an initial injection site as a proxy for sufficient cell filling, and that the methods result in significantly improved cell-filling labeling of fusion proteins relative to previously available methods, which is required for reducing error rates with barcodes when mapping cell connections. The compositions and methods disclosed herein enable intrinsic error correction, permitting larger brain circuits to be accurately mapped due to fewer errors, and enable “targeted” circuit mapping of specific circuits by bridging spatial gaps (e.g., segments of cells in different brain areas can be connected using barcodes without tracing through the intervening volume).Table 1Table 2

[0084] In a specific embodiment, the fluorescent protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 (eGFP).

[0085] The fluorescent protein and the one or more peptide epitope may be directly adjacent in the fusion protein, or may be separated by amino acid linkers. In one embodiment, the fluorescent protein and the one or more peptide epitope are directly adjacent in each fusion protein in the composition, without any intervening amino acid linker. In other embodiments, 1, 2, 3, 4, 5, or more, or all of the fusion proteins further comprise an amino acid linker separating the fluorescent protein and one or more of the peptide epitope. In embodiments where an amino acid linker is present, the linker may be of any length and amino acid composition as suitable foran intended use. In non-limiting embodiments, the linker may comprise a flexible GS linker, including but not limited to GGSGGS (SEQ ID NO: 36), or a kinked linker comprising one or more prolines, or SEQ ID NO: 1 (ALFA). In another embodiment, the amino acid linkers when present are between 1-12 amino acids in length.

[0086] Individual fusion proteins may comprise a single peptide epitope, or multiple (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different peptide epitopes. Fusion proteins with more peptide epitope copies have increased signal per protein expressed. In embodiments where the fusion protein comprises multiple different peptide epitopes, amino acid linkers may be present between all of the domains (i.e., between the fluorescent protein and each peptide epitope), linkers may be present between only some of the domains, or the domains may all be directly adjacent with no amino acid linkers separating them. In embodiments wherein linkers are present between multiple domains and there are at least 2 amino acid linkers, the linkers may be the same or may be different.

[0087] In various embodiments, the composition comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different fusion proteins. In other embodiments, one or more of the fusion proteins comprises 2, 3, 4, 5, or more different peptide epitopes. In further embodiments, 2, 3, 4, 5, or more, or all of the fusion proteins comprises 2, 3, 4, 5, or more different peptide epitopes.

[0088] The domains in the fusion protein may be arranged in any manner appropriate for an intended use. In one embodiment, the fluorescent protein is N-terminal to the one or more peptide epitopes. In another embodiment, the fluorescent protein is C-terminal to the one or more peptide epitopes.

[0089] In another embodiment, 1, 2, 3, 4, 5, or more, or all of the fusion proteins further comprise a localization domain. The addition of localization domains to the fusion protein can increase overall signal in distal regions. Exemplary such localization domains include, but are not limited to, membrane localization domains (e.g. a farnesylation motif), ER localization domains, mitochondrial localization domains, and actin localization domains. In various nonlimiting embodiments, the localization domain may comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:50-54. The amino acid sequence of SEQ ID NO:50-54 are shown in Table 3. In one embodiments, the location of the localization domain in the fusion protein (i.e., N-terminal or C-terminal) is noted in Table 3. In embodiments where there is more than one peptide epitope, the fluorescent protein can be between any two or more peptide epitopes (e.g., peptide epitope — eGFP — peptide epitope, where the peptide epitope can be the same or different). In some embodiments, a fusion protein disclosed herein cancomprise two, three, or more epitopes (e.g., short peptides), wherein an epitope can be linked to a scaffold (e.g., eGFP) via a linker, and any two adjacent epitopes can be linked via one or more linkers. In some embodiments, a fusion protein disclosed herein can comprise two or more copies of an epitope fused to the c terminus of a scaffold. In some embodiments, a fusion protein disclosed herein can have the formula eGFP-epitopel-linker-epitope2-linker-epitope3, where epitope 1, epitope2, and epitope3 can be the same epitope or epitopes. In some embodiments, there are multiple copies of a peptide epitope on a single fusion protein, for example, for example, for signal amplification during detection.Table 3

[0090] The composition may comprise any number of fusion proteins as appropriate for an intended use. In various non-limiting embodiments, the composition comprises between 2 and 500, or between 2 and 250, or between 2 and 100 fusion proteins.

[0091] In some embodiments, a nucleic acid (e.g., in a viral vector) encoding a fusion protein disclosed herein can comprise the formula promoter-scaffold-epitope-enhancer-polyA.

[0092] In another embodiment, the disclosure provides compositions comprising a plurality of antibodies, wherein the plurality of antibodies comprises antibodies that in total selectively bind to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30, or all 31 peptide epitopes comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-31.

[0093] The antibody compositions of the disclosure can be used, for example, in the methods of the disclosure to detect expression of the peptide epitopes in cells of the brain tissue sample. As described below, in the methods of the disclosure, individual cells will express multiple peptide epitopes, and their immunohistochemical detection by the antibodies provides a barcode for the cell and its extensions.

[0094] The antibodies in the composition are detectably distinguishable, either by being directly labeled with distinguishable, detectable labels, such as fluorescent dyes or conjugatednucleic acids or by secondary labeling with secondary antibodies that are distinguishable, detectably labeled, or by tertiary labeling of secondary antibodies with tertiary probes that are distinguishable and detectably labeled. For example, 3 separate compositions of antibodies each comprising 5 antibodies can be provided and used. In some embodiments, each antibody in composition 1 is separately distinguishable from the other composition 1 antibodies, but does not necessarily need to be separately distinguishable from composition 2 or 3 antibodies.

[0095] In some embodiments, a method disclosed herein comprises detection of fluorescent dye labeled primary antibody. In some embodiments, a method disclosed herein comprises detection of fluorescent dye labeled secondary antibody. In some embodiments, a method disclosed herein comprises detection of fluorescent dye labeled tertiary probe (e.g., nanobody). In some embodiments, a method disclosed herein comprises detection using DNA-conjugated primary or secondary antibody, detected with complementary dye-labeled DNA probe. In some embodiments, a method disclosed herein comprises detection using a DNA-conjugated primary or secondary antibody, DNA amplification reaction (e.g. branched DNA assay, rolling circle amplification, hybridization chain reaction), and detection of amplified DNA with tertiary dye- labeled probe. In some embodiments, a method disclosed herein comprises detection using a Horseradish peroxidase labeled primary or secondary antibody, and tyramide signal amplification. In some embodiments, an antibody is preincubated with a DNA-conjugated nanobody that binds to the antibody, and used for detection in a method disclosed herein. Exemplary methods involving the use of DNA-conjugated nanobodies are described in Unterauer et al., Spatial proteomics in neurons at single-protein resolution, bioRxiv 2023.05.17.541210, incorporated herein by reference in its entirety.

[0096] Antibodies against the peptide epitopes are commercially available, as shown below in Table 4.Table 4

[0097] The antibody composition may comprise additional antibodies as appropriate for an intended use. In various non-limiting embodiments, the antibody composition comprises between 2 and 200 antibodies, between 2 and 100 antibodies, or between 2 and 75 antibodies, or between 2 and 50 antibodies, or between 2 and 45 antibodies, or between 2 and 40 antibodies.

[0098] In one embodiment, all antibodies are mixed in the antibody composition. In other embodiments, the composition may comprise multiple (2 or more) separate mixtures, such as multiple mixtures provided in a kit. For example, a first mixture may comprise five antibodies that selectively bind a different peptide epitope selected from the group consisting of SEQ ID NO:l-5 (i.e.: the first antibody selectively binds to the peptide epitope of SEQ ID NO: 1, the second antibody selectively binds to the peptide epitope of SEQ ID NO:2, etc.), a second mixture may comprise ten antibodies that selectively bind a different peptide epitope selected from the group consisting of SEQ ID NO:6-15, and a third mixture may comprise eight antibodies that selectively bind a different peptide epitope selected from the group consisting of SEQ ID NO: 16- 23. It will be clear to those of skill in the art that many such antibody composition mixtures are possible. In these embodiments where the composition comprises multiple mixtures of the antibodies, the antibodies in each individual mixture are detectably distinguishable. In some embodiments where the composition comprises multiple mixtures of the antibodies, the antibodies in different mixtures may be detectably distinguishable, or they may be detectably indistinguishable. The latter embodiment may be used, for example, in methods of the disclosure that involve iterative cycles of immunostaining, fluorescence imaging, and destaining.

[0099] In another aspect, the disclosure provides a composition comprising plurality of nucleic acids encoding different fusion proteins, wherein each nucleic acid in the plurality of nucleic acids encodes a fusion protein that independently comprises:(a) a fluorescent protein comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 40-43; and(b) one or more peptide epitopes comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31;wherein the plurality of nucleic acids in total encodes at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, or all of the peptide epitopes of SEQ ID NO: 1-31.

[0100] The nucleic acid may be DNA, RNA, or modified versions thereof.

[0101] The composition comprises a plurality of nucleic acids encoding different fusion protein, wherein the fusion proteins can be any as disclosed above for the fusion protein compositions of the disclosure. Thus in one embodiment, the encoded fluorescent protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 (eGFP). In other embodiments, the encoded fluorescent protein and the one or more peptide epitope may be directly adjacent in the encoded fusion protein, or may be separated by amino acid linkers. In one embodiment, the encoded fluorescent protein and the one or more peptide epitope are directly adjacent in each encoded fusion protein in the composition, without any intervening amino acid linker. In other embodiments, 1, 2, 3, 4, 5, or more, or all of the encoded fusion proteins further comprise an encoded amino acid linker separating the encoded fluorescent protein and one or more of the encoded peptide epitopes. In embodiments where an encoded amino acid linker is present, the encoded linker may be of any length and amino acid composition as suitable for an intended use. In non-limiting embodiments, the encoded linker may comprise a flexible GS linker, including but not limited to GGSGGS (SEQ ID NO: 36), or a kinked linker comprising one or more prolines, or SEQ ID NO: 1 (ALFA). In another embodiment, the encoded amino acid linkers when present are between 1-12 amino acids in length.

[0102] The encoded fusion proteins may encode a single peptide epitope, or multiple (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different peptide epitopes. In embodiments where the encoded fusion protein comprises multiple encoded peptide epitopes, encoded amino acid linkers may be present between all of the encoded domains (i.e., between the encoded fluorescent protein and each encoded peptide epitope), encoded linkers may be present between only some of the encoded domains, or the encoded domains may all be directly adjacent with no encoded amino acid linkers separating them. In embodiments wherein encoded linkers are present between multiple encoded domains and there are at least 2 encoded amino acid linkers, the encoded linkers may be the same or may be different.

[0103] In various embodiments, the composition comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acids encoding different fusion proteins. In other embodiments, one or more of the encoded fusion proteins comprises 2, 3, 4, 5, or more different encoded peptideepitopes. In further embodiments, 2, 3, 4, 5, or more, or all of the encoded fusion proteins comprises 2, 3, 4, 5, or more different encoded peptide epitopes.

[0104] The domains in the encoded fusion protein may be arranged in any manner appropriate for an intended use. In one embodiment, the encoded fluorescent protein is N- terminal to the one or more encoded peptide epitopes. In another embodiment, the encoded fluorescent protein is C-terminal to the one or more encoded peptide epitopes.

[0105] In another embodiment, 1, 2, 3, 4, 5, or more, or all of the encoded fusion proteins further comprise an encoded localization domain. Exemplary such encoded localization domains include, but are not limited to, membrane localization domains (e.g., a farnesylation motif), ER localization domains, mitochondrial localization domains, and actin localization domains. In various non-limiting embodiments, the encoded localization domain may comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:50-54. In one embodiments, the location of the encoded localization domain in the fusion protein (i.e., N- terminal or C-terminal) is noted in Table 3.

[0106] In one embodiment, each nucleic acid is operatively linked to a promoter. As used herein, “operatively linked” means capable of effecting the expression of the nucleic acid molecules. The promoter need not be contiguous with the nucleic acid sequences, so long as it functions to direct the expression thereof. Any promoter may be used as suitable for an intended purpose. Constitutive promoters drive expression in a largely cell independent fashion, making them useful for generalized barcoding applications. However, different promoters drive different ranges of expression and are useful for tuning the amount of protein produced i.e. in cells that are poor protein factories (and thus need stronger promoters) or cells that produce too much GFP (and therefore need weaker promoters to reduce toxicity). These promoters are typically readily swappable parts, and include, but are not limited to: CAG, CMV, PGK, Efl -alpha, TRE (tet response element). Cell-type dependent promoters allow for barcoding applications to be applied only to specific cell types, or to use a specific epitope as a marker of cell type (e.g. if a specific tag had a CamKII promoter). Such promoters include: CamKII - excitatory neurons; Synapsin - neuron specific; homeobox Dlx5 / 6 - GABAergic neurons; Drdla - dopaminergic neurons; and GluR - glutamic neurons.

[0107] In one embodiment, the promoter independently (i.e., the promoter may be the same or different in different nucleic acids in the composition) comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 60-65. The nucleotide sequences of SEQ ID NO:60-65 are provided in Table 5.Table 5

[0108] The nucleic acids may further comprise any other suitable control sequences, including but not limited to polyadenylation signals, termination signals, and ribosome binding sites. In one embodiment, each nucleic acid further comprises an enhancer operatively linked to the promoter and to the coding region of the fusion protein. Any enhancer may be used as appropriate for an intended use. In one embodiment, the enhancer comprises the nucleotide sequence selected of SEQ ID NO: 70, as shown in Table 6.Table 6

[0109] In another embodiment, the nucleic acids may further comprise recombinase targeting sites, to enable conditional genetic expression. For example, a nucleic acid can be made into a vector (e.g., FLEx vector) by flanking it with recombinase sites to permit expression of thevector only in cells that express the recombinase. Site specific recombinases (e.g. Cre, FlpO, Nigri, Panto, etc) allow for control of the number of cells expressing a signal while keeping the infection rate of the barcoding components at the desired levels. Further, site-specific recombinases allow conditional expression in particular genetically defined cell types. In various embodiments, the encoded fusion protein is flanked on the 5’ and 3’ end with recombinase targeting sites, with the promoter located 5’ to the 5’ flanking region and any enhancer located 3’ to the 3’ flanking region. Any recombinase targeting sites may be used as appropriate for an intended purpose. In non-limiting embodiments, the flanking regions are selected from (a) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO:80 and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO:81 (Cre recombinase targeting sites); (b) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO:82 and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO:83 (FlpO recombinase targeting sites); and (c) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO:84 and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 85 (oNigri recombinase targeting sites). The sequences of SEQ ID NO:80-85 are shown in Table 7.Table 7: The composition according to table 1, which enables conditional expression using recombinases (e.g., the composition can be sparsified using recombinases). The plasmid is of the form - [promoter]-[left flank]-[scaffold, with epitope] -[right flank]-[posttranscriptional enhancer].

[0110] In one embodiment, the nucleic acids comprise expression vectors. Any expression vector may be used as suitable for an intended purpose. In various embodiments, each nucleic acid comprises an expression vector, wherein the expression vector comprises a viral vector selected from the group consisting of an adenoviral vector, an adeno- associated viral (AAV) vector (including but not limited to, AAV1, AAV2, AAV9, AAV.PHP.eB); a Sindbis viralvector, a rabies viral vector, a yellow fever viral vector, a lentivirus viral vector, and an HSV vector. While AAV vectors are exemplified herein, the other listed vectors provide other benefits:• Sindbis virus is a strong RNA virus with a rapid onset of protein expression. Expressing protein barcodes in a short period of time allows for rapid barcoding of unstable or difficult systems e.g. ex vivo human tissue.• Rabies virus can deliver their payload to a starter cell and to upstream cells (retrograde transport), allowing the mapping of functional connections without direct observation of synapses, or complementing anterograde tracing to confirm putative circuits. For an example of this, see Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing. See, e.g., Chen et al., High- Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing, Cell . 2019 Oct 17;179(3):772-786, incorporated herein by reference in its entirety.• Yellow fever vaccine is an anterograde transsynaptic tracer e.g. a starter cell will label post-synaptic partners with the same barcode. This allows projection tracing without directly observing a given synapse. For an example of this, refer to Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D. See, e.g., Li et al., Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D, Nat Methods . 2021 Dec;18(12): 1542-1551, incorporated herein by reference in its entirety.• Lentiviral delivery of barcodes permits random genetic integration and subsequent expression of a larger payload than AAV (up to lOkb). This permits the generation of more complex circuits and enhanced tactics for expression (e.g. loading the lentiviral cassette with multiple GFPs) as well as using more complex cell type specific promoters.• Adenovirus is a larger capacity DNA virus (up to 8.5 kb) that drives rapid strong protein expression. Expressing protein barcodes from such a virus would permit expression in short lived systems (e.g. ex vivo brain tissue).• HSV is a large capacity (wild type genome of 152kb, with a potential payload on the order of 100+ kb) retrograde tracer. Using such a system would permit delivery of multiple exogenous genes and complex genetic circuits to aid in the process of retrograde tracing.[oni] In a specific embodiment, each nucleic acid comprises an AAV expression vector, and each encoded fusion protein comprises eGFP.

[0112] The composition may comprise any number of nucleic acids encoding different fusion proteins as appropriate for an intended use. In various non-limiting embodiments, the composition comprises between 2 and 500, or between 2 and 250, or between 2 and 100 nucleic acids encoding different fusion proteins.

[0113] In another aspect, the disclosure provides a composition, comprising a plurality of viral particles, wherein the plurality of viral particles in total comprises the plurality of nucleic acids encoding different fusion proteins of any embodiment or combination of embodiments herein. In this embodiment, the plurality of nucleic acids are packaged in viral particles, which can be use, for example, to carry out the methods for brain circuit mapping disclosed herein. In one embodiment, viral particles comprising nucleic acids encoding different fusion proteins are present in approximately stoichiometric ratios (i.e., + / - 10% of stoichiometric ratio). In another embodiment, viral particles comprising nucleic acids encoding different fusion proteins are present in non stoichiometric ratios (i.e., + / - 100% of stoichiometric ratio). In a specific embodiment, each encoded fusion protein comprises eGFP.

[0114] In another aspect, the disclosure provides host cells comprising the composition of any embodiment herein. As disclosed herein, the methods of the disclosure involve expressing the fusion proteins in brain cells to permit brain circuit mapping. In one embodiment, host cells comprise the nucleic acid or viral composition of any embodiment disclosed herein. In one such embodiment, the plurality of nucleic acids are stably integrated into the cell genome. In another embodiment, the plurality of nucleic acids are stable within the nucleus as extrachromosomal DNA. In another embodiment, the plurality of nucleic acids are transiently transfected into the host cell. In one embodiment, the host cell is a mammalian host cell. In another embodiment, the mammalian host cell comprises a neuron, glial cell, or oligodendrocyte.

[0115] The disclosure also provides transgenic mammals, comprising a host cell in which the plurality of nucleic acids are stably integrated into the cell genome. In one embodiment, the transgenic mammal is a transgenic mouse. This comprises a strategy to express unique combinations of epitopes from a genetically integrated locus. This would effectively barcode every neuron (and cell) in the mouse, including both central and peripheral nervous systems.

[0116] The disclosure also provides kits, comprising one or more composition of the disclosure. The kits can be used, for example, in carrying out the methods of the disclosure. In one embodiment, the kit comprises (a) any embodiment of the nucleic acid compositions of the disclosure, and (b) any embodiment of the antibody compositions of the disclosure. In another embodiment, the kit comprises (a) any embodiment of the viral particle compositions of the disclosure, and (b) any embodiment of the antibody compositions of the disclosure. In anotherembodiment, the kit comprises (a) any embodiment of the host cell compositions of the disclosure, and (b) any embodiment of the antibody compositions of the disclosure.

[0117] In another embodiment of any of these embodiments, the kits further comprise one or more antibodies that detect synaptic markers. The methods of the certain embodiments of the disclosure include detecting the barcode generated in individual cells by expression of the fusion proteins in cells of brain tissue samples, and staining cells with antibodies that detect synaptic markers to infer circuit connectivity by spatial colocalization of pre- and post-synaptic markers. Any antibodies detecting synaptic markers may be present in the kits as appropriate for an intended use. In some embodiments, the synaptic markers may comprise antibodies that selectively bind synaptic targets selected from the group consisting of Amphiphysin, Ankyrin G, Bassoon, Dynamin 1 / 2 / 3, Gephyrin, Homer 1, MAP2, Munc 13-1, Parvalbumin, Piccolo, RIM1, Synaptophysin, VAMP2, vGluTl, PSD95, and Shank 2. Antibodies against these synaptic targets are commercially available, as noted in Table 8. In some embodiments, the kits include antibodies against both pre-synaptic and post-synaptic markers; the specificity of exemplary epitopes for pre-synaptic or post-synaptic locations is also provided in Table 8. In other embodiments, the kits comprise at least 2, 3, 4, 5, 6, 7, 8, or more antibodies detecting synaptic markers. The antibodies that selectively bind synaptic markers may be directly labeled with distinguishable, detectable labels, or by secondary labeling with secondary antibodies that are distinguishable, detectably labeled. In some embodiments, the antibodies that selectively bind synaptic markers are detectably distinguishable from antibodies that selectively bind to the protein epitopes. In other embodiments, the synaptic markers are not detectably distinguishable from antibodies that selectively bind to the protein epitopes; the methods of the disclosure comprise iterative immunostaining, and the synaptic marker-selective antibodies may be detected separately from the protein epitope selective antibodies.Table 8. Exemplary synaptic markers

[0118] In another aspect, the disclosure provides methods for detecting connectivity between cells in a brain tissue sample, comprising(a) expressing the protein composition and / or the nucleic acid composition of any embodiment in a brain tissue sample;(b) contacting the brain tissue sample with(i) the antibody composition of any embodiment herein under conditions to promote binding of the antibodies to the peptide epitopes to form detectable antibodyepitope complexes; and(ii) antibodies selective for synaptic markers under conditions to promote binding of the antibodies to the synaptic markers to form detectable antibody-synaptic marker complexes;(c) obtaining images of the detectable antibody-epitope complexes and the detectable synaptic markers in the brain sample; and(d) analyzing the images to identify connectivity between cells in the brain tissue sample, e.g., by detecting binding of the antibodies to the peptide epitopes as a barcode, wherein all cell segments sharing the same barcode are defined as connected even if they are spatially separated.

[0119] In some embodiments, the brain tissue sample can comprise cultured neural cells such as neurons. In some embodiments, the brain tissue sample is isolated from a mammalian subject and processed, such as by cryosectioning into tissue sections or tissue blocks.

[0120] In some embodiments, a tissue sample such as a brain tissue sample is chemically fixed (with e.g. paraformaldehyde) before contacting it with the antibody composition and antibodies selective for synaptic markers. In some embodiments, the sample is contacted by antibodies, then embedded in a gel such as a swellable hydrogel. In some embodiments, the sample is embedded in a gel such as a swellable hydrogel, then contacted by antibodies.

[0121] In one embodiment, the brain tissue sample is embedded in a swellable hydrogel. In some embodiments, a swellable hydrogel provided herein comprises a copolymer composition and one or more anchoring reagents. In some embodiments, the polymer composition comprises a copolymer of sodium acrylate, acrylamide, dimethylacrylamide (DMAA), and bis-acrylamide, and the biomolecules in the tissue are anchored to the polymer by inclusion of methacrolein in the polymerization solution. In some embodiments, In some embodiments, the polymer composition comprises a copolymer of sodium acrylate, acrylamide, and bis-acrylamide, and the biomolecules in the tissue are anchored to the polymer by inclusion of methacrolein in the polymerization solution, and dimethylacrylamide is not included. In some embodiments, In some embodiments, the polymer composition comprises a copolymer of acrylamide and bis- acrylamide, and the biomolecules in the tissue are anchored to the polymer by inclusion of methacrolein in the polymerization solution, and dimethylacrylamide and sodium acrylate are not included. In some embodiments, the anchoring reagent comprises methacrolein. In some embodiments, the anchoring reagent comprises any one or more of Acryloyl-X, SE, (6-((acryloyl)amino)hexanoic Acid, Succinimidyl Ester, and / or Methacrylic acid N- hydroxysuccinimide, with or without methacrolein.

[0122] Steps (b)(i) and (ii) may be carried out in any order, or may be carried out at the same time. When steps (b)(i) and (b)(ii) are not carried out at the same time, then steps (c) and (d) may be carried out before either of (b)(i) or (b)(ii).

[0123] In one embodiment, steps (b)-(d) are carried out iteratively. In one non-limiting example, contacting step (b)(i) is carried out first, followed by steps (c) and (d) to obtain fluorescence images of the detectable antibody-epitope complexes and analyze the images, followed by stripping of the sample of the antibodies selective for peptide epitopes. Then the same brain tissue sample is contacted with the antibodies selective for synaptic markers, followed by steps (c) and (d) to obtain fluorescence images of the detectable antibody-synaptic marker complexes and analyze the images.

[0124] In another exemplary embodiment, contacting step (b)(i) comprises two or more iterative steps. By way of non-limiting example, step (b)(i) may comprise contacting the brain tissue sample with embodiments of the antibody composition that are present in two or more mixtures as described above. In this embodiment, contacting step (b)(i) is first carried out with a first mixture of the antibody composition (including a first set of antibodies selective for a subset of the peptide epitopes), followed by steps (c) and (d) to obtain and analyze fluorescence images of the first set of detectable antibody-epitope complexes, followed by stripping the sample of the first mixture of antibodies selective for peptide epitopes. Then the same brain tissue sample is contacted with a second mixture of the antibody composition ((including a first set of antibodies selective for a subset of the peptide epitopes), followed by steps (c) and (d) to obtain and analyze fluorescence images of the second set of detectable antibody-epitope complexes. Other embodiments will be clear to those of skill in the art based on the present disclosure.

[0125] FIG. 2 and FIG. 3 provide exemplary workflows. FIG. 2 provides an example of protein barcode synthesis and delivery, and protein barcode detection using antibodies. In FIG. 3, samples are embedded in a swellable hydrogel and expanded to improve resolution. Sets of proteins in the sample are decoded through N cycles of iterative immunostaining, fluorescence imaging, and destaining. For example, 5 bits may be read out per cycle, defined by the number of spectrally distinct laser lines on the microscope. Protein barcodes are read out across cycles. Endogenous proteins, such as synaptic markers are then read out in the same sample. The joint barcode (morphology) and synaptic data improves reconstructing connectivity.

[0126] Stripping antibody from the samples / destaining can be carried out using any suitable technique.

[0127] As disclosed herein, the methods of the disclosure provide significant improvements in detecting connectivity between cells in a brain tissue sample. Specifically, the inventors have surprisingly discovered that the recited fluorescent protein component of the fusion proteins are far superior than previously used detectable proteins in filling cells in the brain tissue sample, including in axons and dendrites many millimeters from the cell body. The inventors have also identified the peptide epitope targets of SEQ ID NO: 1-31, and antibodies detecting them, as ideally suited for detection in cells in brain tissue samples relative to a starting set of approximately 250 peptide epitopes. For mapping brain circuit connectivity (“connectomics”), detection of marker combinations (such as the compositions of the disclosure), also referred to herein as “barcoding”, the barcodes need to fill the cell, including in axons and dendrites many millimeters from the cell body. The inventors have demonstrated that the compositions and methods of the disclosure can be used to examine expression in distant areas of the brain given an initial injection site as a proxy for sufficient cell filling, and that the barcodes have intrinsic error correction that can be utilized to significantly reduce error rates in mapping cell connections both at the level of projections between brain areas and at the level of single cells relative to previously available methods. The compositions and methods disclosed herein enable intrinsic error correction, permitting larger brain circuits to be accurately mapped due to fewer errors, and enable “targeted” circuit mapping of specific circuits by bridging spatial gaps (i.e., segments of cells in different brain areas can be connected using barcodes without tracing through the intervening volume.

[0128] The compositions for use in the methods may be any embodiment or combination of embodiments disclosed above. In one embodiment, the detectable synaptic markers may comprise antibodies that selectively bind synaptic targets selected from the group consisting of Amphiphysin, Ankyrin G, Bassoon, Dynamin 1 / 2 / 3, Gephyrin, Homer 1, MAP2, Munc 13-1, Parvalbumin, Piccolo, RIM1, Synaptophysin, VAMP2, vGluTl, PSD95, and Shank 2. Antibodies against these synaptic targets are commercially available, as noted in Table 8. In some embodiments, the kits include antibodies that detect both pre-synaptic and post-synaptic markers; the specificity of exemplary antibodies detecting pre-synaptic or post-synaptic proteins is also provided in Table 8. In other embodiments, the kits comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, or more antibodies detecting synaptic markers. The antibodies that selectively bind synaptic markers may be directly labeled with distinguishable, detectable labels, or by secondary labeling with secondary antibodies that are distinguishable, detectably labeled.In some embodiments, the antibodies that selectively bind synaptic markers are detectably distinguishable from antibodies that selectively bind to the protein epitopes. In other embodiments, the synaptic markers are not detectably distinguishable from antibodies that selectively bind to the protein epitopes; the methods of the disclosure comprise iterative immunostaining, and the synaptic marker-selective antibodies may be detected separately from the protein epitope selective antibodies.

[0129] In one embodiment, the brain tissue sample is injected with the viral particle composition of any embodiment herein prior to step (a). In one embodiment, different viral particles comprising nucleic acids encoding different fusion proteins are present in approximately stoichiometric ratios (i.e., + / - 10% of stoichiometric ratio). In a specific embodiment, each encoded fusion protein comprises eGFP. In another embodiment, the plurality of different viral particles comprise AAV particles. In another embodiment, the injecting results in each peptide epitope being expressed in a random subset of cells in the brain tissue sample. In some embodiments, the cells can express unique combinations of small epitope tags on a stable scaffold.Exemplary Embodiments

[0130] Embodiment 1. A composition, comprising a plurality of different fusion proteins, wherein each fusion protein in the plurality of fusion proteins independently comprises:(a) a fluorescent protein comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 40-43; and(b) one or more peptide epitopes comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31; wherein the composition in total comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all of the peptide epitopes of SEQ ID NO:1-31.

[0131] Embodiment 2. The composition of Embodiment 1, wherein the fluorescent protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 (eGFP).

[0132] Embodiment 3. The composition of any one of Embodiments 1-2, wherein 1, 2, 3, 4, 5, or more, or all of the fusion proteins further comprise an amino acid linker separating the fluorescent protein and the one or more peptide epitope.

[0133] Embodiment 4. The composition of Embodiment 3, wherein the linker comprises a flexible GS linker, including but not limited to GGSGGS (SEQ ID NO: 36), or a kinked linker comprising one or more prolines, or SEQ ID NO: 1 (ALFA).

[0134] Embodiment 5. The composition of any one of Embodiments 1-3, wherein the composition comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different fusion proteins.

[0135] Embodiment 6. The composition of any one of Embodiments 1-5, wherein one or more of the fusion proteins comprises 2, 3, 4, 5, or more different peptide epitopes.

[0136] Embodiment 7. The composition of any one of Embodiments 1-6, wherein 2, 3, 4, 5, or more, or all of the fusion proteins comprises 2, 3, 4, 5, or more different peptide epitopes.

[0137] Embodiment 8. The composition of Embodiment 7, wherein two or more peptide epitope in a single fusion protein are separated from each other by an amino acid linker.

[0138] Embodiment 9. The composition of any one of Embodiments 1-8, wherein the fluorescent protein is N-terminal to the one or more peptide epitopes.

[0139] Embodiment 10. The composition of any one of Embodiments 1-8, wherein the fluorescent protein is C-terminal to the one or more peptide epitopes.

[0140] Embodiment 11. The composition of any one of Embodiments 1-10, wherein 1, 2, 3, 4, 5, or more of the fusion proteins further comprise a localization domain.

[0141] Embodiment 12. The composition of Embodiment 9, wherein the localization domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:50-54.

[0142] Embodiment 13. The composition of any one of Embodiments 1-12, wherein the composition comprises between 2 and 500, or between 2 and 250, or between 2 and 100 different fusion proteins.

[0143] Embodiment 14. A composition comprising a plurality of antibodies, wherein the plurality of antibodies comprises antibodies that in total selectively bind to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more peptide epitopes comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-31.

[0144] Embodiment 15. The composition of Embodiment 14, wherein the plurality of antibodies are detectably distinguishable.

[0145] Embodiment 16. The composition of Embodiment 14 or 15, wherein the antibody composition comprises between 2 and 100 antibodies, or between 2 and 75 antibodies, or between 2 and 50 antibodies, or between 2 and 45 antibodies, or between 2 and 40 antibodies.

[0146] Embodiment 17. The composition of any one of Embodiments 14-16, wherein all antibodies in the composition are mixed.

[0147] Embodiment 18. The composition of any one of Embodiments 14-16, wherein the composition comprises two or more separate mixtures of the antibodies.

[0148] Embodiment 19. A composition, comprising a plurality of nucleic acids encoding different fusion proteins, wherein each nucleic acid in the plurality of nucleic acids encodes a fusion protein that independently comprises:(a) an encoded fluorescent protein comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 40-43; and(b) one or more encoded peptide epitopes comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31; wherein the plurality of nucleic acids in total encodes at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all of the peptide epitopes of SEQ ID NO: 1-31.

[0149] Embodiment 20. The composition of Embodiment 19, wherein the encoded fluorescent protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40 (eGFP).

[0150] Embodiment 21. The composition of any one of Embodiments 19-20, wherein 1, 2, 3, 4, 5, or more, or all of the encoded fusion proteins further comprise an encoded amino acid linker separating the encoded fluorescent protein and the encoded one or more peptide epitope.

[0151] Embodiment 22. The composition of Embodiment 21, wherein the encoded linker comprises a flexible GS linker, including but not limited to GGSGGS (SEQ ID NO: 36), or a kinked linker comprising one or more prolines, or SEQ ID NO: 1 (ALFA).

[0152] Embodiment 23. The composition of any one of Embodiments 19-22, wherein the composition comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different encoded fusion proteins.

[0153] Embodiment 24. The composition of any one of Embodiments 19-23, wherein one or more of the encoded fusion proteins comprises 2, 3, 4, 5, or more different encoded peptide epitopes.

[0154] Embodiment 25. The composition of any one of Embodiments 19-24, wherein 2, 3, 4, 5, or more, or all of the encoded fusion proteins comprises 2, 3, 4, 5, or more different encoded peptide epitopes.

[0155] Embodiment 26. The composition of Embodiment 25, wherein two or more encoded peptide epitope in a single fusion protein are separated from each other by an encoded amino acid linker.

[0156] Embodiment 27. The composition of any one of Embodiments 19-26, wherein the encoded fluorescent protein is N-terminal to the one or more encoded peptide epitopes.

[0157] Embodiment 28. The composition of any one of Embodiments 19-26, wherein the encoded fluorescent protein is C-terminal to the one or more encoded peptide epitopes.

[0158] Embodiment 29. The composition of any one of Embodiments 19-28, wherein 1, 2, 3, 4, 5, or more of the encoded fusion proteins further comprise an encoded localization domain.

[0159] Embodiment 30. The composition of Embodiment 29, wherein the encoded localization domain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:50-54.

[0160] Embodiment 31. The composition of any one of Embodiments 19-30, wherein each nucleic acid is operatively linked to a promoter.

[0161] Embodiment 32. The composition of Embodiment 31, wherein the promoter independently comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:60-65.

[0162] Embodiment 33. The composition of Embodiment 32, wherein each nucleic acid further comprises an enhancer operatively linked to the promoter and to the coding region of the fusion protein.

[0163] Embodiment 34. The composition of Embodiment 33, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO: 70.

[0164] Embodiment 35. The composition of any one of Embodiments 19-34, wherein the nucleic acids further comprise recombinase targeting site flanking regions encoded fusion protein further comprises is flanked on the 5’ and 3’ ends with recombinase targeting sites.

[0165] Embodiment 36. The composition of Embodiment 35, wherein the flanking regions are selected from (a) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO:80 and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO:81 (Crerecombinase targeting sites); (b) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO:82 and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO:83 (FlpO recombinase targeting sites); and (c) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO:84 and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO:85 (oNigri recombinase targeting sites).

[0166] Embodiment 37. The composition of any one of Embodiments 19-34, wherein each nucleic acid comprises an expression vector, wherein the expression vector comprises a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral (AAV) vector, a Sindbis viral vector, a rabies viral vector, a yellow fever viral vector, a lentivirus viral vector, and an HSV vector.

[0167] Embodiment 38. The composition of any one of Embodiments 19-37, each nucleic acid comprises an AAV expression vector, and each encoded fusion protein comprises eGFP.

[0168] Embodiment 39. The composition of any one of Embodiments 19-38, comprising between 2 and 500, or between 2 and 250, or between 2 and 100 nucleic acids encoding different fusion proteins.

[0169] Embodiment 40. A composition, comprising a plurality of viral particles, wherein the plurality of viral particles in total comprises the plurality of nucleic acids encoding different fusion proteins of any one of Embodiments 19-39.

[0170] Embodiment 41. A host cell, comprising the composition of any preceding Embodiment.

[0171] Embodiment 42. The host cell of Embodiment 41 comprising the composition of any one of Embodiments 19-39, wherein the plurality of nucleic acids are stably integrated into the cell genome.

[0172] Embodiment 43. The host cell of Embodiment 41 comprising the composition of any one of Embodiments 19-39, wherein the plurality of nucleic acids are transiently transfected into the host cell.

[0173] Embodiment 44. The host cell of any one of Embodiments 41-43, wherein the host cell is a mammalian host cell.

[0174] Embodiment 45. The host cell of Embodiment 44, wherein the mammalian host cell comprises a neuron, glial cell, or oligodendrocyte.

[0175] Embodiment 46. A transgenic mammal, comprising the host cell of Embodiment 42.

[0176] Embodiment 47. The transgenic mammal of Embodiment 46, wherein the transgenic mammal is a transgenic mouse.

[0177] Embodiment 48. A kit comprising one or more composition of any preceding Embodiment.

[0178] Embodiment 49. The kit of Embodiment 48, comprising:(a) the composition of any one of Embodiments 19-39; and(b) the composition of any one of Embodiments 14-18.

[0179] Embodiment 50. The kit of Embodiment 48, comprising:(a) the composition of Embodiment 40; and(b) the composition of any one of Embodiments 14-18.

[0180] Embodiment 51. The kit of Embodiment 48, comprising:(a) the composition of any one of Embodiments 41-45; and(b) the composition of any one of Embodiments 14-18.

[0181] Embodiment 52. The kit of any one of Embodiments 49-51, further comprising one or more detectable synaptic markers.

[0182] Embodiment 53. The kit of Embodiment 52, wherein the one or more detectable synaptic markers comprise antibodies that selectively bind synaptic targets selected from the group consisting of Amphiphysin, Ankyrin G, Bassoon, Dynamin 1 / 2 / 3, Gephyrin, Homer 1, MAP2, Munc 13-1, Parvalbumin, Piccolo, RIM1, Synaptophysin, VAMP2, vGluTl, PSD95, and Shank 2.

[0183] Embodiment 54. The kit of Embodiment 52 or 53, wherein the one or more detectable synaptic markers include both pre-synaptic and post-synaptic markers;

[0184] Embodiment 55. The kit of any one of Embodiments 52-54, wherein the kits comprise at least 2, 3, 4, 5, 6, 7, 8, or more detectable synaptic markers.

[0185] Embodiment 56. A method for detecting connectivity between cells in a brain tissue sample, comprising(a) expressing the protein composition and / or the nucleic acid composition of any embodiment in a brain tissue sample;(b) contacting the brain tissue sample with(i) the antibody composition of any embodiment herein under conditions to promote binding of the antibodies to the peptide epitopes to form detectable antibodyepitope complexes; and(ii) antibodies selective for synaptic markers under conditions to promote binding of the antibodies to the synaptic markers to form detectable antibody-synaptic marker complexes;(c) obtaining images of the detectable antibody-epitope complexes and the detectable synaptic markers in the brain sample; and(d) analyzing the images to identify connectivity between cells in the brain tissue sample.

[0186] Embodiment 57. The method of Embodiment 56, wherein the brain tissue sample is embedded in a swellable hydrogel.

[0187] Embodiment 58. The method of Embodiment 71, wherein steps (b)-(d) are carried out iteratively.

[0188] Embodiment 59. The method of any one of Embodiments 70-72, wherein the brain tissue sample is injected with the composition of Embodiment 40 prior to step (a).

[0189] Embodiment 60. The method of Embodiment 59, wherein the plurality of different viral particles comprise AAV particles.

[0190] Embodiment 61. The method of Embodiment 60, wherein the plurality of different AAV particles are present in the composition at approximately stoichiometric ratios.

[0191] Embodiment 62. The method of Embodiment any one of Embodiments 60 or 61, wherein the injecting results in each peptide epitope being expressed in a random subset of cells in the brain tissue sample.Examples

[0192] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0193] FIG. 1 shows an exemplary workflow of a method disclosed herein. FIG. 2 provides an example of protein barcode synthesis and delivery, showing how individual “bits” in the barcode are synthesized as AAVs, pooled, injected into a mouse brain, and detected with antibodies. FIG. 3 shows exemplary methods for protein barcode readout, describing how bits are detected by high resolution iterative imaging. In particular examples, N (e.g., 29) AAV vectors that contain a scaffold (typically eGFP or derivatives) linked to a small epitope tag at theC-terminal of the GFP were synthesized and created. These AAV vectors were packaged and pooled together at approximately stoichiometric ratios for injection into a mouse brain (via either direct viral injection into the brain or retro-orbital for whole brain expression). The overall concentration of the vector was tuned such that each epitope is expressed in a random 50% of labeled cells, leading to cells expressing unique combinations of small epitope tags on a stable scaffold. These tags can be deconvoluted through antibody detection.DataBarcode balance and estimates of MOI

[0194] The capacity of a given barcoding pool to uniquely label cells is a function of / / , the number of bits in the pool, and k, the number of bits expressed in a given cell. In this system, k correlates to MOI, here defined as the mean number of infections per cell across all infected cells. The total number of possible unique labels in a given animal is equal to the value of the binomial coefficient, nCk. where the highest number of unique labels is achieved when k = nil and each bit is equally likely to be expressed. If MOI is low (k=l), each cell expresses only 1 bit, and the number of uniquely labeled cells is n. If MOI is high (»> ri), each cell expresses all bits, and the number of uniquely labeled cells is 0. With this requirement in mind, assays were developed to run quality control on epitope balance in viral pools post-synthesis, and to estimate MOI based on a single round of unexpanded staining.NGS analysis of barcoding ability

[0195] The effective capacity of an analog barcoding pool is dependent on the balance of each “bit” in the barcode, with optimum diversity of unique labels achieved when all bits are at equal ratios. To assay this, Illumina short read sequencing were used to profile the distribution of short epitope segments in AAVs pooled both before and after packaging, and in the plasmid pools used for packaging. Presented below are probability distributions of such pools, showing the percentage of viral particles with a given epitope. In FIG. 4A, the probability of each epitope in the plasmid pool used for packaging were compared, and the final probability of each epitope in the final AAV pool. In FIG. 4B, the probability of each epitope when each is packaged separately into AAV and pooled afterwards at putatively equal titers were profiled. In both cases, the range of probabilities was acceptably balanced (e.g. no epitope was completely dominant or completely absent) and putative barcoding diversity remained high regardless of pooling and synthesis strategy.Estimates of MOI

[0196] MOI estimation is accomplished in this system by first assuming (a) all viral particles (containing different epitopes) are equally likely to infect and express across labeled cells, (b) the number of infections across labeled cells is best modeled as a Poisson distribution and (c) GFP is expressed in all infected cells. This implies that if the distribution of epitopes is known (as established by NGS analysis of viral pools), the overall infection dynamics can be inferred by counting all GFP+ cells and a single epitope. In practice, even if MOI is not estimated, a well- barcoded sample with a reasonably balanced epitope pool should have a given epitope in a subset of GFP+ cells, ideally close to 50%.

[0197] The assay of counting GFP and the fraction of epitope-labeled cells in a sample using Tyl were performed, in a pool of 24 total epitopes with approximately balanced distribution, and counted approximately 500 eGFP cell bodies. Of these, 42% were also positive for Tyl (see, FIG. 5 in pink). MOI of this sample were estimated at 13, generating a possible 2,496,144 unique epitope combination when all epitopes are sequenced.

[0198] This assay was also performed in samples with predicted low MOIs and found epitope distribution in the sample broadly reflected expectations based on NGS results. These results are consistent with a MOI of ~5 viral genomes per cell. Representative imaging slices are included in FIG. 6.Distal trafficking assays

[0199] For barcoding to be useful for connectomics, the barcodes need to fill the cell, including in axons and dendrites many millimeters from the cell body. An assay was devised to examine expression in distant areas of the brain given an initial injection site as a proxy for sufficient cell filling.

[0200] In mice injected in the motor cortex with the barcoding pools, the presence or absence of individual barcode bits was assayed in tertiary axons of the superior colliculus (see, for example FIG. 7). Any signal has successfully traffic through many axon branches and narrow points. Successful signal readout was established in this location for 15 short epitope tags in expanded and unexpanded tissue.Distal trafficking assay in unexpanded tissue

[0201] This assay can be run in unexpanded tissue using standard immunohistochemistry methods. Here, barcode trafficking and epitope subsampling were qualitatively assessed in a single epitope channel in a distal region. FIGs. 8A-8C show brightfield images showing the anatomical location used for this assay, along with eGFP and an antibody stain for Suntag. FIG. 8A shows brightfield image showing anatomical location of superior colliculus and region of interest. FIG. 8B shows eGFP, a pan-barcode tracer, in many axons. FIG. 8C shows a subsampled stain of Suntag, an epitope tag, brightly visible in a subset of axons.Distal trafficking assay in expanded tissue

[0202] This experiment was performed using expansion microscopy. FIG. 9A shows Top: schematic of experiment. Motor cortex injection site, superior colliculus readout. Bottom: image of motor cortex injection site for a single barcode bit, demonstrating signal. FIG. 9B shows Top: image of signal in superior colliculus. Bottom: examples of individual putative synapses, marked with arrowheads, filled with barcode protein. FIG. 9C shows validation assay was performed independently for each of 15 different barcode bits. FIG. 9D shows 4 additional epitope tags that were validated in mouse brain.

[0203] The image above corresponds to validation for the following short epitope tags. FIG. 9E shows frequency distribution of these 19 epitope tags in a distal region (~4mm) from an injection site (n = 242 axons).Barcode ImagingIteratively imaging and registering 15 bits

[0204] To demonstrate barcode readout, 15 barcode bits were imaged in a gel-embedded and expanded region of motor cortex using 5 rounds of iterative immunostaining and destaining. These images were then computationally stitched and registered to create a 15-channel composite image. FIG. 10A shows example of a single channel and cycle of imaging (i.e., imaging a single barcode “bit”, here ALFA tag). Region of interest is highlighted. FIG. 10B shows merge of 15 channels, collected across 5 imaging cycles. FIG. 10C shows set of all imaging channels, split out over each iterative imaging cycle.Single Barcode Extraction

[0205] To demonstrate single-cell barcode extraction, a cell body in a region of interest (barcoded motor cortex) were examined and manually extracted the barcode by human annotation across 15 imaging channels in a registered volume. FIG. HA shows single imaging channel in the region of interest, with cell body denoted. FIG. 11B shows region of interest across 15 imaging cycles, with human annotation below (1 = stain present, 0 = stain absent).Barcode Codebook Extraction

[0206] To further demonstrate barcode extraction from single cells, every cell body in a barcoded volume of motor cortex were segmented. FIG. 12A shows all somas at imagedvolume of barcode injection site (motor cortex), registered and segmented. FIG. 12B shows bit barcode vector (raw data) corresponding to each cell in the volume. FIG. 12C shows a “Codebook” of barcodes in volume, following human annotation.Jointly imaging barcodes and synaptic markers in expanded tissues

[0207] For connectomics, both barcode and synaptic markers can be read out in the same sample. Here, iterative imaging of 8 synaptic markers and 8 barcode bits were performed in the same volume of motor cortex. FIG. 13A shows positive identification of synapses is shown by colocalization of bassoon and shank2 with a dendritic spine labeled with an epitope tag. FIGs. 13B-13C show examples of individual synapses and barcoding in distal synapses.Variation in generation of epitope diversity

[0208] Epitope diversity can also be generated at the DNA level, by assembling multiple different epitopes onto the same viral particle. In this iteration, scaffolds that contain different sets of epitope tags of length k from a pool of N epitopes were create. Low infection rates (e.g., 1 AAV per cell) or strong founder effects (e.g. RNA viruses, such as sindbis or rabies) can be used to ensure only one virus is expressed in each cell and minimize barcode collisions.

[0209] In a first iteration, oligo pools which incorporated 3 epitopes per construct and a unique DNA barcode per oligo were used, and the oligo pools cloned as a library into pSin-GFP, a constitutive sindbis virus plasmid to generate a library of a putative 2,300 short epitopes, as shown in FIG. 14. 96 of these plasmids were directly sequenced and it was found that 81% of sequences would correctly code for a barcode. The diversity of this pool were assayed by NGS analysis of DNA barcodes to profile the frequency distributions, and a reasonably even distribution (e.g. no barcodes completely dominated sequencing results) was found, as shown in FIG. 15

[0210] A sindbis pool were then injected into mouse motor cortex and stained for epitopes to validate expression and diversity. FIG. 16 shows GFP in green and 2 epitopes stained in red and far red. Epitopes show a distribution across all labeled cells, indicating unique combinations can be generated.

[0211] In a second iteration, Golden Gate assembly was use to perform a shuffled assembly of 6 epitopes chosen from a pool of 23, creating up to 100,000 unique epitope chains, and clone these into the GFP C terminal of 6-bit epitope sequences. This also provides proof of concept for increasing this to 15 bit chains, which provides up to 7.75 E+7 unique sequences.Variations in expression systems

[0212] Reliance on leaky expression can accomplish a similar effect, where only a very small number of cells accumulate sufficient recombinase to invert GFP to then “on” position. FIG. 17 shows eGFP containing 29 short epitope sequences in a Flp-dependent system driven by a Tet response element.Unexpanded multiplexing in tissue

[0213] For some applications, barcodes may be read out without physically expanding the gel i.e. unexpanded multiplexing. Briefly, this involves embedding the tissue in an unexpanded gel and then proceed to stain and strip rounds of antibodies until all epitope bits are imaged. FIGs. 18A-18B show reading out four bits in a gel-stabilized slice of tissue. FIG. 18A shows a 4-color image of epitopes overlaid. Different colors broadly represent different combinations of epitopes or differentials in brightness. FIG. 18B shows individual channels. Clockwise from top left, stains are shown for the epitope tags VSV, TaglOO, NWS, and OLLAS.Materials and MethodsMice

[0214] Mice were purchased from Jackson Laboratory. All mice were hosted in a specific pathogen-free facility. Animals ranged from 4-12 weeks of age during experiments and both male and female animals were used.Vector Construction

[0215] Linear epitope sequences were cloned in frame at the C-terminal of GFP C-terminal in pAAV-CAG-eGFP (https: / / www.addgene.org / 37825 / ) using the BsrGI and EcoRI restriction sites. Epitope sequences were synthesized by either IDT or Twist Biosciences, and codon- optimized for expression in mice. Epitopes were linked to GFP with standard linker composed of a GGSGGS (SEQ ID NO: 36) amino acid sequences. Sequences with more than one copy of the epitope were separated with the same 6 amino acid linker. Plasmid, insert, and amino acid sequences are provided in supplemental data as described in Sequences.

[0216] Assembled AAVs were individually transformed into NEB Stable cells and grown at 30°C. Individual clones were scaled to maxi- or midi-prep scale by Quintara Biosciences (Hayward), Genscript, Azenta (Plainfield, New Jersey), or MCLabs (South San Francisco, CA). Plasmids were verified with whole-plasmid nanopore sequencing from either Plasmidsauraus(Eugene, OR) or Quintara Biosciences (Hayward, CA).AAV prep

[0217] Following sequencing verification, plasmids were pooled at equimass ratios and sent as a pool for pilot AAV packaging with Vector Biolabs or AAVnerGene in multiple serotypes (including, but not limited to, 2 / PHP.eB, AAV2 / 1, and AAV2 / 9). Preps are of suitable purity for injections into animals.Variation: Individual packaging

[0218] Each plasmid is packaged into individual AAV preps, as above, and pooled as needed afterwards.Quality control of epitope pool balance

[0219] To assay viral pool distribution, Illumina short read sequencing was use to profile the distribution of short epitope segments in AAVs pooled both before and after packaging, and in plasmid pools used for packaging. Briefly, genetic material is mechanically fragmented with Covaris shearing, ligated to Illumina adapters, and then further PCR amplified for sequencing. Sequences were aligned to the individual reference maps to count the number of unique epitope sequences present in the plasmid pool and AAV pool.Preparation of vector for infusion

[0220] Viral preps may be used as is (e.g. with no dilution), co-injected with a tracer (e.g. a virus expressing a fluorophore or otherwise stainable signal), co-injected with a dummy virus (empty capsids) or diluted with PBS, ACSF, or a similarly balanced isotonic fluid before injection.Variation: recombination dependent systems

[0221] In the case of recombinase dependent systems, the virus may be co-injected with a viral recombinase ranging from 0% to 100% to sparsify the overall number of labeled cells.Vector infusion

[0222] All procedures were approved by an IACUC board. Briefly, animals are anesthestized and placed in a Stoelting Dual Just for Mouse Stereotaxic Instrument. The surgeon locates and prepares the surgical site with reference to target brain structures derived from GPaxinos, KFranklin: The Mouse Brain in Stereotaxic Coordinates (2012), incorporated herein by reference in its entirety. The pooled viral prep is injected at rates between 5 and 50 nL / min with volumes between 10 and 500 nL total using a Hamilton 10 uL Neuros syringe or .5 uL Neuros syringe, and controlled by a Stoelting QSI. The needle is removed, and the surgical site sutured shut. Animals are monitored for 1 week after surgery and weighed every 3 to assess health.Variations: Pan-neuronal delivery

[0223] Some AAV serotypes, notably Php.EB but also including AAV9, have the ability to cross the blood brain barrier and transduce the vast majority of cells in the mouse brain. In this variation, a virus is injected into the mouse tail vein or retro-orbital sinus. This may be paired with a stereotaxic injection.

[0224] An example of this scheme is a pan-neuronal delivery of a Cre-dependent barcoding pool, and a direct infusion of Cre into a circuit of interest. Another example case is the use of a Cre mouse line that express recombinase in only a subset of cell types. Pan neuronal infection with a Cre-dependent barcoding cassette would lead to barcoding expression in only that cell type.Sample perfusion and fixation

[0225] As early as one day and as late as years post-infusion, the animal is transcardially perfused with 10 mLs of ice-cold PBS and 80 mLs fresh, ice-cold 4% paraformaldehyde using a 27 gauge needle at a rate of 5-10 mLs / minute. The brain is extracted and post-fixed for 16-24 hours in ice-cold 4% paraformaldehyde, and then washed 3 times lx PBS with 100 millimolar glycine to inactivate the fixative.Sample sectioning

[0226] After washing, samples are hemi- sectioned to provide a flat surface and subselect the region of interest, and mounted on a Leica plate for sectioning on a Leica VT1200S. Samples are sectioned at 50 uM at a speed of 40 mm / s and collected as free floating well plates for imaging. Samples may also be processed for cryosectioning or other forms of tissue processing.Swellable hydrogel embedding

[0227] Brain slices were pre-incubated with monomer solution (34% sodium acrylate, 10% acrylamide, 4% dimethylacrylamide, 1% NaCl, and 0.01% bis-acrylamide in lx PBS) at 4°C for 30 minutes. The slices were exchanged into activated monomer solution (monomer solution, 0.001% 4-hydroxy-TEMPO, 0.2% methacrolein, 0.2% TEMED, 0.2% APS) and incubated at 4°C for 30 minutes. Incubated brain slices were transferred to polymerization chambers and incubated overnight at 37°C until polymerization was complete.Hydrogel homogenization

[0228] Following polymerization, samples were exchanged into homogenization buffer (10% sodium dodecyl sulfate, 8M urea, 25 mM EDTA in 2x PBS, adjusted to pH 7 with 5 M HC1 at 80°C). Samples were incubated in homogenization buffer overnight at 80°C. Samples were thenwashed 3x 10 min with IxPBS, once with 1% decaethylene glycol monododecyl ether in lx PBS for Ih at 60°C, and then 3x 10 min with lx PBSExpansion

[0229] Samples were washed 3x 20 min in ultrapure water until gel was fully expanded.Iterative Immunostaining and Imaging

[0230] Sample was blocked with PBS + 2.5% normal donkey serum + 0.2% Triton-X 100. Sample was washed 3x 10 min in lx PBS. Sample was incubated with primary antibodies prepared in PBS + 2.5% normal donkey serum + 0.2% Triton-X 100 overnight with shaking at 30 rpm. Sample was then washed 3x 10 min in lx PBS. Sample was incubated with dye-labeled secondary antibodies prepared in PBS + 2.5% normal donkey serum + 0.2% Triton-X 100 for 6h at RT with shaking at 30 rpm. Sample was then washed in 3x 10 min lx PBS. Sample was then imaged on a Yokogowa CSU-W1 spinning disc confocal microscope with a Nikon 40x 1.15NA LWD water immersion objective lens in the following fluorescence channels: 488nm, 561nm, 640nm. Following imaging, antibodies were stripped from the sample with incubation in homogenization buffer overnight at 80°C, followed by 3x 20 min washes of 1% decaethylene glycol monododecyl ether in lx PBS, followed by 3x 10 min washes in lx PBS. To read out 15 epitopes, this process was iterated 5 times, with 3 epitopes imaged per iteration.

[0231] FIG. 19 shows a frequency distribution of epitopes in cells in two biological conditions. This serves as a demonstration of the pooled labeling and presence of up to 26 epitope bits in the same sample; and a demonstration of doing this more than once in many cells simultaneously.

[0232] FIG. 20 shows multiplexing of 20 peptide bits in the motor cortex in non-expanded tissue. This demonstrates detection of 20 peptide tags in a single sample. Detection of tags occurred over 7 imaging cycles.

[0233] FIG. 21 shows epitope distribution in a site ~4 mm distal from the injection site. 24 epitopes are shown, n= 242 axons.

[0234] FIGs. 22A-22B show characterization of the sensitivity of the Cre-dependent systems to recombinases, while demonstrating stable barcode lengths across multiple density labels. FIG. 22A shows percentage of labeled GFP+ neurons (GFP+ cells / NeuN+ cells) based on the percent Cre in the injection. FIG. 22B shows the percentage of GFP+ cells that are epitope-labeled, demonstrating that epitope occupancy is uncorrelated with overall labeling density in a tunable system.

[0235] FIG. 23A shows labeled images showing epitope expression from a sindbis virus using a GFP scaffold and a 6-bit shuffled assembly at the C terminal. FIG. 23B shows frequency distribution of epitope expression from a sindbis virus using a GFP scaffold and a 6-bit shuffled assembly at the C terminal.

[0236] FIG. 24A shows labeled images showing epitope expression from an AAV virus using an mNeonGreen scaffold and a 3 -bit shuffled assembly at the C terminal. FIG. 24B shows frequency distribution of epitope expression from an AAV virus using an mNeonGreen scaffold and a 3 -bit shuffled assembly at the C terminal. This reduces the number of infections required to generate long barcodes, and increases the overall length of barcodes.

[0237] FIGs. 25A-25B show detection of cytosolic and mitochondrially-targeted barcodes in the same sample (FIG. 25A shows merged image; FIG. 25B shows split images). This demonstrated that a COX8 motif effectively localizes in neurons such that it is uniquely distinguishable from cytosolically expressed versions of the epitope in cell bodies and local regions. This effectively doubles the bit information per round of imaging.

[0238] FIGs. 26A-26B show the detection of synaptically-targeted scaffold in MOp (primary motor cortex, FIG. 26A) and superior colliculus (FIG. 26B). We demonstrated that a synaptically targeted epitope trafficks and is detectable at pre-synapses >4 mm away from the target region, potentially reducing the resolution requirements required for synaptic assignment and increasing trafficking.

[0239] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

CLAIMS1. A method for analyzing a mammalian tissue, comprising:(a) contacting the mammalian tissue with a plurality of different vectors for expressing multiple different epitope tags in cells of the mammalian tissue, wherein each different vector encodes a different epitope tag, and wherein two or more cells in the mammalian tissue each expresses a different combination of different epitope tags;(b) contacting the mammalian tissue with a first plurality of binders recognizing a first subset of the multiple different epitope tags;(c) detecting first signals associated with the first plurality of binders in the mammalian tissue;(d) contacting the mammalian tissue with a second plurality of binders recognizing a second subset of the multiple different epitope tags which is different from the first subset;(e) detecting second signals associated with the second plurality of binders in the mammalian tissue; and(f) generating a codeword for each of the two or more cells in the mammalian tissue, wherein the codeword comprises signal codes corresponding to the presence or absence of the first signals and signal codes corresponding to the presence or absence of the second signals, and the codeword for a particular cell corresponding to the combination of different epitope tags expressed in the cell.

2. The method of claim 1, wherein the mammalian tissue is a neural tissue, and the each of two or more cells is independently selected from the group consisting of a neuron, an oligodendrocyte, an astrocyte, an ependymal cell, a microglia, a Schwann cell, and a satellite cell.

3. The method of claim 1 or claim 2, wherein the mammalian tissue is a brain tissue or a spinal cord tissue, and the two or more cells are neurons.

4. The method of any one of claims 1-3, wherein: i) the mammalian tissue is a cell culture comprising cultured neurons, optionally wherein the cell culture is a patient derived cell culture; ii) the mammalian tissue is a cultured tissue, optionally wherein the cultured tissue is a cultured brain tissue; or iii) the mammalian tissue is in a live mammalian individual and the plurality of different vectors are introduced into the individual to contact with the mammalian tissue.

5. The method of any one of claims 1-4, wherein the plurality of different vectors are viral vectors.

6. The method of any one of claims 1-5, wherein the plurality of different vectors are AAV vectors.

7. The method of any one of claims 1-6, wherein the plurality of different vectors comprises stoichiometric ratios of the different vectors or non-stoichiometric ratios of the different vectors.

8. The method of any one of claims 1-7, wherein the plurality of different vectors comprises more than 10 different vectors.

9. The method of any one of claims 1-8, wherein the plurality of different vectors comprises about 30 different vectors.

10. The method of any one of claims 1-8, wherein the plurality of different vectors comprises about 100 different vectors.

11. The method of any one of claims 1-10, wherein the multiple different epitope tags comprise more than 10 different epitope tags.

12. The method of any one of claims 1-11, wherein the multiple different epitope tags comprise about 30 different epitope tags.

13. The method of any one of claims 1-12, wherein the multiple different epitope tags comprise about 50 different epitope tags.

14. The method of any one of claims 1-13, wherein the multiple different epitope tags comprise peptide tags.

15. The method of any one of claims 1-14, wherein the multiple different epitope tags are each between about 6 and about 30 amino acid residues in length.

16. The method of any one of claims 1-15, wherein each different vector encodes a different fusion protein comprising the epitope tag linked to a scaffold protein.

17. The method of claim 16, wherein the scaffold protein is common among the different fusion proteins encoded by the plurality of different vectors, or wherein the scaffold protein is different among the different fusion proteins encoded by two or more different vectors of the plurality of different vectors.

18. The method of claim 16 or claim 17, wherein the scaffold protein is a fluorescent protein, optionally wherein the scaffold protein is an eGFP, a mNeonGreen, mGreenLantern, or a momomeric GFP.

19. The method of any one of claims 1-18, wherein each different vector encodes a different fusion protein comprising the epitope tag linked to a localization domain, optionally wherein the localization domain is common among the different fusion proteins encoded by the plurality of different vectors, or optionally wherein the localization domain is different among the different fusion proteins encoded by two or more different vectors of the plurality of different vectors.

20. The method of any one of claims 1-19, wherein each different vector comprises a promoter operably linked to a sequence encoding the epitope tag and / or scaffold protein.

21. The method of claim 20, wherein the promoter is a CAG promoter or a Sindbis virus subgenomic promoter.

22. The method of any one of claims 1-21, wherein the mammalian tissue is the brain of an mammalian individual, wherein: each different epitope tag is expressed randomly in about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the neurons labeled by the different epitope tags, or each different epitope tag is expressed randomly in over 50% of the neurons labeled by the different epitope tags, optionally wherein each different epitope tag is expressed randomly in about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the neurons labeled by the different epitope tags.

23. The method of any one of claims 1-22, wherein at least or about 10, at least or about 100, at least or about 103, at least or about 104, at least or about 105, at least or about 106, at least or about 107, at least or about 108, at least or about 109, at least or about 1010, or at least or about 1011neurons in the mammalian tissue each expresses a unique combination of different epitope tags.

24. The method of any one of claims 1-23, comprising: contacting the mammalian tissue with a third plurality of binders recognizing a third subset of the multiple different epitope tags; detecting third signals associated with the third plurality of binders in the mammalian tissue,wherein the codeword further comprises signal codes corresponding to the presence or absence of the third signals.

25. The method of claim 24, comprising: contacting the mammalian tissue with a fourth plurality of binders recognizing a fourth subset of the multiple different epitope tags; detecting fourth signals associated with the fourth plurality of binders in the mammalian tissue, wherein the codeword further comprises signal codes corresponding to the presence or absence of the fourth signals.

26. The method of claim 25, comprising: contacting the mammalian tissue with a fifth plurality of binders recognizing a fifth subset of the multiple different epitope tags; detecting fifth signals associated with the fifth plurality of binders in the mammalian tissue, wherein the codeword further comprises signal codes corresponding to the presence or absence of the fifth signals.

27. The method of any one of claims 1-26, wherein each plurality of binders comprises binders recognizing two, three, four, five, six, seven, eight, nine, ten, up to fifteen, up to twenty, up to twenty-five, up to fifty, or more different epitope tags.

28. The method of claim 27, wherein in each cycle of binder recognition and signal detection, a signal associated with each different epitope tag detected at a particular neuron, or the absence of the signal at the neuron, is recorded as a signal code at a bit in the codeword for the neuron.

29. The method of claim 28, wherein the method comprises two, three, four, five, or more cycles of binder recognition and signal detection, and in each cycle the plurality of binders comprises two, three, four, five, or more different binders each recognizing a different epitope tag of the multiple different epitope tags.

30. The method of claim 29, wherein the method comprises five cycles of binder recognition and signal detection, and in each cycle the plurality of binders comprises three different binders each recognizing a different epitope tag of the multiple different epitope tags.

31. The method of claim 29 or claim 30, wherein in a particular cycle of binder recognition and signal detection, each different binder is detected in a different channel of fluorescent microscopy.

32. The method of any one of claims 28-31, wherein the codeword for the neuron is between 2 and 100 bits, optionally wherein the codeword for the neuron is 15 bits, 30 bits, 50 bits, or 100 bits.

33. The method of any one of claims 28-32, wherein prior to a particular cycle of binder recognition and signal detection, the method comprises a step of removing the plurality of binders of a previous cycle from the mammalian tissue, and / or extinguishing signals associated with the plurality of binders of the previous cycle.

34. The method of any one of claims 1-33, wherein each plurality of binders comprises primary antibodies or epitope-binding fragments thereof that bind to the epitope tags, optionally wherein the primary antibodies or epitope-binding fragments thereof are detectably labeled.

35. The method of claim 34, wherein each plurality of binders further comprises secondary antibodies or epitope-binding fragments thereof that bind to the primary antibodies or epitopebinding fragments thereof, optionally wherein the secondary antibodies or epitope-binding fragments thereof are detectably labeled.

36. The method of claim 34 or claim 35, wherein each of the primary antibodies or epitopebinding fragments thereof or the secondary antibodies or epitope-binding fragments thereof is conjugated to a nucleic acid tag, optionally wherein the nucleic acid tag comprises one or more barcode sequences.

37. The method of any one of claims 1-36, wherein the mammalian tissue is a brain tissue and the method comprises detecting a pre-synaptic marker, a post-synaptic marker, and / or a neurotransmitter marker in the mammalian tissue.

38. The method of claim 37, wherein the pre-synaptic marker is selected from the group consisting of piccolo, bassoon, CASK, one or more SNARE types, SNAP25, VAMP, and syntaxin.

39. The method of claim 37 or claim 38, wherein the post-synaptic marker is selected from the group consisting of Homer, post-synaptic density-95 (PSD95), neuroligin, SAP 102, SAPAP, SHANK, and calcium-dependent protein kinase II.

40. The method of any one of claims 37-39, wherein the neurotransmitter marker is selected from the group consisting of a marker for glutamatergic transmission, a marker for GABAergic transmission, a marker for dopaminergic transmission, a marker for cholinergic transmission, and a marker for serotonergic transmission.

41. The method of any one of claims 37-40, wherein the neurotransmitter marker is selected from the group consisting of VGAT, GABRA1, gephyrin, NMDA-1, and vGluTl.

42. The method of any one of claims 1-41, wherein the mammalian tissue is expanded or not expanded.

43. The method of any one of claims 1-42, wherein the mammalian tissue is a brain tissue and the method comprises generating a plurality of different codewords at cellular structures in the brain tissue.

44. The method of claim 43, comprising identifying two or more cellular structures having the same codeword as belonging to the same neuron.

45. The method of claim 44, comprising identifying two or more cellular structures each having a different codeword as belonging to different neurons.

46. The method of any one of claims 43-45, wherein the cellular structures are selected from the group consisting of a nucleus or a portion thereof, a cell body or a portion thereof, an axon or a portion thereof, and a dendrite or a portion thereof.

47. The method of any one of claims 43-46, comprising embedding the brain tissue in a swellable polymer matrix and expanding the swellable polymer matrix and the brain tissue embedded therein.

48. A composition comprising a plurality of different vectors at stoichiometric ratios, wherein each different vector encodes a fusion protein comprising a different epitope tag linked to a scaffold protein, and wherein the plurality of different vectors are configured to express the fusion proteins in cells of a mammalian tissue.

49. The composition of claim 48, wherein the plurality of different vectors are viral vectors.

50. The composition of claim 49, wherein the plurality of different vectors are AAV vectors or sindbis virus vectors.

51. The composition of any one of claims 48-50, comprising more than 10 different vectors each expressing a fusion protein comprising a different epitope tag, and two or more or all of the different epitope tags are linked to the same scaffold protein.

52. The composition of claim 51, comprising about 30 different vectors each expressing a fusion protein comprising a different epitope tag linked to a common scaffold protein.

53. The composition of any one of claims 48-52, wherein the different epitope tags are peptide tags between about 6 and about 30 amino acid residues in length.

54. The composition of any one of claims 48-53, wherein the scaffold protein is a fluorescent protein.

55. A plurality of different fusion proteins each comprising a different peptide tag linked to a common scaffold protein, wherein the different peptide tags are between about 6 and about 30 amino acid residues in length and the common scaffold protein is a fluorescent protein.

56. The plurality of different fusion proteins of claim 55, comprising more than 10 different fusion proteins each comprising a different peptide tag.

57. The plurality of different fusion proteins of claim 56, comprising about 30 different fusion proteins each comprising a different peptide tag.

58. A mammalian tissue comprising a plurality of different vectors in contact with cells of the mammalian tissue, wherein each different vector encodes a fusion protein comprising a different epitope tag linked to a scaffold protein, and wherein the plurality of different vectors are configured to express the fusion proteins in cells of the mammalian tissue.

59. The mammalian tissue of claim 58, comprising more than 10 different vectors each encoding a different peptide tag.

60. The mammalian tissue of claim 59, comprising about 30 different vectors each encoding a different peptide tag.

61. A mammalian tissue comprising a plurality of different fusion proteins expressed in cells of the mammalian tissue, wherein each different fusion protein comprises a different peptide tag linked to a scaffold protein, and wherein two or more cells in the mammalian tissue each expresses a different combination of peptide tags selected from the plurality of different fusion proteins.

62. The mammalian tissue of claim 61, wherein the mammalian tissue is a brain tissue, and wherein: each different peptide tag of the plurality of different fusion proteins is expressed randomly in about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the neurons labeled by the different peptide tags, or each different peptide tag of the plurality of different fusion proteins is expressed randomly in over 50% of the neurons labeled by the different peptide tags, optionally wherein each different peptide tag is expressed randomly in about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the neurons labeled by the different peptide tags.

63. The mammalian tissue of claim 61 or claim 62, comprising more than 10 different fusion proteins each comprising a different peptide tag linked to a common scaffold protein expressed in cells of the mammalian tissue.

64. The mammalian tissue of claim 63, comprising about 30 different fusion proteins each comprising a different peptide tag linked to a common scaffold protein expressed in cells of the mammalian tissue.

65. A set of binders, comprising: i) a first plurality of binders recognizing a first subset of multiple different epitope tags, wherein each binder in the first plurality is configured to be detected in a different channel of fluorescent microscopy, and ii) a second plurality of binders recognizing a second subset of the multiple different epitope tags, wherein the second subset is different from the first subset, and wherein each binder in the second plurality is configured to be detected in a different channel of fluorescent microscopy, wherein the multiple different epitope tags are peptide tags between about 6 and about 30 amino acid residues in length.

66. The set of binders of claim 65, further comprising: iii) a third plurality of binders recognizing a third subset of the multiple different epitope tags, wherein each binder in the third plurality is configured to be detected in a different channel of fluorescent microscopy, iv) a fourth plurality of binders recognizing a fourth subset of the multiple different epitope tags, wherein the second subset is different from the first subset, and wherein each binderin the fourth plurality is configured to be detected in a different channel of fluorescent microscopy, and v) a fifth plurality of binders recognizing a fifth subset of multiple different epitope tags, wherein each binder in the fifth plurality is configured to be detected in a different channel of fluorescent microscopy.

67. The set of binders of claim 65 or claim 66, wherein each subset comprises two, three, four, five, or more different epitope tags and is nonoverlapping with another subset.

68. The set of binders of any one of claims 65-67, wherein each binder is an antibody or epitope binding fragment thereof.

69. A mammalian tissue comprising a plurality of about 30 different fusion proteins expressed in cells of the mammalian tissue, wherein each of the plurality of fusion proteins comprises a different epitope tag linked to a common scaffold protein, wherein two or more cells in the mammalian tissue each expresses a different combination of epitope tags selected from the plurality of different fusion proteins, wherein the mammalian tissue is a brain tissue and each different epitope tag of the plurality of different fusion proteins is expressed randomly in about 50% of the neurons labeled by the different epitope tags, and wherein the mammalian tissue is in contact with a plurality of binders recognizing a subset of the different epitope tags, wherein each binder in the plurality is configured to be detected in a different channel of fluorescent microscopy.

Images