Methods and systems for physical expansion and imaging of biological samples

Abstract

Described herein are methods and systems for physical expansion and imaging of biological samples. In one aspect of the disclosure, a method of preparing a biological sample for generating an image of its ultrastructure with an imaging instrument includes a) physically expanding the sample by a factor of at least 2 in at least one dimension; and b) bulk labeling a plurality of components of the sample with at least one reagent to introduce contrast.

Description

[0001] Statement regarding federally funded research or development

[0002] This invention was completed with government support under license numbers DK045735 and OD020142 granted by the National Institutes of Health in the United States. The government enjoys certain rights in this invention.

[0003] Cross-references to related applications

[0004] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 989,158, filed March 13, 2020. The entire contents of that application are incorporated herein by reference. Background of the Invention

[0006] Fluorescence microscopy offers the benefits of high-contrast imaging and high-precision labeling. However, fluorescence microscopy is limited in its ability to image samples, particularly proteins against the backdrop of cellular ultrastructure. Individual protein types can be labeled, which creates contrast and allows the user to image them. However, the remainder of the sample remains unlabeled and therefore invisible under the fluorescence microscope. Labeling the remainder of the sample to reveal this background does not provide a solution, as it cannot label at a sufficiently high density to reveal the fine structure (ultrastructure) of the cells. Furthermore, the optical resolution of fluorescence microscopy is insufficient to reveal these fine structures. Similar limitations have been experienced in related microscopy fields. In electron microscopy (EM), three-dimensional images of samples are feasible; however, three-dimensional images require continuous data acquisition over days to weeks to generate images of these samples. Summary of the Invention

[0007] The disclosure described herein provides an optical microscopy imaging method for revealing the ultrastructure of cells. The method described herein combines the physical expansion of the sample with non-specific labeling of the largely expanded sample. Expanded samples allow users to label de-crowded samples at a much higher density in bulk than before, and if the expansion factor is large enough, magnify fine structures to a scale where structures can be resolved without the need for electron microscopy (EM) or specific labeling (e.g., immunofluorescence).

[0008] In one aspect of this disclosure, a method for preparing a biological sample for generating an image of its ultrastructure using an imaging instrument includes (a) physically expanding the sample by a factor of at least 2 in at least one dimension; and (b) batch labeling multiple components of the sample with at least one reagent to introduce contrast.

[0009] This aspect can include various implementation methods. In one implementation, the sample includes cellular components, cells, tissue sections, biofilms, patient-derived samples, or combinations thereof.

[0010] In another embodiment, the sample is chemically fixed, cryopreserved, unfixed, or a combination thereof.

[0011] In another embodiment, the imaging instrument includes a fluorescence optical microscope, a transmitted light microscope, a reflected light microscope, a scattered light microscope, a super-resolution microscope, a mobile phone camera, a camera, an ultrasound, an X-ray, a magnetic resonance, an electron microscope, or a combination thereof.

[0012] In another embodiment, the reagents that introduce contrast include fluorescent dyes, non-fluorescent dyes, metal particles, quantum dots, dielectric particles, or combinations thereof.

[0013] In another embodiment, the reagent that introduces contrast is an initiator or catalyst for the amplification reaction. In some cases, the amplification reaction is based on in situ hybridization, click chemistry, enzyme-mediated, peroxidase-based, polymerization-based, or uses chromogenic or chemiluminescent substrates.

[0014] In another embodiment, at least one of the reagents is used to label proteins, post-translational protein modifications, amino acids, synthetic amino acids, anabolic metabolites, lipids, nucleotides, nucleic acids, carbohydrates, or combinations thereof.

[0015] In another embodiment, at least one of the reagents is an amine-reactive, thiol-reactive, carboxyl-reactive, tyrosine-reactive, glutamine-reactive, lipophilic probe, or a combination thereof.

[0016] In another embodiment, at least one of the reagents is a succinimide ester (including N-hydroxysuccinimide (NHS) ester), isocyanate, isothiocyanate, benzoyl fluoride, carboxylic acid ester, tetrafluorophenyl (TFP) ester, thiodichlorophenol (SDP) ester, carbonyl azide, or sulfonyl chloride; or an aldehyde-containing reagent, including coumarin, pyrene, o-phthalaldehyde (OPA), iodoacetamide, maleimide, 2-thiopyridine, 3-arylpropynitrile, benzyl halide, bromomethyl ketone, hydrazine, hydroxylamine, amine; or a combination thereof.

[0017] In another implementation, at least two batch labels are used to create multichannel images.

[0018] In another implementation, sample features are automatically identified from the image using computational methods.

[0019] In another embodiment, the sample is expanded by a factor of 12 to 24 in each direction.

[0020] In another embodiment, at least one additional reagent specifically labels the cellular components.

[0021] In another aspect, methods for preparing biological samples include (a) immobilizing the sample; (b) embedding the biological sample in a swellable polymer containing a fixative-modified monomer; (c) breaking chemical bonds within the biological sample; (d) swelling the sample in a solvent; and (e) labeling the sample with at least one batch label and swelling the sample in another solvent.

[0022] This aspect can be implemented in various ways. In one embodiment, at least one of the reagents is an amine-reactive fixative, and the swellable polymer comprises at least one amine-functionalized monomer. In some cases, the amine-reactive fixative is thermally reversible, such as formaldehyde (FA); or cleavable, such as dimethyl octyldiimide (DMS), dimethyl heptaethylenediimide (DMP), and dimethyl hexamethylenediimide (DMA); or combinations thereof. In some cases, at least one amine-functionalized monomer is acrylamide (AAm), allylamine (ADP), 2-vinylpyridine (2-VP), N-(2-aminoethyl)acrylamide hydrochloride, 2-aminoethyl methacrylate hydrochloride, or combinations thereof.

[0023] In another embodiment, at least one of the reagents is thiol-degradable, and the swellable polymer contains at least one thiol-reactive monomer. In some cases, the thiol-degradable fixative is dithiobis(succinimide propionate) (DSP), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), succinimide 3-(2-pyridinedithio)propionate (SPDP), succinimide 6-(3(2-pyridinedithio)propionamide)hexanoate (LC-SPDP), or 4-succinimide oxycarbonyl-α-methyl-α-(2-pyridinedithio)toluene (SMPT) or combinations thereof. In some cases, the thiol-reactive monomer is ethyl pyridyl disulfide methacrylate (PDSMA), ethyl pyridyl disulfide acrylamide (PDSAAm), or combinations thereof.

[0024] In another embodiment, the swellable hydrogel is crosslinked with a polymer crosslinking agent (such as piperazine diacrylamide, at a concentration between 0.001% and 0.2%).

[0025] In another embodiment, the sample is denatured using an anionic detergent (such as sodium dodecyl sulfate) or a reagent with a high ionization sequence (such as urea and guanidine hydrochloride), or a combination thereof.

[0026] In another aspect, a method for preparing biological samples includes (a) embedding the sample in a non-swellable polymer, wherein the polymer chains are covalently linked to the molecules of the sample; (b) embedding the sample in a swellable hydrogel, wherein the polymer chains are not anchored to the molecules of the sample; (c) breaking chemical bonds within the sample; (d) swelling the sample in a solvent, wherein most of the sample’s proteome is retained primarily through entanglement between the protein-polymer hybrid of step (a) and the swellable network of step (b); and (e) labeling the sample with at least one batch label and swelling the sample in another solvent.

[0027] This aspect can include various implementation methods. In one implementation method, the non-swellable polymer is not cross-linked.

[0028] In another embodiment, the non-swellable polymer is a polymer between 0.01% and 80% w / w. In another embodiment, the non-swellable polymer is crosslinked with a non-degradable crosslinking agent at a concentration of less than 0.5% w / w.

[0029] In another embodiment, the non-swellable polymer is crosslinked with a pyrolytic crosslinking agent dissolved before, during, or after step (c).

[0030] In another embodiment, the non-swellable polymer contains an α,β-unsaturated aldehyde polymer.

[0031] In another embodiment, the swellable hydrogel is crosslinked with a polymer crosslinking agent (such as piperazine diacrylamide, at a concentration between 0.001% and 0.2%).

[0032] In another embodiment, the sample is denatured using an anionic detergent (such as sodium dodecyl sulfate) or a reagent with a high ionization sequence (such as urea and guanidine hydrochloride), or a combination thereof.

[0033] In another aspect, methods for preparing biological samples may include (a) immobilizing the sample; (b) embedding the sample in a swellable polymer containing a fixative-modified monomer; (c) breaking chemical bonds within the sample; (d) swelling the sample in a solvent; (e) re-embedding the sample in a neutral polymer crosslinked with a cleavable crosslinking agent; (f) re-embedding the sample in another swellable polymer crosslinked with a crosslinking agent different from the crosslinking agents in the swellable polymer and the neutral polymer; (g) cleaving first and second hydrogel crosslinking agents; and (h) labeling the sample with at least one batch label and swelling the sample in a solvent.

[0034] This aspect can include various implementation methods. In one implementation, most of the sample's proteome is preserved primarily through protein-polymer hybrid entanglement in the polymer network rather than covalent cross-linking.

[0035] In another aspect, a method for preparing a biological sample includes (a) embedding the sample in a non-swellable polymer, wherein the polymer chains are covalently linked to the molecules of the sample; (b) embedding the sample in a swellable hydrogel, wherein the polymer chains are not linked to the molecules of the sample; (c) breaking chemical bonds within the sample; (d) swelling the sample in a suitable solvent; (e) re-embedding the sample in a neutral polymer crosslinked with a cleavable crosslinking agent; (f) re-embedding the sample in a swellable polymer crosslinked with a crosslinking agent different from the crosslinking agent in the polymer of step (e); (g) cleaving the hydrogel crosslinking agent of steps (e) and (f); and (h) labeling the sample with at least one batch label and swelling the sample in a suitable solvent.

[0036] This aspect can include various implementation methods. In one implementation, most of the sample's proteome is preserved primarily through protein-polymer hybrid entanglement in the polymer network rather than covalent cross-linking.

[0037] In another aspect, methods for preparing biological samples may include (a) immobilizing the sample; (b) embedding the sample in a polymer containing a fixative-modified monomer; (c) breaking chemical bonds within the sample; (d) modifying the polymer network by inserting additional molecules into the polymer network to increase its size; and (e) labeling the sample with at least one batch label and swelling the sample in a solvent.

[0038] This aspect can include various implementations. In one implementation, the polymer in step (b) is swellable.

[0039] In another embodiment, the polymer chains are grown via reversible addition-fragmentation chain transfer (RAFT) polymerization of hydrophilic monomers. In some cases, the polymer chains are grown via RAFT photopolymerization of a thiocarbonate-crosslinked polymer network in the presence of hydrophilic monomers, a photocatalyst, and a light source.

[0040] In another embodiment, the polymer of step (b) is synthesized using a pyrolytic crosslinking agent.

[0041] In another embodiment, the polymer of step (b) contains a monomer, a crosslinking agent, or a polymer insertion site, or a combination thereof. In some cases, the insertion site is a polymerizable molecule that enables in-situ polymerization of the monomer insertion. In some cases, the insertion site is conjugated with a bifunctional linear polymer.

[0042] In another embodiment, the in-situ formed polymer or the inserted bifunctional linear polymer contains new monomer or polymer insertion sites, allowing for successive rounds of in-situ polymerization or bifunctional linear polymer insertion, or combinations thereof.

[0043] In another embodiment, the polymer chains formed in situ or inserted are swellable.

[0044] In another implementation, step (d) occurs before step (c) or after step (e).

[0045] In another aspect, methods for preparing biological samples may include (a) embedding the sample in a non-swellable polymer, wherein the polymer chains are covalently linked to the molecules of the sample; (b) embedding the sample in a hydrogel, wherein the polymer chains are not linked to the molecules of the sample; (c) breaking chemical bonds within the sample; (d) modifying the polymer network by inserting additional molecules into the polymer chains; and (e) labeling the sample with at least one batch label and swelling the sample in a suitable solvent.

[0046] This aspect can include various implementations. In one implementation, the polymer in step (d) is swellable.

[0047] In another embodiment, the polymer chains are grown via reversible addition-fragmentation chain transfer (RAFT) polymerization of hydrophilic monomers. In some cases, the polymer chains are grown via RAFT photopolymerization of a thiocarbonate-crosslinked polymer network in the presence of hydrophilic monomers, a photocatalyst, and a light source.

[0048] In another embodiment, the polymer of step (d) is synthesized using a pyrolytic crosslinking agent.

[0049] In another embodiment, the polymer of step (d) contains a monomer, a crosslinking agent, or a polymer insertion site, or a combination thereof. In some cases, the insertion site is a polymerizable molecule that enables in-situ polymerization of the monomer insertion. In some cases, the insertion site is conjugated with a bifunctional linear polymer.

[0050] In another embodiment, the in-situ formed polymer or the inserted bifunctional linear polymer contains novel monomer or polymer insertion sites, allowing for successive rounds of in-situ polymerization or bifunctional linear polymer insertion, or combinations thereof.

[0051] In another embodiment, the polymer chains formed in situ or inserted are swellable.

[0052] In another implementation, step (d) occurs before step (c) or after step (e).

[0053] In another aspect, a method for preparing biological samples for visualizing cellular and subcellular structures below human eye resolution using unaided human eye may include: a) physically expanding the sample by a factor of at least 10 in at least one dimension; and b) batch labeling multiple components of the sample with at least one reagent to introduce contrast visible to the human eye.

[0054] This aspect can include various implementations. In one implementation, the visible contrast is organic or inorganic pigments, synthetic or natural visible dyes, or metal particles or combinations thereof.

[0055] In another embodiment, visible contrast is a substance with a refractive index greater than 1.33, wherein the substance includes polymers, plastics, glass, liquids, solids, or combinations thereof.

[0056] In another embodiment, the reagent introducing contrast is an initiator or catalyst of the amplification reaction, or a combination thereof. In some cases, the amplification reaction is based on in situ hybridization, click chemistry, enzyme-mediated, peroxidase-based, polymerization-based, or uses chromogenic or chemiluminescent substrates, or combinations thereof. In some cases, the chromogenic substrate is a peroxidase substrate, such as tyramide conjugates, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), 3-amino-9-ethylcarbazole (AEC), and 2,2'-azo-di-[3-ethylphenylthiazoline-6-sulfonic acid] (ABTS), or combinations thereof. In some cases, the reagent initiates polymerization-based signal amplification.

[0057] In some cases, polymerization-based signal amplification is photoinitiated, enzyme-mediated, or a combination thereof. In some cases, the polymer formed is based on acrylamide, acrylate, or polyethylene glycol (PEG), or a combination thereof. In some cases, the photoinitiator is eosin dye. In some cases, the refractive index of the polymer formed by polymerization is higher than that of the swellable polymer used for sample expansion. In some cases, the polymer formed by polymerization-based signal amplification is stained with a visible dye. In some cases, the visible dye is an azo dye, such as Evans Blue (T-1824) or Direct Red 81 (disodium 7-benzamide-4-hydroxy-3-[[4-[(4-sulfonated phenyl)azo]phenyl]azo]naphthalene-2-sulfonate) or a combination thereof.

[0058] In another implementation, the visualized structure is a single cell, organelle, or ultrastructural feature, or a combination thereof.

[0059] In another implementation, the visualized sample is a microorganism.

[0060] In another embodiment, the sample expands more than 25 times.

[0061] In another implementation, at least two batch labels are used to create multichannel images.

[0062] In another embodiment, at least one additional reagent specifically labels the cellular components.

[0063] In another embodiment, the imaging instrument used for imaging the sample is one of a fluorescence optical microscope, a transmitted light microscope, a reflected light microscope, a scattered light microscope, a super-resolution microscope, a mobile phone camera, a camera, an ultrasound, an X-ray, a magnetic resonance, an electron microscope, or a combination thereof.

[0064] In another aspect, the method of preparing a sample may include (a) embedding the sample in a polymer network such that at least some molecules of the sample are covalently crosslinked with the polymer network; (b) physically expanding the polymer network in at least one dimension by a factor of at least 2; and (c) marking the interface between the polymer network and the sample with a reagent to create an expanded representation of at least some sample components.

[0065] This aspect can include various implementation methods. In one implementation method, the sample components include individual proteins, protein complexes, bacteria, viruses, or combinations thereof.

[0066] In another embodiment, the sample is immobilized with aldehydes during the polymer embedding step.

[0067] In another embodiment, the sample is cross-linked with a polymer network via cleavable molecules, and its polymer-anchored components are labeled to represent the interface between the polymer network and the sample. In some cases, the cleavable molecules comprise amino acid reactive groups, hydrogel reactive groups, and cleavable linkers. In some cases, the cleavable linkers are disulfide bridges.

[0068] In another aspect, the method of preparing the sample includes (a) embedding the sample in a polymer network such that at least some molecules of the sample are covalently crosslinked with the polymer network; (b) modifying the polymer network by inserting additional molecules into the polymer network to expand the polymer network; and (c) marking the interface between the polymer network and the sample with a reagent to create an expansion diagram of at least some sample components.

[0069] This aspect can include various implementations. In one implementation, the polymer in step (c) is swellable.

[0070] In another embodiment, the polymer chain is grown via reversible addition-fragmentation chain transfer (RAFT) polymerization of hydrophilic monomers. In some cases, the polymer chain is grown via RAFT photopolymerization of a thiocarbonate-crosslinked polymer network in the presence of hydrophilic monomers, a photocatalyst, and a light source. In some cases, the polymer of step (c) is synthesized using a cleavable crosslinking agent.

[0071] In another embodiment, the polymer of step (c) contains a monomer, a crosslinking agent, or a polymer insertion site, or a combination thereof. In some cases, the insertion site is a polymerizable molecule that enables in-situ polymerization of the monomer insertion. In some cases, the insertion site is conjugated with a bifunctional linear polymer.

[0072] In another embodiment, the in-situ formed polymer or the inserted bifunctional linear polymer contains novel monomer or polymer insertion sites, allowing for successive rounds of in-situ polymerization or bifunctional linear polymer insertion, or combinations thereof.

[0073] In another embodiment, the polymer chains formed in situ or inserted are swellable.

[0074] In another aspect, the method of preparing the sample includes (a) embedding the sample in a polymer network such that at least some molecules of the sample are covalently crosslinked with the polymer network; (b) physically expanding the polymer network by a factor of at least 2 in at least one dimension; (c) re-embedding the sample in a neutral polymer crosslinked with a cleavable crosslinking agent; (d) re-embedding the sample in a swellable polymer crosslinked with a crosslinking agent different from the crosslinking agent in the polymers of steps (a) and (c); (e) cleaving the hydrogel crosslinking agent of steps (a) and (c); and (f) labeling the interface between the polymer network and the sample with a reagent to create an expansion pattern of at least some sample components.

[0075] This aspect can include various implementations. In one implementation, the sample is crosslinked with a polymer network in step (a) via cleavable molecules, and its polymer-anchored components are labeled to represent the interface between the polymer network and the sample. In some cases, the cleavable molecules comprise amino acid reactive groups, hydrogel reactive groups, and cleavable linkers. In some cases, the cleavable linkers are disulfide bridges.

[0076] In another embodiment, the sample is cross-linked with the polymer network in step (a) by cleavable molecules that are subsequently cleaved after step (a), and its polymer-anchored components are re-cross-linked with the polymer network in step (d), subsequently cleaved and labeled to represent the interface. In some cases, the molecules contain amino acid reactive groups, hydrogel reactive groups, and cleavable linkers. In some cases, the cleavable linkers are disulfide bridges. Attached Figure Description

[0077] To more fully understand the nature and desired objectives of the invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings, wherein similar reference characters denote corresponding portions throughout the several views.

[0078] Figure 1 Images of samples expanded using an iterative expansion technique according to an embodiment of the invention are provided. (a) Unexpanded HeLa cells. (b) HeLa cells expanded once. (c) PanExM expanded HeLa cells. The area within the orange box is shown in the inset, revealing Golgi vesicles (arrow heads). Cells in (a)-(c) are labeled with NHS ester. Enlarged boxes in (d) and (c) show cristae in mitochondria. (e) MitoTracker Orange staining of the same area. (f) SYTOX TM Green staining, showing DNA in the mitochondrial nucleoid. (g)(d)-(f) superposition. (h) Cross-section along the dashed line shown in (g). (i) NHS-ester-rich region in the HeLa cell nucleus, showing the nucleolus. (j) SYTOX Green DNA staining, corresponding to the region shown in (i). (k)(i)-(j) superposition. (l) Cross-section along the dashed line shown in (k). (m) NHS-ester-labeled mitochondria. (n) Same region as in (m), showing anti-TOM20 immunostaining and revealing the outer membrane of the mitochondria. (o) Superposition of MN. The light-colored scale bar shows the values ​​corrected for expansion. The shaded scale bar is not corrected for the expansion coefficient.

[0079] Figure 2Images of samples expanded using an iterative expansion technique according to an embodiment of the present invention. (a) NHS-ester-labeled mitochondria in HeLa cells, revealing cristae. (b) Distance distribution between adjacent cristae (n = 123, N = 4 experiments). (c) NHS ester channels in HeLa cells expressing ER membrane-localized Sec61β-GFP. (d) Anti-GFP tags in the same region, revealing ER. (e) Overlay of (c)-(d). (f) Magnified anti-GFP image of the region indicated by the orange box in D, revealing individual ER tubules—clearly resolved as hollow tubules (arrow heads), and a dense network of ER tubules (arrows). (g) Distribution of ER tubule diameters (n = 142, N = 2 cells). (h)-(s) Images from a 3D image overlay characterized by Golgi complexes close to the nuclei of HeLa cells expressing Golgi-localized ManII-GFP. (h)-(k) Images of NHS esters at axial positions of 0.23, 0.61, 0.84, and 1.18 μm, respectively. (l)-(o) Anti-GFP images with the same field of view as HK. (p)-(s) superimposed of (h)-(o). (t) STED super-resolution image showing NHS ester channels in the Golgi apparatus layer of ManII-GFP-expressing HeLa cells. (u) Anti-GFP STED image of the same region. (v) superimposed of (t)-(u). (w)-(y) Perinuclear region of HeLa cells labeled with NHS esters and maleimide (nucleus on the left), revealing the cysteine-rich Golgi complex. (w) NHS ester channels. (x) Maleimide channels. (y) superimposed of (w)-(x). Inset shows magnified white boxes. (z) Linear plot along the dashed line in y, revealing the change from NHS ester to maleimide staining through the Golgi apparatus and nucleus. All scales are corrected for a given coefficient of expansion. For box plots, the median and interquartile ranges are displayed using whiskers stretched to the minimum and maximum values.

[0080] Figure 3 A schematic diagram depicting sample expansion according to an embodiment of the present invention is shown.

[0081] Figure 4 This is an image of HeLa cells labeled with NHS ester, displayed using a colorimetric table similar to that used in EM images.

[0082] Figure 5Measurements of protein retention in samples according to embodiments of the present invention are depicted. A) Comparison of unexpanded samples with samples expanded once. (Unexpanded: n = 2515 cells; Expanded once: n = 294 cells). b) Comparison of samples expanded once with samples expanded twice. (Expanded once: n = 60 cells; Expanded twice: n = 67 cells). The median and quartile ranges must be displayed using values ​​pulled to the minimum and maximum.

[0083] Figure 6 A table depicting the expansion coefficient results of samples according to embodiments of the present invention is provided.

[0084] Figure 7 This is an image of an expanded sample according to an embodiment of the present invention.

[0085] Figure 8-10 This is an image of a palmitoylated proteome labeled according to an embodiment of the present invention.

[0086] Figure 11 These are images of samples using different microscopy techniques according to embodiments of the present invention.

[0087] Figure 12 and 13 The image shows a comparison between a sample obtained using an embodiment of the present invention (left side) and a conventional microscope image (right side).

[0088] Figure 14-17 This is the workflow according to an embodiment of the present invention.

[0089] Figure 18 This describes a sample expansion control system according to an embodiment of the present invention.

[0090] Figure 19 A schematic diagram illustrating the expansion of a sample imprint according to an embodiment of the present invention.

[0091] Figure 20This is a schematic diagram of a pan-expansion microscopy concept according to an embodiment of the present invention. Panel A depicts the procedure: in step (1), the sample is physically expanded more than twice in each dimension. In step (2), the sample is batch-labeled, referred to herein as “pan-staining”. Panel B depicts examples of pan-staining: (1) the reaction of a primary amine (-NH2) on a protein with an N-hydroxysuccinimide (NHS) ester-conjugated dye (star-shaped) produces a protein with a stable protein-dye amide bond. (2) the reaction of a maleimide-conjugated dye (star-shaped) on a protein with a thiol group (-SH) after sample reduction with tris(2-carboxyethyl)phosphine (TCEP) produces a protein with a stable protein-dye thioether bond. (3) the copper-catalyzed azidopalmitoylation of a protein with an alkyne-conjugated dye (star-shaped) produces a protein with a stable triazole bond.

[0092] Figure 21This is a schematic diagram of a pan-dilatational microscopy scheme according to an embodiment of the present invention. Panels A1-A2 depict two different sample-hydrogel embedding procedures. In panel A1, the sample is first chemically fixed and then embedded in a hydrogel containing fixative-modified monomers. In panel A1-a, the sample is fixed with an amine-reactive fixative (e.g., 4% formaldehyde (FA) or 2% FA + 2% dimethyl octyl diimide (DMS)) and then embedded in a swellable hydrogel containing amine-functionalized monomers (e.g., a hydrogel monomer solution of 10% acrylamide (AAm) or 0.1% N-(2-aminoethyl)acrylamide hydrochloride (AEM) added to 20% sodium acrylate (SA) and 0.1% N,N'-methylenebis(acrylamide) (BIS). In plates A1-b, the samples were fixed with a thiol-degradable fixative (e.g., 2% FA + 0.5% dimethyl dithiodipropionylimine (DTBP)) and then embedded in a swellable hydrogel containing thiol-reactive monomers (e.g., a hydrogel monomer solution of 1% pyridyl disulfide methyl methacrylate (PDSMA) added to 20% sodium acrylate (SA) and 0.1% N,N'-methylenebis(acrylamide) (BIS)). In panel A2, the sample is first embedded in a non-swellable polymer, which can be linear (e.g., 15% polyacrylamide (pAAm) or 0.2% α,β-unsaturated aldehyde polymer) or crosslinked (e.g., 15% pAAm + 0.005% BIS or 15% pAAm + 0.05% N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA)), and then embedded in a swelling hydrogel (e.g., 10% AAm + 20% SA + 0.1% BIS for 5-fold expansion or 10% AAm + 18% SA + 0.01% piperazine diacrylamide (PDA) for 8-fold expansion), so that most of the sample components are retained through the entanglement of the non-swellable polymer and the swelling hydrogel. Panels B1-B3 depict three different sample expansion mechanisms for sample denaturation. Sample homogenization can take many forms. It can be in the form of detergent and thermal denaturation (e.g., 5% sodium dodecyl sulfate (SDS) + 50 mM tris(hydroxymethyl)aminomethane (Tris) + 200 mM NaCl, pH 6.8, 75 °C) or in the form of high ionization sequence and thermal denaturation (e.g., 6 M guanidine hydrochloride + 50 mM Tris, pH 6.8, 75 °C). In plate B1, the hydrogels from plates A1-A2 were denatured and swelled directly in deionized water without further modification. In plate B2, the hydrogels from plates A1-A2 were denatured and iteratively swelled to achieve a higher coefficient of expansion. The loss of sample components was minimal because the sample was retained through the re-embedding of polymer entanglements in the gel.More specifically, a hydrogel that expands by approximately 5 times (synthesized using a cleavable crosslinking agent such as DHEBA) is re-embedded in a neutral hydrogel (e.g., 10% AAm + 0.05% DHEBA) to maintain it in its expanded form, and then re-embedded again in a swellable hydrogel (e.g., 10% AAm + 20% SA + 0.1% BIS). The crosslinks of the first and second hydrogels are cleaved to allow the second swellable hydrogel to expand by another approximately 5 times, resulting in a total sample expansion of approximately 25 times. In Plate B3, the hydrogel with the embedded sample is modified and chemically altered, resulting in an increase in both polymer content and size. In Plate B3-a, this modification is in the form of polymer chain elongation. The polymer chains of the hydrogel are crosslinked with trithiocarbonate (e.g., 0.5% bis[(2-propionate)ethyl methacrylate]trithiocarbonate (bisPEMAT; orange dot)) and polymerized and elongated (M; where M = 20% AAm + 8% SA) via reversible addition-fragmentation chain transfer (RAFT) polymerization of hydrophilic monomers. This polymerization reaction occurs in the presence of a photocatalyst (C; where C = 0.035% phenothiazine (PTH)) and is exposed to 400 nm LED light in a deoxygenated environment for more than 6 h. The resulting modified hydrogel is larger in size than that in the original hydrogel, and its coefficient of expansion depends on the nature and concentration of the selected monomers and the conversion rate of the polymerization reaction. In Plate B3-b, the polymer network modification is in the form of polymer chain insertion. In this example, the sample-embedded hydrogel is crosslinked with a cleavable crosslinking agent (e.g., N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA); light blue dot) and synthesized with potential polymer insertion sites (e.g., the clickable monomer propargyl acrylate (PA); orange dot). In step (1), the potential polymer insertion sites are either converted into polymerizable molecules via a click reaction with an azide-acrylate (e.g., azide-PEG-acrylate) or remain intact. In step (2), in the case of polymer insertion via in-situ polymerization, the polymer insertion sites are made polymerizable, and the hydrogel is incubated in a solution of monomers (e.g., 10% AAm + 10% SA) that polymerize in situ with the polymerizable insertion sites. In the case of bifunctional linear polymer insertion, the insertion sites remain intact, and new polymer chains are inserted into the hydrogel using a bifunctional linear polymer (e.g., a poly(acrylamide-co-acrylic acid) polymer with two azide groups at each end). In both cases, the crosslinking of the original hydrogel is broken after polymer chain insertion, allowing the modified hydrogel to extend in water. In plate C, the swollen hydrogels from plates B1-B3 were pan-stained (e.g., using 20 mg / mL N-hydroxysuccinimide (NHS) ester dye in 100 mM NaHCO3) and washed with deionized water to achieve maximum sample swelling.

[0093] Figure 22 The mechanism of iterative expansion through polymer entanglement according to an embodiment of the invention is depicted. Panel A shows a schematic diagram of two proteins embedded in a first expanding gel before sample denaturation and expansion. Dots represent acrylamide monomer modification of the sample fixative. Panel B depicts a schematic diagram of denatured and expanded proteins in the first expanding hydrogel. Panel C depicts a schematic diagram representing the dissolution of the first expanding hydrogel: alkaline hydrolysis of N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) transforms the crosslinked first expanding hydrogel network (dashed box) into long and linear polymer chains capable of forming entanglements. Panel D shows a schematic diagram of proteins embedded in the final expanding hydrogel and re-embedded in a neutral polyacrylamide hydrogel (not shown for simplicity) after the first expansion. Panel E depicts a schematic diagram of protein-polymer hybrids entangled in the final expanding gel after the dissolution of the crosslinking agent in the first expanding gel. Panel F illustrates the definition of polymer entanglement: a polymer chain is likely entangled if it traverses any plane three times (asterisk).

[0094] Figure 23 These are images of samples expanded using a pan-expansion technique according to an embodiment of the invention, revealing the ultrastructure of EM-type cells. Plate A depicts non-expansioned HeLa cells pan-stained with NHS ester dye. Plate B depicts HeLa cells expanded 4 times and pan-stained with NHS ester dye. Plate C depicts pan-ExM expanded HeLa cells pan-stained with NHS ester dye. Plate D depicts the same image as C, but shown in reverse color. Plate E depicts the area within the box in D, revealing landmark cellular ultrastructural features such as mitochondrial cristae (two upper right arrows) and Golgi vesicles (the remaining arrows). Plate F is the same image as in E, but shows the superposition of NHS ester pan-stained channels and maleimide pan-stained channels. Representative images from 3 (A, B) and 11 (CF) independent experiments are shown. Plate G depicts a line cross-section along the dashed line in the lower left corner of Plate E, revealing Golgi vesicles. Panel H depicts a line cross-section along the central dashed line in Panel E, revealing the mitochondrial cristae. Panel I depicts a line cross-section along the dashed line in Panel F, revealing the changes in NHS ester and maleimide staining across the Golgi apparatus layers. Panels AC are displayed using a black-to-white color chart. Panels D and E are displayed using a white-to-black color chart. The scale bar in the upper right corner is not corrected for the coefficient of thermal expansion. Scale bars: (A) 10 μm, (B) 40 μm, (C, D) 100 μm, (E, F) 20 μm.

[0095] Figure 24These are images of samples expanded using a pan-expansion technique according to an embodiment of the present invention, showing conventional immunofluorescence images of mitochondria superimposed on a novel EM-type background image. Plate A depicts anti-COX-IV immunostaining of mitochondria. Plate B depicts the same area as Plate A, showing HeLa cells pan-stained with NHS esters. Plate C depicts a superposition of Plates A and B, revealing the localization of COX-IV in the inner membrane of mitochondria. Plate D depicts anti-TOM20 immunostaining of mitochondria. Plate E depicts the same area as Plate D, showing HeLa cells pan-stained with NHS esters. Plate F depicts a superposition of Plates D and E, revealing the localization of TOM20 on the outer membrane of mitochondria. The scale bar in the upper right corner is not corrected for the expansion coefficient. Scale bar, (AC) 15 μm, (DF) 10 μm.

[0096] Figure 25 Images of samples expanded using a pan-expansion technique according to an embodiment of the invention are shown as conventional Golgi immunofluorescence images superimposed on a novel EM-type background image. Panels A and D depict anti-GFP Golgi immunostaining, revealing different Golgi vesicles. Panels B and E, the same areas as A and D, show HeLa cells pan-stained with NHS esters, revealing the ultrastructural background (e.g., mitochondria, Golgi vesicles, NPCs, nuclei, nucleoli, vacuoles). Panels C and F are superimposed images. The scale bar in the upper right corner is not corrected for the expansion coefficient. Scale bars: (AC) 45 μm, (DF) 20 μm.

[0097] Figure 26 These are images of samples expanded using a pan-expansion technique according to an embodiment of the present invention, showing conventional chromosome staining superimposed on a novel EM-type background image. Plates A and D depict SYTOX Green staining, showing chromosomes. Plates B and E depict HeLa cells stained with NHS ester pan-staining, showing protein-dense microtubules and centromeres. Plates C and F depict superimposed images of plates A and B, and plates D and E, respectively. Scale bars are used to correct for the expansion coefficient. Scale bars: (AC) 1 μm, (DF) 1.5 μm.

[0098] Figure 27Images depicting samples expanded using a pan-expansion technique according to embodiments of the present invention, showing specific proteins against a cellular ultrastructural background. Plate A depicts HeLa cells stained with NHS ester pan-staining. Plate B depicts anti-α-tubulin immunostaining in the same region. Plate C depicts an overlay of Plates A and B. Plate D depicts mitochondria stained with NHS ester pan-staining. Plate E, in the same region as Plate D, shows anti-TOM20 immunostaining and reveals the outer membrane of the mitochondria. Plate F depicts an overlay of Plates D and E. Plate G depicts mitochondria stained with NHS ester pan-staining. Plate H depicts MitoTracker Orange staining in the same region as Plate G. Plate I depicts DNA in the mitochondrial nucleoid stained with SYTOX Green. Plate J depicts an overlay of Plate G1. Plate K depicts a line profile along the dashed line shown in Plate J. Representative images from 3-fold (AF) and 5-fold (GJ) independent experiments are shown. Panel L plots the root mean square (RMS) error relative to distance for comparing pre- and post-exam images of microtubules (n = 5 cells) using pan-ExamM. Lines correspond to the mean, and error bars correspond to the standard deviation. Panels A, D, and G are displayed using a white-to-black color chart. Panels B, E, H, and I are displayed using a black-to-white color chart. Scale bars show the values ​​for dilation correction. Scale bars: (AC) 2 μm, (DF) 1 μm, (GJ) 500 nm.

[0099] Figure 28 These are images of nuclear ultrastructure obtained using the pan-expansion technique according to an embodiment of the present invention. Panel A depicts an image of U-2OS cells in interphase stained with NHS ester pan-staining. Panel B depicts a SYTOX Green nucleic acid staining image of the same region as in Panel A. Panel C depicts a superposition of Panels A and B. Panels DF depict magnified views of the regions outlined by the boxes in Panels AC, showing amine-rich regions corresponding to the nuclear pore complex (NPC), which correspond to circular channels in the SYTOX Green image that do not include chromatin. Arrows point to an NPC and the corresponding chromatin channel. Panels GI depict magnified views of the regions outlined by the green boxes in AC, showing ultrastructural details of the nucleolus. Arrows (from left to right) point to the fibrous center (FC), dense fibrous component (DFC), and granular component (GC), respectively. Representative images from 5 independent experiments (AI) are shown. Panel J depicts a line cross-section along the dashed line shown in Panel F. Panel K depicts a line cross-section along the dashed line shown in Panel I. Panels A, B, D, E, G, and H are displayed using a white-to-black color chart. All scales are corrected for a defined coefficient of thermal expansion. Scales: (AC) 5 μm, (DF) 250 nm, (GI) 1 μm.

[0100] Figure 29 These are images of the ultrastructure of mitosis expanded using a pan-expansion technique according to an embodiment of the present invention. Plate A depicts the SYTOX Green channels of a mitotic U-2OS cell, revealing chromosomes. Plate B depicts anti-α-tubulin immunostaining in the same region as Plate A. Plate C depicts NHS ester pan-staining in the same region as Plates A and B. Plate D depicts a superposition of Plates A and C. Plate E depicts a magnified image of the region outlined by the box in Plate C. Plate F depicts a magnified image of the region outlined by the box in Plate D. The rightmost arrow highlights individual microtubules within the microtubule bundle, and the leftmost arrow points to the centromere. Representative images from three independent experiments (A–F). Plate G depicts a line cross-section along the dashed line shown in Plate E. Plate H depicts a line cross-section along the dashed line shown in Plate F. Plates A, C, and E are displayed using a white-to-black color table. Plate B is displayed using a black-to-white color table. All scales are corrected for a defined coefficient of thermal expansion. Scale: (AD) 2 μm, (E, F) 300 nm.

[0101] Figure 30 Images depicting the ultrastructure of centrioles expanded using a pan-expansion technique according to an embodiment of the invention are shown. Panel A depicts a side view of mature centrioles labeled with NHS-ester pan-ExM in U-2OS cells, revealing subdistal appendages (arrows). Panel B depicts anti-polyglutamate chain (polyE) immunostaining in the same region as Panel A, revealing three distinct polyglutamate-labeled microtubule triplets. Panel C depicts a superposition of Panels A and B. Panel D depicts an axial view of mature centrioles stained with different NHS-ester pan-staining techniques, revealing microtubule triplets (top arrows) and pericentriolar material (PCM) (bottom arrows). Representative images from four (A, D) and one (B, C) independent experiments are included. Panels A, B, and D are displayed using a white-to-black color table. All scale bars are corrected for a defined expansion coefficient. Scale bars: (AC) 200 nm, (D) 100 nm.

[0102] Figure 31These are images of organelle ultrastructures expanded using a pan-expansion technique according to an embodiment of the present invention. Plate A depicts NHS ester-pan-stained mitochondria in HeLa cells. Plate B depicts NHS ester-pan-stained HeLa cells expressing Sec61β-GFP, which is membrane-localized to the ER, in NHS ester-pan-stained cells. Plate C depicts the anti-GFP tag in the same region of Plate B, revealing the ER. Plate D depicts a superposition of Plates A and B. Plate E depicts a magnified image of the region outlined by the box in Plate B, revealing individual ER tubules—clearly resolved as hollow tubules (leftmost arrow) and a dense network of ER tubules (rightmost arrow). Plate F depicts an STED super-resolution image showing NHS ester-pan-stained Golgi apparatus stacks in HeLa cells expressing ManII-GFP. Arrows indicate five distinct Golgi vesicles. Plate G depicts an anti-GFP STED image of the same region as Plate F. Arrows indicate three distinct Golgi vesicles. Plate H depicts a superposition of Plates F and G. Three leftmost arrows point to ManII GFP-positive Golgi vesicles, and two rightmost arrows point to two ManII GFP-negative Golgi vesicles. Representative images from 11 (A), 2 (BE), and 3 (FH) independent experiments. Plate I depicts the distribution of distances between adjacent mitochondrial cristae calculated from cross-sections similar to those shown along the dashed lines in Plate A (n = 123 line sections, N = 4 independent experiments). Plate J depicts the distribution of ER tubule diameters (n = 142 cross-sections, N = 2 cells from 1 independent experiment). Plate K depicts the distribution of intervesicular distances in the Golgi stack (n = 193 line sections, N = 3 independent experiments). Median and interquartile ranges are shown using the must, pulled to the minimum and maximum values. Report mean ± standard deviation. Plates A, B, and F are shown in a white-to-black color chart. Plates C, E, and G are shown in a black-to-white color chart. All scales are corrected for a given coefficient of thermal expansion. Scales: (A) 500 nm, (BE) 1 μm, (FH) 250 nm.

[0103] Figure 32Images of samples expanded using a pan-expansion technique according to an embodiment of the invention are shown, demonstrating compatibility with 3D imaging. Panel AL depicts images from a 3D image overlay, characterized by the Golgi complex adhering close to the nucleus in HeLa cells expressing Golgi-localized ManII-GFP. Panels A, D, G, and J depict NHS ester images at axial positions of 0.23, 0.61, 0.84, and 1.18 μm, respectively, displayed using a white-to-black color table. Panels B, E, H, and K depict anti-GFP images at the same field of view as Panels A, D, G, and J, displayed using a black-to-white color table. Panels C, F, I, and L depict overlays of NHS ester and anti-GFP images. Representative images from five independent (AL) experiments are included. Insets show magnified boxes and reveal individual ManII-positive Golgi vesicles. Panels B, E, H, and K are corrected for crosstalk (see Method). Scale bars and axial positions are corrected for the determined expansion coefficients. Scale bar (AL) 2μm.

[0104] Figure 33 These are images of samples expanded using a pan-expansion technique according to an embodiment of the invention, demonstrating differential pan-staining of the palmitoylated proteome. Plate A depicts HeLa cells with NHS ester pan-staining. Plate B depicts palmitate pan-staining corresponding to the same region shown in Plate A. Plate C depicts an overlay of Plates A and B. The areas within the boxes are shown in the inset and reveal sac-like structures resembling clathrin-coated pits. The rightmost arrow points to the palmitate-rich nuclear envelope. The bottommost arrow points to tubular structures resembling ER tubules near the mitochondria. Plate D depicts HeLa cells with NHS ester pan-staining. Plate E depicts palmitate pan-staining corresponding to the same region shown in Plate D. Plate F depicts an overlay of Plates D and E. The areas within the boxes are shown in the inset and reveal ER tubules (arrows). Plate G depicts an image of HeLa cells with NHS ester pan-staining, showing mitochondria. Plate H depicts palmitate pan-staining corresponding to the same region shown in Plate G. Arrows point to mitochondrial cristae with two palmitate-rich membranes. Plate I depicts a superposition of plates G and H. The two topmost arrows point to mitochondrial cristae, and the two bottommost arrows point to tubular structures resembling ER tubules. Plate J depicts an NHS ester pan-stained image of HeLa cells showing the Golgi apparatus stacks. Plate K depicts palmitate pan-stained areas corresponding to those shown in Plate J. Plate L depicts a superposition of plates J and K. Arrows point to palmitate-rich Golgi vesicles. Representative images from two independent (AL) experiments. Plates A, B, D, E, G, H, J, and K are shown in a white-to-black color chart. Scale bars are not corrected for expansion coefficients. Scale bars: (AC) 30 μm, (DF) 50 μm, (GI) 20 μm, (JL) 10 μm.

[0105] Figure 34 The image shown is of HeLa cells stained with NHS esters using an embodiment of the present invention (Panel A), which is displayed using a reverse color table similar to an EM image (Panel B).

[0106] Figure 35 This is an image of a HeLa cell nucleus expanded ~40 times using a pan-expansion technique, according to an embodiment of the present invention, showing conventional chromosome staining superimposed on a novel EM-type background image. Panel A depicts the cell nucleus stained with NHS ester pan-staining, showing the nucleolar ultrastructure (top arrow: dense fibrous center; rightmost arrow: fibrous component; leftmost arrow: granular component). Panel B depicts the same area as in Panel A, showing granular SYTOXGreen staining corresponding to nucleosomes. Panel C depicts a superposition of Panels A and B. The scale bar is corrected for the expansion coefficient. Scale bar, (AC) 800 nm.

[0107] Figure 36 This is an image of the HeLa cell nuclear surface stained with NHS ester pan-staining, obtained according to an embodiment of the present invention, showing hollow nuclear pore complexes (NPCs). The scale bar is corrected for the coefficient of thermal expansion. Scale bar, 70 nm.

[0108] Figure 37 This is an image of a 50 μm thick mouse brain tissue section, expanded 14 times and stained with NHS ester pan-staining, obtained according to an embodiment of the present invention. The scale bar is not corrected for the expansion coefficient. Scale bar, (AD) 50 μm.

[0109] Figure 38 Images are of 70 μm thick mouse brain tissue sections expanded 5 times and stained with a lipophilic dye, obtained according to embodiments of the present invention. Plate A depicts a BODIPY-TR methyl ester-stained mouse brain tissue section, showing lipophilic structures. Plate B depicts enlarged boxes from Plate A, highlighting membranous structures in neuronal cell bodies (top arrow: ER tubules; middle arrow: lipid droplets; bottom arrow: nuclear membrane). The scale bar is not corrected for the expansion coefficient. Scale bar, (A) 50 μm.

[0110] Figure 39This is a measurement of the expansion coefficient across different structures according to an embodiment of the invention. Panel A depicts the expansion coefficients determined from images of the registered nuclei, mitochondria, and microtubules before and after expansion (nuclei: n = 5 cells; mitochondria: n = 5 cells; microtubules: n = 5 cells, N = 1 experiment). The figure also shows the estimated expansion coefficients determined from average nuclear cross-sectional measurements in the same experiment (n = 39 nuclei; N = 1 experiment). Panel B depicts the average expansion coefficient achieved using pan-ExM (n = 6 experiments). Panel C depicts a table showing the expansion coefficients calculated from average nuclear cross-sectional measurements from 7 independent experiments. In Experiment 3, the crosslinking agent N,N'-cystamine bisacrylamide (BAC) was used in the final hydrogel at a concentration of 0.1% (w / v) instead of N,N'-methylenebisacrylamide (BIS). The expansion coefficients determined from Experiment 3 are not included in the determination of the average expansion coefficient (Panel B). Show the median and interquartile ranges by pulling to the minimum and maximum values. Report the mean ± standard deviation.

[0111] Figure 40 Measurements of protein retention in samples according to embodiments of the present invention are depicted. Panel A depicts a comparison of ER signals in unexpanded samples versus samples expanded once. (Unexpanded: n = 2515 cells, N = 10 FOVs; Expanded once: n = 294 cells, N = 10 FOVs). Panel B depicts a comparison of Golgi apparatus signals in samples expanded once versus samples expanded twice. (Expanded once: n = 60 cells; Expanded twice: n = 67 cells). For each distribution, the median and quartile ranges are shown using dragged-to-minimum and maximum values.

[0112] Figure 41A schematic diagram of a panception according to an embodiment of the invention is depicted, which is a polymerization-based panstaining signal amplification technique applied to swollen cells. Plate A depicts a biological sample (e.g., cells) chemically fixed (e.g., using 4% FA and 0.1% glutaraldehyde (GA)). In Plate B, the sample is swollen more than 10-fold (e.g., 20-fold swollen using the panstaining ExM protocol). The swollen gel has a refractive index (n) close to that of water (n ~ 1.33). In Plate C, cells are panstained with a photoinitiator (e.g., 200 μM amine-reactive eosin-5-isothiocyanate). In Plate D, the sample is incubated in a photopolymerizable monomer solution (e.g., 40% AAm + 2% BIS + 210 mM N-methyldiethanolamine (MDEA) + 35 mM N-vinyl-2-pyrrolidone (VP), where AAm and VP are monomers, MDEA is a tertiary amine, and BIS is a crosslinking agent). The sample was then irradiated with light of an appropriate wavelength to initiate free radical photopolymerization (e.g., 530 nm light at 55 mW / cm²). 2 (Continued for 30 minutes). The resulting photopolymer typically has a higher refractive index (n>1.33) than the expanded hydrogel, making it visible to the naked eye. The photopolymer is optionally stained with a visible dye (e.g., 1 mg / mL Evans blue) to enhance visible contrast.

[0113] Figure 42 The diagram illustrates the chemicals and reactions included in the panception according to embodiments of the present invention. Panel A depicts the chemicals used in the photopolymerization reaction. Panel B depicts the mechanism of photoinitiated free radical generation via eosin and MDEA. Panel C depicts the eosin regeneration reaction in atmospheric oxygen.

[0114] Figure 43 The rationale for using Evans Blue as a visible dye in panception according to an embodiment of the invention is illustrated. Figures AB show that Evans Blue—an anionic dye—effectively stains neutral polyacrylamide hydrogels instead of anionic poly(acrylamide / sodium acrylate) copolymers, regardless of the hydrogel crosslinking agent concentration. Figures A and C depict a photopolymer composed of 210 mM MDEA + 35 mM VP + 40% AAm + 0.1% (A) or 2% (C) BIS % w / w + 12.5 μM eosin, which is used on a 35 mm × 10 mm glass cover glass slide with a 530 nm LED at 12 mW / cm². 2The polymers were polymerized under light for 10 min and stained with 1 mg / mL Evans blue for 20 min. Because these hydrogels are neutral, they were effectively stained with Evans blue. Plates B and D depict polymers composed of 10% AAm + 19% SA + 0.1% (B) or 2% (D) BIS % w / v + 0.25% APS / TEMED, which were polymerized at RT for 3 h and stained with 1 mg / mL Evans blue for 20 min. Because these hydrogels are anionic, they were not stained with Evans blue. Plate E depicts the chemical structure of the polyacrylamide polymer. Plate F depicts the chemical structure of the poly(acrylamide / sodium acrylate) copolymer (blue: negatively charged carboxyl groups). Plate G depicts the chemical structure of the Evans blue dye (blue: negatively charged sulfate groups that repel carboxyl groups in the anionic poly(acrylamide / sodium acrylate) copolymer). The sulfate groups in Evans blue repel the carboxyl groups in the poly(acrylamide / sodium acrylate) copolymer, preventing their staining.

[0115] Figure 44 The rationale for using Direct Red 81 as a visible dye in panception is illustrated according to an embodiment of the present invention. Figure AD depicts the... Figure 43 The same experiments detailed in [the text] show that Direct Red 81 stains neutral hydrogels rather than anionic hydrogels. Plate F depicts the chemical structure of the poly(acrylamide / sodium acrylate) copolymer (shaded: negatively charged carboxyl groups). Plate G depicts the chemical structure of Evans Blue dye (shaded: negatively charged sulfate groups, which repel carboxyl groups in the anionic poly(acrylamide / sodium acrylate) copolymer). The sulfate groups in Direct Red 81 repel the carboxyl groups in the poly(acrylamide / sodium acrylate) copolymer, preventing them from staining.

[0116] Figure 45 Images of a sample processed according to an embodiment of the present invention, showing individual HeLa cells with the naked eye. The HeLa cells were swollen 20-fold and stained with 200 μM eosin-5-isothiocyanate. The sample was swollen in deionized water and sectioned to ~1 mm thickness. The gel was incubated in a photopolymer solution (40% AAm + 2% w / w BIS + 210 mM MDEA + 35 mMVP) and illuminated with a 530 nm LED at ~55 mW / cm². 2Photopolymerization was performed for 15 minutes. The gel was rinsed in water, imaged on an iPhone 11 smartphone, stained with 1 mg / mL Evans blue for 20 min, rinsed in water, and imaged again. Panel A depicts the panception, revealing a visible cluster of ~45 cells stained with Evans blue dye, placed on a 14 mm MatTek disk and imaged against a dark background. Panel B depicts the inset from Panel A, showing single cells with distinguishable nuclei and cytosol. Panel C depicts the same field of view as Panel B after photopolymerization but before staining with Evans blue, showing the visible photopolymer because its refractive index is higher than that of the expanded hydrogel. Images were acquired using an iPhone 11, and contrast was adjusted. The scale bar was not corrected for sample magnification. Scale bar, (A) 1.5 mm, (BC) 0.5 mm.

[0117] Figure 46 These are images of panception-treated samples imaged using phase-contrast microscopy according to an embodiment of the present invention. Plate A depicts an image of the nucleus in HeLa cells that have been expanded 20 times and panception-treated, revealing the nucleolus (arrow). Plate B depicts an image revealing the cytosol in the mitochondria (arrow). Plate C depicts a magnified image of the nucleolus. Plate D depicts an image of the linear pseudopodia (arrow). The scale bar is not corrected for the expansion coefficient. Scale bar, (A, C, D) 50 μm, (B) 20 μm.

[0118] Figure 47 Images of panception-treated samples imaged using both phase-contrast and fluorescence microscopy. Panel A depicts a phase-contrast image of the cytosol of panception-treated HeLa cells, revealing mitochondria. Panel B depicts a fluorescence image of the same region corresponding to Panel A. Panel C depicts a superposition of Panels A and B. Arrows indicate the correlation of mitochondria in the images. The scale bar is not corrected for the coefficient of thermal expansion. Scale bar, (AC) 20 μm.

[0119] Figure 48A schematic diagram of panception in cells expanded 100 times according to an embodiment of the invention is depicted. Panel A depicts a biological sample (e.g., cells) being chemically fixed (e.g., using 4% FA and 0.1% GA). In Panel B, the sample is expanded 20 times using a pan-ExM scheme. The expanded gel has a refractive index (n) close to that of water (n ~ 1.33). In Panel C, the cells are pan-stained with biotin (e.g., 200 μM amine-reactive NHS ester-PEG4-biotin). In Panel D, the sample is expanded another 5 times for a final expansion coefficient of 100 because the second swellable hydrogel (hydrogel, in Panel B) is synthesized with a cleavable crosslinking agent orthogonal to the crosslinking agent used to synthesize the first swellable hydrogel (e.g., N,N'-bis(acryloyl)cysteine ​​(BAC)). In panel E, the sample is labeled with streptavidin conjugated with a photoinitiator (e.g., eosin), and biotin dendritic molecules (e.g., 8-arm PEG-biotin) are optionally used to amplify streptavidin staining. Pan-staining with amine-reactive chemicals can occur after 20-fold expansion (instead of 100-fold expansion) because at higher sample dilutions, the reaction of isothiocyanates (e.g., eosin-5-isothiocyanate) with primary amines on proteins is significantly less efficient (100-fold expansion corresponds to a 1,000,000-fold sample dilution, while 20-fold dilution corresponds to an 8,000-fold sample dilution). On the other hand, the biotin-streptavidin reaction has a higher reaction dissociation constant (K0). d ~10e-14M), which can effectively pan-stain even at very low reactant concentrations. Therefore, a two-step pan-staining program can be used, in which high K d Enzymatic reactions (such as biotin-streptavidin labeling) were used after 100-fold sample expansion. In Plate F, the samples were incubated in a photopolymerizable monomer solution (e.g., 40% AAm + 2% BIS + 210 mM MDEA) at 35 mW / cm². The samples were then irradiated with light of an appropriate wavelength to initiate free radical photopolymerization (e.g., 530 nm light at 55 mW / cm²). 2 (30 minutes later). The resulting photopolymer typically has a higher refractive index (n>1.33) than the expanded hydrogel, making it visible to the naked eye. The photopolymer is optionally stained with a visible dye (e.g., 1 mg / mL of Evans blue) to enhance visible contrast.

[0120] Figure 49A schematic diagram illustrating sample panblotting expansion according to an embodiment of the present invention is shown. In panel A, cells are first treated with N-hydroxysuccinimide disulfide acrylate (NHSDSA). In panel B, the sample is embedded in a swellable hydrogel (e.g., 20% SA + 10% AAm + 0.1% BIS) such that the position of the amino acid lysine is imprinted onto the polymer network. In panel C, the template proteome is removed by proteolytic hydrolysis (e.g., proteinase K) and denaturing agent (e.g., SDS), and the anchor is dissociated with a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)). In panel D, the sample interface (i.e., the imprinted positions of the amino acids) is labeled with a thiol-reactive dye (e.g., maleimide dye), and the hydrogel swells ~5 times in water and is imaged.

[0121] Figure 50 This diagram illustrates the iterative form of generalized imprint expansion. The steps in the drawing board AD are... Figure 49 The steps in Plate D are identical to those in Plate E, except that the swellable hydrogel in Step 2 is crosslinked with alkali-cleavable DHEBA, and the sample interface is not marked in Plate D. In Plate E, the swellable sample is embedded in a neutral polyacrylamide polymer crosslinked with DHEBA to retain it within its swellable volume, and an excess of pyridyl disulfide ethyl methacrylate (PDSMA) is introduced to participate in and reduce the disulfide exchange of thiol groups. The disulfide exchange process involves attacking the thiol at the disulfide, breaking the -SS- bond, and subsequently forming a new disulfide polymer 'anchor' molecule that includes a portion of the original disulfide compound. In this way, the position of the lysine residues is re-imprinted in the subsequent swellable hydrogel with nanometer precision. In Plate F, the sample is embedded in a second swellable hydrogel, and the anchor is dissociated by TCEP reduction. This process (the steps in Plate CF) can be repeated iteratively using hydrogels with a reversible acrylamide crosslinker orthogonal to DHEBA. In panel G, the reduced thiol molecules are conjugated with maleimide-functionalized dyes, and the hydrogel is expanded ~5 times in pure water for a final expansion coefficient of ~25 times.

[0122] definition

[0123] The invention is best understood by referring to the following definitions.

[0124] Bulk labeling

[0125] As used herein, the terms "bulk labeling" or "pan-staining" refer to labeling a large number of non-uniform molecules in a biological sample. This contrasts with specific labeling achieved through techniques such as immunofluorescence, immunohistochemistry, or immunocytochemistry. Typically, pan-staining is applied after sample swelling to densely label newly exposed binding surfaces on biomolecules. Typically, pan-staining labels more than one type of cell compartment. Preferably, imaging of the pan-stained, swollen sample reveals underlying sample ultrastructure (e.g., organelles) without requiring specific labeling.

[0126] As used in this text, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0127] As used herein, unless otherwise specified or obvious from the context, the term "about" is understood to mean within the range of normal tolerances in the art, such as within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless explicitly stated otherwise from the context, all numerical values ​​provided herein are modified by the term "about".

[0128] As used in the specification and claims, the terms “comprising,” “including,” “containing,” “having,” etc., may have the meanings assigned to them under U.S. patent law and may mean “including,” “comprising,” etc.

[0129] As used herein, unless otherwise specified or obvious from the context, the term “or” is to be understood as inclusive.

[0130] The ranges provided in this document are to be understood as abbreviations of all values ​​within that range. For example, the range 1 to 50 is to be understood as including any number, combination of numbers, or subrange of numbers in a group consisting of 1, 2, 3, 4, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (and their fractions, unless the context clearly specifies otherwise). Detailed Implementation

[0131] Iterative dilatation microscopy

[0132] The methods and systems described herein relate to the spatial expansion of samples for microscopy. A sample can be embedded in a first polymer network. This polymer network can spatially expand the sample, for example, by a factor of 4 from its original size. The expanded sample can then be further embedded in a second polymer network, which can further expand the sample spatially. The components of the sample can be preserved through the entanglement of polymer chains caused by the first and second polymer networks. Thus, the sample can be significantly expanded in size relative to its original form (e.g., by a factor of 21). This significant expansion can result in high resolution and high clarity of the entire sample structure using fluorescence microscopy.

[0133] Sample marking

[0134] Samples can be labeled using a variety of reagents. For example, samples can be labeled using specific reagents or global (e.g., pan-) reagents, or combinations thereof. Specific reagents can label specific molecules or proteomic-specific components of a sample. Examples of specific reagents include, but are not limited to, lipid-specific reagents, DNA-specific reagents, reagents for covalently or electrostatically conjugated proteins, amine-reactive probes, etc. Similarly, global reagents can label many sample components indiscriminately.

[0135] In some cases, samples can be labeled using bulk labeling. Bulk labeling can include reagents that react with a class of chemical groups (such as hydroxyl, aldehyde, amino, etc.). In other cases, samples can be labeled using specific or molecular-specific labeling. For example, specific or molecular-specific labeling can utilize reagents that react in a highly targeted manner with predetermined molecules in the sample (such as direct and indirect immunofluorescence and fluorescent proteins).

[0136] In some cases, at least one of the reagents is a succinimide ester (including N-hydroxysuccinimide (NHS) ester), isocyanate, isothiocyanate, benzoyl fluoride, carboxylic acid ester, tetrafluorophenyl (TFP) ester, thiodichlorophenol (SDP) ester, carbonyl azide, or sulfonyl chloride. In some cases, at least one of the reagents is an aldehyde-containing reagent, including coumarin, pyrene, o-phthalaldehyde (OPA), and naphthiadicarboxaldehyde (NDA). In some cases, at least one of the reagents is thiol-reactive, including iodoacetamide, maleimide, 2-thiopyridine, 3-arylpropynitrile, benzyl halide, and bromomethyl ketone. In some cases, at least one of the reagents is an aryl chemical, such as NBD halides. In some cases, at least one of the reagents reacts with tyrosine residues (such as diazonium salts and PTAD). In some cases, at least one of the reagents can bind to the N- or C-terminus of a protein. In some cases, at least one of the reagents can perform bisorthogonal modification on proteins containing non-natural amino acids such as L-azidohomalanine and L-homopropylglycine.

[0137] In some cases, at least one of the reagents reacts with a carboxylic acid on a protein, such as hydrazine, hydroxylamine, or an amine. In some cases, at least one of the reagents reacts with glutamine residues via transglutaminase-catalyzed transamidation. In some cases, at least one of the reagents binds to a protein through hydrophobic interactions, such as SYPRO Orange. In some cases, at least one of the reagents is a lipophilic dye that binds to the cell membrane, such as BODY TR methyl ester. In some cases, at least one reagent is an antibody. In some cases, at least one reagent is applied to the sample—while it is alive.

[0138] In some cases, multiple reagents can be used to label a sample. For example, at least two reagents can be used to label a sample, which can then be recorded in at least two channels of a sample image. In some cases, at least two reagents are represented by different colors.

[0139] Furthermore, the sample can be labeled with one or more reagents at different steps of the expansion technique. For example, the sample can be labeled before it is embedded in the polymer network. In some cases, the reagent can be embedded within the polymer network so that the sample can be labeled while being embedded in the polymer network. In some cases, the sample can be labeled after expansion. When iterative expansion is performed, the sample can be labeled after the first, second, third, etc. expansions.

[0140] In some cases, batch labeling of samples after 10-fold or more expansion allows for the identification of organelles based on their morphological characteristics, resulting in pan-staining with optical contrast equivalent to heavy metal staining in electron microscopy (EM).

[0141] Sample fixation

[0142] In some cases, enzymatic reactions in a sample can be inactivated (e.g., chemically) before the sample is embedded in a polymer network, while retaining its structure; this process is called immobilization. Chemical immobilization of biological samples can be reversed if the immobilized molecules readily degrade under certain conditions, or if altering temperature, pH, pressure, and / or adding specific reactants to equilibrium favors dissociation reactions. Examples of thermally reversible immobilizers include commonly used formaldehyde (FA). Examples of degradable immobilizers include diimine esters, such as thiol-crackable dithiodipropionylimine dimethyl ester (DTBP), base-crackable dimethyl octyldiimine (DMS), dimethyl heptaethylenediimine (DMP), and dimethyl hexamethylenediimine (DMA); and diamine-reactive crosslinking agents, including thiol-crackable dithiodibis(succinimide propionate) (DSP) and hydroxylamine-crackable ethylene glycol bis(succinimide succinate) (EG). S), thiol-crackable 1,5-difluoro-2,4-dinitrobenzene (DFDNB); and isobifunctional N-hydroxysuccinimide-pyridine dithiol crosslinking agents, including thiol-crackable succinimide 3-(2-pyridinedithio)propionate (SPDP), succinimide 6-(3(2-pyridinedithio)propionamide)hexanoate (LC-SPDP) and 4-succinimide oxycarbonyl-α-methyl-α(2-pyridinedithio)toluene (SMPT).

[0143] Sample embedding

[0144] The sample can be contacted with a hydrogel composed of a variety of synthetic monomers in the form of olefinically unsaturated polymerizable molecules. These monomers include, but are not limited to, electrolyte monomers, which cause the hydrogel to swell in low-ionic solutions such as sodium acrylate (SA).

[0145] If the sample is fixed with a reversible and amine-reactive crosslinking agent, the embedded hydrogel preferably contains an amine-functionalized monomer in excess of the fixing material to simultaneously quench the fixation and the modification reaction formed by the fixative. Examples of amine-reactive crosslinking agents include formaldehyde (FA), dimethyl octyldiimide (DMS), dimethyl heptaethylenediimide (DMP), and dimethyl hexamethylenediimide (DMA). Examples of amine-reactive monomers include acrylamide (AAm), allylamine (ADP), 2-vinylpyridine (2-VP), N-(2-aminoethyl)acrylamide hydrochloride (AEM), and 2-aminoethyl methacrylate hydrochloride (AMA).

[0146] If the sample is fixed with a thiol-reactive fixative (such as a crosslinking agent with disulfide bridges), the embedded hydrogel preferably contains a thiol-reactive monomer in addition to the fixative material to react with the fixation modification via disulfide exchange. Examples of thiol-reactive fixatives include dithiobis(succinimide)propionate (DSP), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), succinimide 3-(2-pyridinedithio)propionate (SPDP), succinimide 6-(3(2-pyridinedithio)propionamide)hexanoate (LC-SPDP), and 4-succinimide oxycarbonyl-α-methyl-α-(2-pyridinedithio)toluene (SMPT). Examples of thiol-reactive monomers include ethyl pyridyl disulfide methacrylate (PDSMA) and ethyl pyridyl disulfide acrylamide (PDSAAm).

[0147] In some cases, the sample is conjugated with a non-swellable polymer material before contact with the swelling polymer material. The aim is to allow the hybrid formed between the non-swellable polymer and the sample to entangle with the swelling polymer while retaining and expanding the sample content within the swelling polymer network. In one embodiment, the non-swellable polymer is linear (e.g., non-crosslinked) and consists of polymerizable monomers such as acrylamide, acrylate, methacrylamide, methacrylate, allylamine, allyl alcohol, acryloyl, and methacryloyl. In one embodiment, the non-swellable polymer is linear and consists of α,β-unsaturated aldehyde polymers. In one embodiment, the non-swellable polymer is crosslinked with a non-degradable crosslinking agent such as N,N'-methylenebis(acrylamide) (BIS), N,N'-methylenediacrylamide, and piperazinediacrylamide. In one embodiment, the non-swellable polymer is crosslinked with a degradable crosslinking agent, such as N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), N,N'-(bisacryloyl)cystamine (BAC), and N,N'-diallyl L-tartrate diamide (DATD)). In a later embodiment, the crosslinking is typically degraded after embedding in the swelling polymer material and before the sample swells.

[0148] Sample expansion

[0149] Samples can be swelled by embedding them in a polymer network. The polymer network can be swellable, allowing it to expand when a liquid (e.g., water) is absorbed. As the polymer network expands, the components of the embedded sample can separate proportionally. Therefore, the sample size can increase linearly. For example, swelling using a first polymer may result in cell size expansion by a factor of 3 to 5. In some cases, the polymer network may be part of a hydrogel. For example, standard direct swelling may result in cell expansion by a factor of 3 to 5. Using low concentrations of a rigid crosslinking agent (such as piperazine-acrylamide), sample swelling can be increased to 8 to 10 times without compromising the mechanical stability of the hydrogel. Example compositions of hydrogels may include, but are not limited to, polyacrylamide, polyacrylates, or combinations thereof.

[0150] In some cases, polymer networks can take the form of a polymer mesh. The mesh can comprise interwoven polymer chains. The spacing between the interwoven polymer chains can be on the nanoscale. For example, before expansion, the mesh size of the polymer network can include 3 nm.

[0151] Without being constrained by theory, the applicant believes that the dilation method described herein reduces the emission density of the labeled sample (through spatial distribution). This allows for imaging using relatively simple imaging equipment.

[0152] Sample expansion

[0153] Samples can be swelled by embedding them in a polymer network. The polymer network is dialyzable, allowing it to swell when a liquid (e.g., water) is absorbed. As the polymer network swells, the components of the embedded sample can be separated proportionally. Therefore, the sample size can increase linearly. For example, standard direct swelling can result in cells swelling by a factor of 3 to 5. Using low concentrations of a rigid crosslinking agent (such as piperazine-acrylamide), sample swelling can be increased to 8 to 10 times without compromising the mechanical stability of the hydrogel. Examples of hydrogel compositions may include, but are not limited to, polyacrylamide, polyacrylates, or combinations thereof.

[0154] In some cases, polymer networks can take the form of a polymer mesh. The mesh can comprise interwoven polymer chains. The spacing between the interwoven polymer chains can be on the nanoscale. For example, before expansion, the mesh size of the polymer network can include 3 nm.

[0155] Iterative expansion of samples through polymer entanglement

[0156] When introduced together, polymer chains from one polymer network can become entangled with polymer chains from another polymer network. For example, embedding a sample within a first polymer network and subsequently embedding the sample and the first polymer network together into a second polymer network can result in polymer chain entanglement. This chain entanglement can physically interlock protein-polymer hybrids within the second polymer network. This interlocking prevents the proteome of the embedded sample from being washed away during iterative expansion of the hydrogel. Therefore, the sample expansion technique described herein can iteratively expand the sample size by a factor significantly larger than that of conventional sample expansion techniques (e.g., a factor of 20).

[0157] A combination of at least two orthogonal reversible crosslinkers with polymer entanglement can provide multiple expansion iterations by moving back and forth between the crosslinkers.

[0158] Sample expansion through modification of polymer networks

[0159] In one embodiment, the sample may be embedded in a swellable or non-swellable hydrogel. The hydrogel in which the original sample is embedded may be further modified to increase its three-dimensional dimensions. In some embodiments, modification of the polymer network may be the elongation of its polymer chains, or the insertion and / or formation of new polymer chains therein, or a combination thereof.

[0160] Hydrogels can undergo polymer chain elongation to increase their size. Polymer chain elongation is the insertion of monomers and / or linear polymers into existing polymer chains to increase their length. The elongation factor can be on the order of 2, 5, or higher. In some embodiments, the polymer chains are elongated via reversible addition-fragmentation chain transfer (RAFT) polymerization of hydrophilic monomers. In some cases, the polymer network embedded in the original sample is crosslinked with a trithiocarbonate (e.g., bis[(2-propionate)ethyl methacrylate]trithiocarbonate (bisPEMAT)). In some cases, the RAFT polymerization of the trithiocarbonate-crosslinked polymer network occurs in the presence of polymerizable monomers, a photocatalyst, and light irradiation. In some cases, the photocatalyst is phenothiazine (PTH), the light source is a 400 nm LED, and the incorporated monomers are acrylamide and acrylate. In one embodiment, the sample-embedded hydrogel consists of 10% acrylamide (AAm) + 10% sodium acrylate (SA) + 0.5% bisPEMAT. In some cases, the hydrogels were incubated in a monomer solution of 20% AAM + 8% SA + 0.035% PTH and then exposed to 400 nm LED light in an anaerobic atmosphere for more than 6 hours. In other cases, the resulting modified hydrogels were washed in deionized water and allowed to reach their maximum size.

[0161] In one embodiment, the sample-embedded hydrogel can be modified by polymer chain insertion. Polymer chain insertion can occur through in-situ polymerization of monomer inserts or through the insertion of bifunctional linear polymers. The inserted polymer chains can act as molecular spacers, physically separating the original polymer network chains from each other, thus expanding the sample hybridization.

[0162] In one embodiment, the sample-embedded hydrogel is synthesized using potential monomer insertion sites. In some cases, the potential insertion sites are chemical groups capable of being converted into olefinically unsaturated polymerizable groups. In some cases, the sample-embedded hydrogel is swellable and synthesized using a cleavable crosslinking agent. In some cases, the potential insertion sites are clickable stems (e.g., alkynyl groups) or protected thiols (e.g., pyridine disulfides). These sites can be converted into polymerizable groups by reacting with clickable monomers (e.g., azidoacrylates) or thiol-reactive monomers (e.g., maleimide acrylates or ethyl pyridyl disulfide methacrylate (PDSMA)). In some cases, the potential insertion sites are not converted into polymerizable molecules.

[0163] In one embodiment, in the presence of a polymerization initiator, the sample-embedded hydrogel with polymerizable intercalation sites has new polymer chains formed therein through in-situ polymerization of monomer intercalations. In some cases, these monomers form polyelectrolytes. In some cases, linear polymers are inserted in place of the monomers (or together with the monomers). Examples of linear polymers are polyacrylamide (pAAm) and low-durability-length polyethylene glycol (PEG) polymers. In some cases, the crosslinking of the original hydrogel is broken down after in-situ polymerization and before sample expansion. The modified hydrogel with the new molecular spacers can be expanded in water. In some cases, the embedded sample is expanded 3 times or more.

[0164] In one embodiment, the sample-embedded hydrogel with potential insertion sites has new polymer chains formed therein through the insertion of a bifunctional linear polymer. Preferably, the inserted bifunctional linear polymer is conjugated to the insertion site. Examples of conjugation reactions include, but are not limited to, covalent bonding, enzymatic reactions, and electrostatic interactions. In some cases, the conjugation reaction is based on click chemistry. In some cases, the inserted bifunctional linear polymer is a polyelectrolyte. One example is the insertion of a poly(acrylamide-co-acrylic acid) polymer having two azide groups into a hydrogel with alkyne polymer insertion sites. In some cases, the crosslinking of the original hydrogel is broken after polymer insertion and before sample swelling.

[0165] In one embodiment, the inserted monomer or polymer contains new potential insertion sites, allowing for another round of in-situ polymerization or insertion of bifunctional linear polymers, and thus increasing sample size.

[0166] Sample Panception (Visual Perception)

[0167] In one embodiment, the sample can be expanded, and its bulk signal (i.e., panstaining) is amplified to the point that its outline and structure become visible to the naked eye. In some cases, cells with a diameter of ~50 mm are expanded 20 times in each direction to a size of ~1 mm. The human eye can distinguish structures spaced ~200 mm apart. If the signal has sufficient visible contrast, the enlarged cells are visible to the naked eye. Typically, cells amplified 20 times by a 1:1 ratio of labeled components are not visible to the naked eye. Preferably, the sample is bulk labeled with a reagent capable of initiating or catalyzing a signal amplification reaction. In some cases, the initiator initiates a polymerization-based signal amplification reaction in which bulk staining is amplified up to 1,000,000 times. In some cases, the sample is bulk labeled with an amine-reactive polymerization initiator (e.g., eosin-5-isothiocyanate). In some cases, the formed polymer has a higher refractive index than the background substrate, making it visible to the naked eye. In some cases, the formed polymer is labeled with a visible dye (such as Evans blue) to enhance its visible contrast. Typically, cells that expand 20 to 100 times—with their main signals amplified through aggregation—have structures and potential organelles (e.g., the nucleus and mitochondria) that are visible to the naked eye.

[0168] Sample overprint swelling

[0169] In some cases, it is desirable to reveal the structure of a single untargeted protein against its native ultrastructural background. It is possible to expand the sample into nanoscale protein imprints via a polymer network (one or more), rather than expanding the protein in a swellable hydrogel. In some of these cases, the sample may be too small to allow for proper expansion (e.g., viruses, protein complexes, proteins, etc.). However, imprints (e.g., amino acid positions) created by the sample on the polymer network can be expanded. The polymer network can be expanded after sample embedding. While the sample itself may not expand, the imprint left by the sample on the polymer network will expand proportionally to the size of the polymer network. These imprints (e.g., expanded interfacial surfaces originating from the contact surfaces between the polymer network and the original sample) can then be batch-labeled, which can then be used to identify structural features or components of the original sample. In some cases, the imprints can be iteratively expanded as described above. Figure 49 The diagram illustrates the expansion and marking of the surface interface.

[0170] Additional sample preparation

[0171] Additional sample preparation steps can be implemented to perform or facilitate the sample expansion techniques described herein. In some cases, molecular interactions between sample components are inactivated to allow for homogeneous dissociation of the sample components from each other. In others, molecular interactions are electrostatic, or based on van der Waals forces, hydrogen bonding, and hydrophobic binding. For example, after being embedded in a polymer network, the sample can be defatted and / or denatured using heat and sodium dodecyl sulfate. Defatting and / or denaturation can extract lipids and / or unfolded protein chains from the sample. In some cases, the sample is denatured but not defatted after being embedded in a polymer network. Examples of denaturing agents that are not effective at extracting lipids are reagents with high ionotropic sequences, such as urea, thiourea, lithium perchlorate, lithium acetate, magnesium chloride, formamide, trimethylamine, and guanidine hydrochloride. In some cases, the sample is homogenized by protein hydrolysis with proteases (such as serine, aspartic, metatalo, and cysteine ​​proteases) as well as amino and carboxyl peptidases.

[0172] sample

[0173] Various samples can be expanded using the techniques described herein. For example, samples may include, but are not limited to, at least one cell, at least one tissue section, biofilm, cellular components, patient-derived samples, chemically fixed samples, cryopreserved samples, etc.

[0174] microscope

[0175] Samples can be prepared for microscopic imaging. The microscope can be any type of microscope capable of detecting emitted, transmitted, reflected, or scattered light. For example, microscopes can include fluorescence microscopes, super-resolution microscopes, etc.

[0176] Sample imaging

[0177] Sample expansion can lead to higher-quality imaging of the sample's proteome, especially when imaging the proteome against an ultrastructural background. Traditional microscopy techniques cannot provide the sharpness, resolution, or depth required for imaging the sample proteome against an ultrastructural background. While electron microscopy (EM) can provide three-dimensional imaging of the sample's ultrastructural background, EM requires specialized microscopy techniques to produce images that combine the ultrastructural background with specific tags, resulting in highly specialized instrumentation and continuous data acquisition over days to weeks.

[0178] Conversely, imaging of samples expanded using the techniques described herein can be performed using conventional optical microscopy, which significantly reduces equipment costs, computer processing requirements, and the time required to generate images of ultrastructural samples.

[0179] application

[0180] Images of these expanded samples can be used in a variety of industries and applications. For example, sample expansion can be used for diagnostic imaging. In these cases, reagents may include differential protein markers. Differential protein markers may include markers for at least one post-translational modification. In some cases, a post-translational modification may include acetylation, glycosylation, phosphorylation, ubiquitination, alkylation, ubiquitin-like modification, biotinylation, gamma-glutamylation, glycosylation, isopreneation, esterification, phosphopanylthioethylamineation, phosphorylation, sulfation, selenization, C-terminal amidation, or hydroxylation.

[0181] Another example of using sample dilation for diagnostic imaging is revealing the spatial distribution of candidate drugs within a sample. Another example could include tracing compartmentalized signals in a signal cascade. Another example could include structural analysis of misfolded protein aggregates involved in neurodegenerative diseases. Another example could include visualization of the spatial distribution of different bacterial populations in biofilms. Another example could include identifying chromatin abnormalities in cancer detection.

[0182] Software control

[0183] In some cases, at least a portion of the techniques described herein can be implemented using software control. The control system 1800 can be an electronic device programmed to control the expansion and labeling of a sample. The control system 1800 can be programmed to autonomously perform a sample expansion regimen without requiring input (from a feedback device or user) or can incorporate such input. For example, in Karl Johan Astrom & Richard M. Murray, Feedback Systems: An Introduction for Scientists & Engineers The principle of how to use feedback (e.g., from a sensor) to regulate the operation of a component is described in (2008).

[0184] The control system 1800 can be a computing device, such as a microcontroller (e.g., a...). or IOIO TM Trademarks are available), general-purpose computers (e.g., personal computers or PCs), workstations, mainframe computer systems, etc. Exemplary control systems are in... Figure 18 As described in the description. The control system 1800 may include a processor device (e.g., a central processing unit or "CPU") 1802, a memory device 1804, a storage device 1806, a user interface 1808, a system bus 1810, and a communication interface 1812.

[0185] The processor 1802 can be any type of processing device used to execute instructions, process data, etc.

[0186] The memory device 1804 can be any type of memory device, including any one or more of random access memory (“RAM”), read-only memory (“ROM”), flash memory, electrically erasable programmable read-only memory (“EEPROM”), etc.

[0187] Storage device 1806 can be any data storage device for reading from or writing to any removable and / or integrated optical, magnetic, and / or optical-magnetic storage media, such as hard disks, optical disc read-only memories (CD-ROMs), rewritable CD-DRWs, digital versatile discs (DVD-ROMs), DVD-RWs, etc. Storage device 1806 may also include a controller / interface for connection to system bus 1810. Therefore, memory device 1804 and storage device 806 are suitable for storing data and instructions for a programming process executed on processor 1802.

[0188] The user interface 1808 may include a touch screen, control panel, keyboard, buttons, display, or any other type of interface, which may be connected to the system bus 1810 via a corresponding input / output device interface / adapter.

[0189] The communication interface 1812 can be adapted and configured to communicate with any type of external device or with other components of the gas chromatography system. The communication interface 1812 can be further adapted and configured to communicate with any system or network, such as one or more computing devices on a local area network (“LAN”), wide area network (“WAN”), or the Internet. The communication interface 1812 can be directly connected to the system bus 1810, or it can be connected via a suitable interface.

[0190] Therefore, the control system 1800 can execute processes independently and / or in cooperation with one or more additional devices, and may include algorithms for controlling components of the microscope and / or the sample expansion system according to the invention. The control system 1800 can be programmed or instructed to execute these processes according to any communication protocol and / or programming language on any platform. Thus, the processes can be embodied in data and instructions stored in memory device 1804 and / or storage device 1806, or received at user interface 1808 and / or communication interface 1812 for execution on processor 1802.

[0191] Machine Learning

[0192] In some cases, machine learning can be trained to identify components of the proteome of a sample image or sample expanded according to the expansion technique described herein. Machine learning algorithms can be... Figure 18 It is part of the control system 1800. Machine learning algorithms or estimators can be trained to identify organelles or spatial features of labeled and expanded samples.

[0193] For example, one or more images of samples stained with a specific labeling reagent can be fed into a machine learning algorithm or estimator. Additionally, one or more of the same samples stained with a globally labeled reagent can also be fed into a machine learning algorithm or estimator. The machine learning algorithm or estimator can identify different components of the samples (e.g., by labeling specific samples) and determine characteristics of the identified components (e.g., patterns of emission intensity from the globally labeled reagent). From these identified characteristics, the machine learning algorithm or estimator can compare these characteristics with the components of globally labeled sample images and identify the components of the globally labeled samples (one or more) based on the comparison (and without requiring organelle-specific labels).

[0194] Although machine learning algorithms or estimators are particularly useful for the space dilation method described herein, the applicant believes that machine learning algorithms or estimators (e.g., after being trained on samples space dilated according to the method described herein) can be applied to conventional samples, whether dilated or undilated using the conventional ExM protocol.

[0195] Example process flow #1

[0196] Figure 14 An example workflow process for preparing biological samples for identifying structural features of samples, according to an embodiment of the present invention, is described.

[0197] At step 1405, the sample may physically expand by a factor of at least 2 in at least one dimension. Expansion may include embedding the sample into at least one polymer network. The polymer network may include polymer chains crosslinked with a crosslinking agent. A liquid, such as deionized water, may be introduced into the polymer network, and its size may increase linearly. The embedded sample may also increase in size with the introduction of the liquid.

[0198] In some cases, the embedded sample can then be re-embedded in a second polymer network. The second polymer network may include polymer chains cross-linked with other cross-linking agents. In some cases, these cross-linking agents may be orthogonal to the cross-linking agents of the first polymer network.

[0199] Re-embedding the sample into a second polymer network allows the polymer chains of the first polymer network to become entangled with those from the second polymer network. This entanglement can preserve or retain the sample's proteome during expansion.

[0200] After re-embedding, liquid can be introduced into the second polymer network, causing it to swell. Therefore, the swollen sample can expand further, such as to 4... 2 The coefficient. Furthermore, because the entanglement of the polymer network preserves the sample's proteome, the swollen sample does not significantly degrade during swelling.

[0201] At step 1410, most of the multiple components of the sample can be labeled with at least one reagent. The reagent can be a component-specific reagent, a global reagent, or a combination thereof. Because the different components of the expanded sample are spatially distant from each other, these components can be identified by labeling.

[0202] Example process flow #2

[0203] Figure 15 An example workflow process for preparing biological samples for identifying structural features of samples, according to an embodiment of the present invention, is described.

[0204] In step 1505, the chemically fixed sample can be incubated with a solution of formaldehyde and acrylamide to prevent protein-protein crosslinking while maximizing the formation of protein-acrylamide conjugates.

[0205] At step 1510, the sample can be embedded in a swellable dense hydrogel crosslinked with a cleavable crosslinking agent. At step 1515, the sample can be degreased and denatured using a surfactant and heat. At step 1520, the sample can swell in water. At step 1525, the sample can be re-embedded in a neutral hydrogel crosslinked with a cleavable crosslinking agent. At step 1530, the sample can be re-embedded in a swellable dense hydrogel crosslinked with a crosslinking agent orthogonal to the crosslinking agents in the first and second hydrogels.

[0206] At step 1535, the first and second hydrogel crosslinking agents can be cleaved. At step 1540, the sample can swell in water.

[0207] Example process flow #3

[0208] Figure 16 An example workflow process for preparing biological samples for identifying structural features of samples according to an embodiment of the present invention is described.

[0209] At step 1605, the sample may physically expand by a factor of at least 2 in at least one dimension. Expansion may include embedding the sample into at least one polymer network. The polymer network may include polymer chains crosslinked with a crosslinking agent. A liquid, such as deionized water, may be introduced into the polymer network, and its size may increase linearly. The embedded sample may also increase in size with the introduction of the liquid.

[0210] At step 1610, the protein polymer hybrid of another polymer network can be entangled to retain most of the sample's proteome. The embedded sample can be re-embedded in the second polymer network. The second polymer network may include polymer chains cross-linked with other cross-linking agents. In some cases, these cross-linking agents may be orthogonal to the cross-linking agents of the first polymer network.

[0211] Re-embedding the sample into a second polymer network allows the polymer chains of the first polymer network to become entangled with those from the second polymer network. This entanglement can preserve or retain the sample's proteome during expansion.

[0212] After re-embedding, liquid can be introduced into the second polymer network, causing it to swell. Therefore, the swollen sample can expand further, such as to 4... 2 The coefficient. Furthermore, because the entanglement of the polymer network preserves the sample's proteome, the swollen sample does not significantly degrade during swelling.

[0213] Example process flow #4

[0214] Figure 17 An example workflow process for preparing biological samples for identifying structural features of samples, according to an embodiment of the present invention, is described.

[0215] At step 1705, the sample may be embedded in a polymer network. The polymer network may include polymer chains crosslinked with a crosslinking agent.

[0216] At step 1710, the polymer network can physically expand by a factor of at least 2 in at least one dimension. A liquid, such as deionized water, can be introduced into the polymer network, and its size can increase linearly. The embedded sample can also increase in size with the introduction of liquid.

[0217] At step 1715, the interface between the polymer network and the sample can be labeled with reagents to create an expanded diagram of the structural features of the embedded sample. The reagents can be component-specific, global, or a combination thereof. Because the different components of the expanded sample are spatially distant from each other, these components can be identified through labeling.

[0218] equivalent

[0219] Although preferred embodiments of the invention have been described using specific terminology, such descriptions are for illustrative purposes only, and it should be understood that changes and variations may be made without departing from the spirit or scope of the appended claims.

[0220] By incorporating references

[0221] All patents, published patent applications and other references cited herein are expressly and entirely incorporated herein by reference.

Claims

1. A method for preparing a biological sample for generating an image of its ultrastructure using an imaging instrument, the method comprising: (a) Physically expanding the sample by a factor of at least 2 in at least one dimension to generate an expanded sample; and (b) Labeling the expanded sample with at least one batch labeling reagent to produce a batch labeled sample, wherein the at least one batch labeling reagent labels multiple portions of non-uniform molecules in the biological sample.

2. The method of claim 1, wherein the sample comprises cellular components, cells, tissue sections, biofilms, patient-derived samples, or combinations thereof.

3. The method according to claim 1, wherein the sample is chemically fixed, cryopreserved, unfixed, or chemically fixed and cryopreserved.

4. The method according to claim 1, wherein the imaging instrument includes a fluorescence optical microscope, a transmitted light microscope, a reflected light microscope, a scattered light microscope, a super-resolution microscope, a mobile phone camera, a camera, an ultrasound, an X-ray, a magnetic resonance, an electron microscope, or a combination thereof.

5. The method according to claim 1, wherein the batch labeling reagent comprises fluorescent dyes, non-fluorescent dyes, metal particles, quantum dots, dielectric particles, or combinations thereof.

6. The method according to claim 1, wherein the batch labeling reagent is an initiator or catalyst for the amplification reaction.

7. The method of claim 6, wherein the amplification reaction is in situ hybridization, a click chemistry-based reaction, an enzyme-mediated reaction, a peroxidase-based reaction, a polymerization-based reaction, or uses a chromogenic or chemiluminescent substrate.

8. The method according to claim 1, wherein the batch labeling reagent comprises at least one batch-labeled protein, amino acid, anabolic metabolite, lipid, nucleic acid, carbohydrate or combination thereof.

9. The method according to claim 1, wherein at least one of the batch labeling reagents is an amine-reactive, thiol-reactive, carboxyl-reactive, tyrosine-reactive, glutamine-reactive, lipophilic probe or a combination thereof.

10. The method according to claim 1, wherein at least one of the batch labeling reagents is succinimide ester, isocyanate, isothiocyanate, benzoyl fluoride, carboxylic acid ester, tetrafluorophenyl (TFP) ester, thiodichlorophenol (SDP) ester, carbonyl azide or sulfonyl chloride; aldehyde-containing reagent, coumarin, pyrene, o-phenylenedialdehyde (OPA), iodoacetamide, maleimide, 2-thiopyridine, 3-arylpropynitrile, benzyl halide, bromomethyl ketone, hydrazine, hydroxylamine, amine or a combination thereof.

11. The method of claim 1, wherein at least two batch labeling reagents are used to create multichannel images.

12. The method of claim 1, wherein sample features are automatically identified from the image by computational means.

13. The method of claim 1, wherein the sample is expanded by a factor of 12 to 24 in each direction.

14. The method of claim 1, wherein the sample comprises cells, and wherein the method comprises contacting the sample with at least one additional reagent that specifically labels cellular components.

15. The method of claim 1, further comprising: (a) Fixing the biological sample with a reagent; (b) The biological sample is embedded in a swellable polymer containing a fixative-modified monomer; (c) Prior to the step of physically swelling the sample in a solvent, breaking the chemical bonds within the biological sample; and (d) Further expand the batch of labeled samples in another solvent.

16. The method of claim 15, wherein the batch labeling reagent is an amine reactive fixative, and the swellable polymer contains at least one amine-functionalized monomer.

17. The method of claim 16, wherein the amine reactive fixative comprises: Thermally reversible fixatives or alkali-degradable fixatives, or combinations thereof.

18. The method according to claim 16, wherein the at least one amino-functionalized monomer is acrylamide (AAm), allylamine (ADP), 2-vinylpyridine (2-VP), N-(2-aminoethyl)acrylamide hydrochloride, 2-aminoethyl methacrylate hydrochloride, or a combination thereof.

19. The method of claim 15, wherein the batch labeling reagent is thiol-degradable, and the swellable polymer contains at least one thiol-reactive monomer.

20. The method of claim 19, wherein the thiol-degradable fixative is dithiobis(succinimide propionate) (DSP), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), succinimide 3-(2-pyridinedithio)propionate (SPDP), succinimide 6-(3(2-pyridinedithio)propionamide)hexanoate (LC-SPDP), or 4-succinimide oxycarbonyl-α-methyl-α-(2-pyridinedithio)toluene (SMPT), or a combination thereof.

21. The method of claim 19, wherein the thiol reactive monomer is ethyl pyridyl disulfide methacrylate (PDSMA), ethyl pyridyl disulfide acrylamide (PDSAAm), or a combination thereof.

22. The method of claim 15, wherein the swellable polymer comprises a swellable hydrogel crosslinked with a polymer crosslinking agent at a concentration between 0.001% and 0.2%.

23. The method of claim 15, further comprising denaturing the biological sample with an anionic detergent, a reagent with a high ionization sequence, or a combination thereof prior to sample swelling.

24. The method of claim 1, further comprising: (a) The biological sample is embedded in a non-swellable polymer, wherein the polymer chains of the non-swellable polymer are covalently linked to the molecules of the sample; (b) The sample is embedded in a swellable hydrogel, wherein the polymer chains of the swellable hydrogel are not anchored to the molecules of the sample; and (c) Prior to the step of physically swelling the sample in a solvent, breaking the chemical bonds within the sample, wherein a large portion of the sample's proteome is retained primarily through the entanglement of the protein-polymer hybrid of step (a) with the swellable network of step (b); and (d) Expand the sample in another solvent.

25. The method of claim 8, wherein the protein comprises post-translational protein modifications, the amino acids comprise synthetic amino acids, and the nucleic acid comprises nucleotides.

26. The method of claim 10, wherein the succinimide ester comprises N-hydroxysuccinamide ester.

27. The method of claim 17, wherein the thermally reversible fixative comprises formaldehyde, and wherein the alkali-degradable fixative comprises dimethyl octyl diimide or dimethyl heptyl diimide.

28. The method of claim 23, wherein the anionic detergent comprises sodium dodecyl sulfate, and wherein the reagent with a high ionization sequence comprises urea, thiourea, lithium perchlorate, lithium acetate, magnesium chloride, formamide, trimethylamine, or guanidine hydrochloride.

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