Microscopic imaging method and kit

Abstract

An imaging method is described herein that includes first marking a sample and acquiring a first image. Performing the first labeling includes applying a first target to the sample; applying a first joint to the sample; and applying a first imaging molecule comprising a first detection motif onto the sample. The step of obtaining the first image comprises obtaining the first image of the first detection motif. The first target labels a point of interest, such as a molecule, a complex, a structure, an organelle or a cell, in the sample. The first linker mediates a specific, indirect, and reversible interaction between the first imaging molecule and the first target.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 427,212, filed on November 22, 2022, the entire contents of which are incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Contract No. P30 DK045735 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] An ASCII text file named "047162-7393WO1_Sequence Listing" created on November 7, 2023, containing 116KB KB bytes, is hereby incorporated by reference in its entirety. Background Art

[0007] DNA probes, as programmable probes, have revolutionized certain aspects of fluorescence microscopy. For example, DNA-PAINT has become one of the most promising super-resolution microscopy methods in the past few years. However, conventional DNA-PAINT techniques suffer from relatively slow imaging speeds, are susceptible to background artifacts, and may have limited multiplexing potential.

[0008] Therefore, there is a need for an imaging technique that can enjoy the advantages of DNA-PAINT without suffering from its problems. The present invention addresses this need. Summary of the Invention

[0009] In some aspects, the present invention relates to the following non-limiting embodiments:

[0010] Microscope imaging methods

[0011] In some aspects, the present invention relates to microscopy imaging methods.

[0012] In some embodiments, the method comprises: exposing a sample having a plurality of targets to a plurality of transient single-stranded nucleic acid adaptor molecules; exposing the sample to a plurality of single-stranded nucleic acid imaging molecules; and exposing the sample to an illumination source having a wavelength capable of interacting with the plurality of single-stranded nucleic acid imaging molecules.

[0013] In some embodiments, the transient single-stranded nucleic acid adaptor molecule comprises: a first region having a target-complementary sequence; and a second region having a single-stranded nucleic acid imaging molecule-complementary sequence.

[0014] In some embodiments, the number of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the estimated or actual number of targets.

[0015] In some embodiments, the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules.

[0016] In some embodiments, the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the plurality of single-stranded nucleic acid imaging molecules by a ratio selected from at least about 1, at least about 10, and at least about 100 times.

[0017] In some embodiments, the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the plurality of single-stranded nucleic acid imaging molecules by a factor of about 500.

[0018] In some embodiments, the target-complementary sequence is less than 11 nucleotides.

[0019] In some embodiments, the target-complementary sequence is selected from 6 to 10 nucleotides and 8 to 10 nucleotides.

[0020] In some embodiments, the method further comprises: exposing the sample to an eraser molecule adapted and configured to quench the transient single-stranded nucleic acid adaptor molecule; exposing the sample to a second plurality of transient single-stranded nucleic acid adaptor molecules having a second, different, target-complementary sequence; exposing the sample to a plurality of single-stranded nucleic acid imaging molecules; and exposing the sample to an illumination source having a wavelength capable of interacting with the plurality of single-stranded nucleic acid imaging molecules.

[0021] In some embodiments, the method is performed without washing the plurality of transient single-stranded nucleic acid adaptor molecules from the sample.

[0022] In some embodiments, the eraser molecule and the second plurality of transient single-stranded nucleic acid adaptor molecules are introduced simultaneously.

[0023] In some embodiments, the eraser molecule and the second plurality of transient single-stranded nucleic acid adaptor molecules are introduced sequentially.

[0024] In some embodiments, the plurality of single-stranded nucleic acid imaging molecules comprises a speed-optimized sequence.

[0025] In some embodiments, the plurality of single-stranded nucleic acid imaging molecules are fluorogenic.

[0026] In some embodiments, the plurality of single-stranded nucleic acid imaging molecules are fluorescent; and the detected light change is fluorescence emitted by the single-stranded nucleic acid imaging molecules.

[0027] In some embodiments, single-stranded nucleic acid imaging molecules are individually detected to generate single-molecule localization super-resolution microscopy images.

[0028] In some embodiments, the sample is a biological tissue section.

[0029] In some embodiments, the multiple targets are antibodies or binding ligands that bind to multiple specific proteins in the sample; and each type of antibody or binding ligand is conjugated to a different single-stranded nucleic acid.

[0030] In some embodiments, the single-stranded nucleic acid is an RNA or DNA molecule.

[0031] In some embodiments, a single-stranded nucleic acid imaging molecule comprises a single-stranded nucleic acid coupled to a molecule that exhibits a Raman signature detectable by Raman microscopy.

[0032] In some embodiments, the single-stranded nucleic acid imaging molecule comprises a single-stranded nucleic acid coupled to a nanoparticle.

[0033] In some embodiments, the nanoparticles are gold nanoparticles.

[0034] In some embodiments, the interaction is scattering.

[0035] Reagent test kit

[0036] In some aspects, the invention relates to a kit.

[0037] In some embodiments, a kit comprises: a plurality of transient single-stranded nucleic acid adaptor molecules; and a plurality of single-stranded nucleic acid imaging molecules.

[0038] In some embodiments, the transient single-stranded nucleic acid adaptor molecule comprises: a first region having a target-complementary sequence; and a second region having a single-stranded nucleic acid imaging molecule-complementary sequence.

[0039] In some embodiments, the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules.

[0040] In some embodiments, the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the plurality of single-stranded nucleic acid imaging molecules by a ratio selected from at least about 1, at least about 10, and at least about 100 times.

[0041] In some embodiments, the number or concentration of the plurality of transient non-fluorescent single-stranded nucleic acid adaptor molecules is greater than the plurality of fluorescent imaging molecules by a factor of about 500.

[0042] Imaging methods

[0043] In some aspects, the invention relates to a method of imaging.

[0044] In some embodiments, the method includes making a first mark; and acquiring a first image.

[0045] In some embodiments, performing a first labeling comprises applying to the sample one or more targets comprising a first target comprising a first target single-stranded nucleic acid; applying to the sample a first linker comprising a first linker single-stranded nucleic acid; and applying to the sample a first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif.

[0046] In some embodiments, acquiring a first image comprises acquiring a first image of a first detection motif.

[0047] In some embodiments, the first linker single-stranded nucleic acid comprises: a first region having sufficient sequence complementarity to bind to the target single-stranded nucleic acid; and a second region having sufficient sequence complementarity to bind to the first imaging molecule single-stranded nucleic acid.

[0048] In some embodiments, the first linker binds the target and the first imaging molecule.

[0049] In some embodiments, the first target comprises a first target single-stranded nucleic acid attached to an antibody or polypeptide that specifically binds to a site of interest in a sample, optionally a protein, protein complex, nucleic acid, cellular structure, organelle, or cell.

[0050] In some embodiments, the first target comprises a first target single-stranded nucleic acid attached to a targeting nucleic acid that specifically binds to or is complementary to a point of interest, optionally a nucleic acid, in a sample.

[0051] In some embodiments, the first detection motif is a fluorescent motif, optionally a fluorescent protein, a fluorescent small molecule, or a quantum dot.

[0052] In some embodiments, the first detection motif is a metal nanoparticle, optionally a gold nanoparticle.

[0053] In some embodiments, the first detection motif is a Raman scattering motif, optionally a Raman dye, optionally a Raman dye suitable for stimulated Raman scattering microscopy.

[0054] In some embodiments, the first detection motif is an isotope.

[0055] In some embodiments, the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 1-30.

[0056] In some embodiments, the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 5 to 20.

[0057] In some embodiments, the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 8 to 12.

[0058] In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is on The range is 1*10 4 1 / M*s and 1*10 7 1 / M*s.

[0059] In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is off The range is between 1 1 / s and 0.0001 1 / s.

[0060] In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is d The range is between 10 μM and 1 nM.

[0061] In some embodiments, the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 1 to 30.

[0062] In some embodiments, the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 5 to 20.

[0063] In some embodiments, the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 8 to 12.

[0064] In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is on The range is 1*10 4 1 / M*s and 1*10 7 1 / M*s.

[0065] In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is off The range is between 1000 1 / s and 0.0001 1 / s.

[0066] In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is d The range is between 10 μM and 1 nM.

[0067] In some embodiments, performing the first labeling includes: applying a plurality of first targets to the sample, each first target comprising a first target single-stranded nucleic acid; applying a plurality of first linkers to the sample, each first linker comprising a first linker single-stranded nucleic acid; and applying a plurality of first imaging molecules to the sample, each first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif.

[0068] In some embodiments, a first image of a plurality of first detection motifs of a plurality of first imaging molecules is acquired.

[0069] In some embodiments, each first linker mediates the association of each of the plurality of first targets with each of the plurality of first imaging molecules in a sequence-specific manner.

[0070] In some embodiments, the plurality of first detection motifs do not interfere with each other during acquisition of the first image.

[0071] In some embodiments, the method further comprises: applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker; performing a second labeling; and acquiring a second image of the second detection motif. In some embodiments, performing the second labeling comprises: applying a second target comprising a second target single-stranded nucleic acid to the sample; applying a second linker comprising a second linker single-stranded nucleic acid to the sample; and applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample.

[0072] In some embodiments, the one or more targets applied in the first labeling further include a second target comprising a second single-stranded nucleic acid. In some embodiments, the method further includes: applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker; performing a second labeling; and acquiring a second image of the second detection motif. In some embodiments, performing the second labeling includes: applying a second linker comprising a second linker single-stranded nucleic acid to the sample; and applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample.

[0073] In some embodiments, the second linker single-stranded nucleic acid comprises: a third region having sufficient complementarity to bind to the second target single-stranded nucleic acid; and a fourth region having sufficient complementarity to bind to the second imaging molecule single-stranded nucleic acid.

[0074] In some embodiments, the second linker mediates the association between the second target and the second imaging molecule.

[0075] In some embodiments, the eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first linker, and the eraser molecule prevents hybridization between the first target single-stranded nucleic acid and the first region of the linker, and / or prevents hybridization between the first imaging molecule single-stranded nucleic acid and the second region of the linker.

[0076] In some embodiments, the first target, first linker, first imaging molecule, and eraser molecule are not washed from the sample prior to applying the second target, second linker, and second imaging molecule.

[0077] In some embodiments, one or more of the target, the first linker, the first imaging molecule, and the eraser molecule are not washed from the sample prior to applying the second linker and the second imaging molecule.

[0078] In some embodiments, the signal of the first detection motif and the signal of the second detection motif overlap or are the same.

[0079] In some embodiments, four or more different detection motifs with different signals are used in each of the first label and the second label.

[0080] In some embodiments, the sample is expanded according to expansion microscopy techniques.

[0081] Device

[0082] In some aspects, the present invention relates to an apparatus.

[0083] In some embodiments, the apparatus comprises: a sample holder for holding a sample; a computer-operated liquid applicator for applying liquid to the sample; a computer-operated microscope; and a computer.

[0084] In some embodiments, the computer is programmed to operate the liquid applicator to perform a first application of: one or more targets comprising a first target for specifically binding to a first component in a sample; a first imaging molecule comprising a first detection motif detectable by a microscope; and a first linker for mediating association between the first target and the first imaging molecule.

[0085] In some embodiments, the computer is further programmed to operate the microscope to record a first signal from the first detection motif,

[0086] In some embodiments, the computer is further programmed to operate the liquid applicator to make a second application.

[0087] In some embodiments, the second application includes applying: an eraser molecule for interrupting the first linker-mediated interaction between the first target and the first linker; a second target for specifically binding to a second component in the sample; a second imaging molecule comprising a second detection motif detectable by a microscope; and a second linker for mediating the association between the second target and the second imaging molecule.

[0088] In some embodiments, the one or more targets further comprise a second target for specifically binding to a second component in the sample, and the second application comprises applying: an eraser molecule for interrupting the interaction between the first target and the first linker mediated by the first linker; a second imaging molecule comprising a second detection motif detectable by a microscope; and a second linker for mediating the association between the second target and the second imaging molecule.

[0089] In some embodiments, the computer is further programmed to operate the microscope to record a second signal from a second detection motif.

[0090] In some embodiments, the first application, the recording of the first signal, the second application, and the recording of the second signal are performed sequentially in this order.

[0091] In some embodiments, the device does not remove the liquid applied in the first application before making the second application and / or recording the second signal.

[0092] In some embodiments, the first signal and the second signal overlap or are identical to each other.

[0093] In some embodiments, the first detection motif and the second detection motif are either the first detection motif or the second detection motif is a fluorescent motif, optionally a fluorescent protein, a fluorescent small molecule, or a quantum dot.

[0094] In some embodiments, the first detection motif and the second detection motif are either the first detection motif or the second detection motif are metal nanoparticles, optionally gold nanoparticles.

[0095] In some embodiments, the first detection motif and the second detection motif is the first detection motif or the second detection motif is a Raman scattering motif, optionally a Raman dye, optionally a Raman scattering dye suitable for stimulated Raman scattering microscopy.

[0096] In some embodiments, the first detection motif and the second detection motif are either the first detection motif or the second detection motif is an isotope.

[0097] In some embodiments, the device further comprises a reservoir for storing one or more of a target, a first linker, a first imaging molecule, an eraser, a second target, a second linker, and a second imaging molecule.

[0098] In some embodiments, the method further comprises at least one selected from the group consisting of a first target, a first linker, a first imaging molecule, an eraser, a second target, a second linker, and a second imaging molecule.

[0099] In some embodiments, the first target comprises a first target single-stranded nucleic acid.

[0100] In some embodiments, the first adaptor comprises a first adaptor single-stranded nucleic acid.

[0101] In some embodiments, the first imaging molecule comprises a first imaging molecule single-stranded nucleic acid attached to a first detection motif.

[0102] In some embodiments, the eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first linker.

[0103] In some embodiments, the second target comprises a second target single-stranded nucleic acid.

[0104] In some embodiments, the second adaptor comprises a second adaptor single-stranded nucleic acid.

[0105] In some embodiments, the second imaging molecule comprises a second imaging molecule single-stranded nucleic acid attached to a second detection motif.

[0106] In some embodiments, the first linker ssN comprises: a first region having sufficient sequence complementarity to bind to the first target ssN; and a second region having sufficient sequence complementarity to bind to the first imaging molecule ssN.

[0107] In some embodiments, the second linker single-stranded nucleic acid comprises: a third region having sufficient sequence complementarity to bind to the second target single-stranded nucleic acid; and a fourth region having sufficient sequence complementarity to bind to the second imaging molecule single-stranded nucleic acid.

[0108] In some embodiments, the device comprises a first target, a first linker, a first imaging molecule, an eraser, a second target, a second linker, and a second imaging molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] For a fuller understanding of the nature and intended objectives of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings, wherein like reference characters designate corresponding parts throughout the several views.

[0110] Figures 1A-1B A schematic diagram of an imager according to an embodiment of the present invention being bound to a target of interest (T1) via a transient linker is provided.

[0111] Figures 2A-2BA schematic diagram of an eraser strand according to an embodiment of the present invention is provided.

[0112] Figure 3 (Top panel) Depicts how the same imager can be used with multiple adapters to image multiple targets of interest (over time, e.g., sequentially), according to embodiments of the present invention. Figure 3 (Bottom panel) Depicts how linkers share a common target-complementary region and support multiple imagers, according to embodiments of the present invention.

[0113] Figures 4A-4D , 5A-5B, and 6A-6G depict the concentrations of various linker binding lengths.

[0114] Figures 7A-7D 8A-8D are super-resolution microscopy images using an embodiment of the present invention.

[0115] Figure 9 A single molecule imaging method according to an embodiment of the present invention is described.

[0116] Figure 10 Depicted are a system 1000 and kit 1000a for single molecule imaging according to embodiments of the present invention.

[0117] Figure 11A is a time-lapse series of diffraction-limited imaging of cells according to an embodiment of the present invention.

[0118] Figure 11B is a time-lapse series of diffraction-limited imaging of mouse spleen tissue showing erasure of the CD45 protein signal, according to an embodiment of the present invention.

[0119] Figures 12A-12D A six-target DNA-PAINT image of the Golgi apparatus demonstrating spectrally unlimited super-resolution multiplexed imaging according to an embodiment of the present invention.

[0120] Figures 13A-13E : Proof of concept of FLASH-PAINT according to some embodiments. Figure 13A Schematic diagram of "classical" DNA-PAINT using imagers directly binding to docking sites and FLASH-PAINT using transient linkers. Figure 13B Proof-of-concept DNA origami nanostructures: Sampling of framework-patterned DNA origami by direct DNA-PAINT and 20 nm grid DNA origami by transient linker (FLASH-PAINT). Figure 13C: DNA origami experiments comparing the association rates of direct binding and binding through a transient linker. Figure 13D : Measured (data points) and calculated (curves) association rates for direct and linker-mediated binding at different instantaneous linker concentrations. Figure 13E : 4-plex imaging of a 5nm grid DNA origami, featuring binding sites arranged into letters ("Y," "A," "L," "E"). Each round of data was acquired in 25 minutes, for a total imaging time of approximately 100 minutes. Between 121 and 246 super-resolution images of individual letters were averaged to generate the displayed letters. Figure 28-32 A representative field of view and a single DNA origami letter are shown. Scale bar: Figures 13A-13C :100nm; Figure 13E :20nm.

[0121] Figures 14A-14C : According to some embodiments, molecular target switching by a transient linker-eraser combination. Figure 14A Figure 3: Schematic diagram of molecular target switching. Eraser E1 is neutralized by transient linker 1 in round 2, while the newly added transient linker 2 guides the imager probe to the new target. Figure 14B Quantification of switching efficiency using DNA origami (in the example of linker sequence A19). In the first round of imaging, only the imager was introduced without the transient linker. In the second round, both the transient linker (20 nM) and the imager were applied. Finally, the solution was replaced with the eraser (100 nM) and the imager. After a 3-minute incubation period, a third round of imaging was performed. Figure 14C Time course of switching the labeled molecular target from Tom20 on mitochondria to α-tubulin (microtubules) in U-2OS cells. Samples were placed in medium containing transient adapter 1 to visualize mitochondria and imager pre-acquisition. At the start of acquisition, eraser 1 (to erase the mitochondrial signal) and transient adapter 2 were added to the medium. Scale bar: 5 μm.

[0122] Figures 15A-15B : 9-target FLASH-PAINT images of U-2OS cells, according to some embodiments. Nine different protein targets located in the Golgi complex, mitochondria, and nucleus were imaged at super-resolution. The yellow, red, and green sub-images zoom in on the mitochondria (yellow box), a portion of the Golgi complex and nuclear envelope (red box), and the nucleolus (green box), respectively. For clarity, only a subset of the proteins are shown in these sub-images; labels marked in gray are not shown. Scale bars: 5 μm (overview), 500 nm (zoom in).

[0123] Figures 16A-16G: According to some embodiments, 9-target FLASH-PAINT imaging of normal and bulbous-tipped cilia in RPE pHSmo cells. Figures 16A-16B :Normal cilia ( Figure 16A ; cilia 1) and bulbous tip ( Figure 16B FLASH-PAINT images of nine different protein targets at the cilium 2). Quartiles along the length of the cilium are represented by square boxes. The nine target subsets of the enlarged proximal and distal regions are shown in blue and yellow boxes, respectively. The enlarged box marked "Δz = 50 nm" shows a 50 nm thick cross-section of the acquired 3D super-resolution dataset to highlight the distribution of acetylated tubulin (Actub) within the cilium. The yellow and blue arrows point to the basal distal appendage CEP164 and the transition zone (TZ) Rpgrip1l marker, respectively. At the bulbous tip ( Figure 16B asterisks in ) and varicose veins ( Figure 16B Between the two middle boxes (white arrows), thinning of the ciliary membrane can be observed ( Figure 16B White arrow in yellow box). Figure 16C The bar graph summarizes the axial distribution of nine different targets in two cilia. For each target, the median (line) and the 25% and 75% quartiles (bottom and top of the bar graph) are indicated. Figure 16D : The number of target clusters in each of the four regions of two cilia. Figure 16E : Median distance of target clusters to the central acetylated tubulin (Actub) filament in each of the four regions of two cilia. In the proximal region, Actub and Glutub axonemal targets have the shortest distance to the filament (black dashed rectangle). Figures 16F-16G :With bulbous tip cilia (cilia 2) ( Figure 16F Compared to the black dashed rectangle in &16G), the proximal ends of the two ciliary Sept2 clusters ( Figure 16F ) and remote ( Figure 16G The median distance between clusters of two different targets in the ) region is closer to all other targets in normal cilia (ciliary body 1). In the distal region, Ift88 ( Figure 16G Similar observations were made (see magenta dashed rectangle in the figure). Scale bars: 1 μm (overview); 300 nm (magnification).

[0124] Figures 17A-17P : 12-target FLASH-PAINT imaging of the Golgi complex in untreated and nocodazole-treated HeLa cells according to some embodiments. Figures 17A-17C : Overview of the Golgi complex and secretory pathway in interphase HeLa cells. Different subsets of protein targets are shown in the three panels as indicated by the color markers (targets marked in gray are not shown). Figure 17D: 3D surface reconstruction of the cis, intermediate, and trans cisternae of the Golgi complex. Figures 17E-17G :Zoom in separately Figures 17A-17C The white boxed area highlights a side view of the Golgi stack, revealing the sequential organization of the stacks into cis cisternae, intermediate cisternae, trans cisternae, and the TGN. Figure 17E , the spatial relationship between ERES, ERGIC, and COPI and II vesicles Figure 17F , and showing that megalin is located at the front of the Golgi stack at the edge of the Golgi apparatus Figure 17G . Figure 17H : Median distance between localization events for different targets. Only localization events with a distance less than 500 nm from each other were considered. Median distances > 100 nm are shown in blue. Figures 17I-17P :and Figures 17A-17H Representation of Golgi microstacks in equal nocodazole-treated interphase HeLa cells. Scale bar: 5 μm ( Figures 17A-17C and 17I-17K), 500nm( Figures 17D-17G )、1μm( Figures 17L-17O ).

[0125] Figures 18A-18I : According to some embodiments, FLASH-PAINT is used for super-resolution volumetric multiplexed organelle imaging. Figures 18A-18D : In the 4-channel FLASH-PAINT dataset of HeLa cells (about 2.5 μm thick), ER ( Figure 18A ;Sec61β), mitochondria ( Figure 18B ; Tom20), lysosome ( Figure 18C ; Lamp1) and the Golgi complex ( Figure 18D ; ManII-GFP) depth projection. Figures 18E-18F : 3D rendering of the positioning data of the four markers ( Figure 18E ) and surface rendering ( Figure 18F ). Figures 18G-18H : The number of contact sites (g; defined as distance <100 nm) and the median area of ​​contact sites ( Figure 18H ). Figure 18I : Bar graph of individual contact site areas. Data points represent the contact areas of all identified individual contacts between organelles in the cell. For each type of contact, the median (line) and the 25th and 75th percentile quartiles (bottom and top of the bar) are indicated. Scale bar: 5 μm.

[0126] Figures 19A-19I: Experimental workflow for kinetic measurements using DNA origami according to some embodiments. Two different DNA origami species are used simultaneously. The first DNA origami species has a single docking site for the imager probe bound by a transient linker chain and orthogonal docking sites arranged in a framework pattern ( Figure 20 The second DNA origami species has a single docking site for direct imager probe binding and the same framework pattern, but with a docking sequence that is orthogonal to the docking sequence used in the first DNA origami species. Figure 19A : In the first round of imaging, single docking sites on two DNA origami species are imaged; the same imager probe can be bound to the first DNA origami species through a transient linker and directly bound to the second DNA origami species. Figure 19B : In the second round of imaging, the frame of the first DNA origami species is imaged. Figure 19C : In the third round of imaging, the frame of the second DNA origami species is imaged. Figure 19D : In the final round of imaging, both frames of the two DNA origami species were imaged. Figure 19E : After applying standard single-molecule localization-based super-resolution microscopy post-processing techniques (i.e., localization fitting and drift correction), the first three imaging rounds were aligned with the final round. Figures 19F-19G Using box images of two DNA origami species, we identified individual docking sites and extracted binding kinetics for transient linker-mediated ( Figure 19F ) and directly ( Figure 19G ) Kinetic analysis of binding. Figure 19H : Cross-section of an exemplary field of view. Colors are assigned according to the imaging wheel that records the signal. Figure 19I : Schematic illustration of direct and transient linker-mediated imager binding. Scale bar 100 nm.

[0127] Figure 20 Figure 2: DNA origami designs used in accordance with some embodiments. Schematic representation of all DNA origami designs used in this study. Hexagons represent 3'-staple positions. Blue and red hexagons represent two different staples extended with docking sites for transient binding of imager probes or transient linkers. Orange hexagons depict a staple extended with biotin modification for immobilization on a coverslip surface.

[0128] Figures 21A-21C According to some embodiments, three independent replicates of kinetic experiments were performed to measure the effective association rate of imager probes binding to DNA origami docking sites directly or through transient linkers as a function of transient linker concentration. Figures 19A-19I The workflow described in is used. Figure 13DThe data points shown are the average of these three experiments.

[0129] Figures 22A-22B : Measured off-rate of transient linkers using Imager R2 (high-speed imager) according to some embodiments. Figures 19A-19I Figure 3. Workflow of the 5xR2 imager docking site. Dissociation rates for direct and transient linker-mediated binding of all 12 transient linkers to the 5xR2 imager docking site sequence.

[0130] Figures 23A-23B : Measurement of association rate of transient linkers using Imager R2 (high-speed imager) according to some embodiments. Figures 19A-19I Figure 3. Workflow of the association rates of all 12 transient linkers for direct and transient linker-mediated binding to the 5xR2 imager docking site sequence.

[0131] Figures 24A-24B : Measured off-rates of transient linkers using Imager P1 (classical imager). Figures 19A-19I Figure 3. Workflow of the dissociation rates of direct and transient linker-mediated binding of all 12 transient linkers to the P1 imager docking site sequence.

[0132] Figures 25A-25B : Measured association rate of transient linkers for Imager P1 (classical imager) according to some embodiments. Figures 19A-19I Figure 3. Workflow of the association rates of all 12 transient linkers for direct and transient linker-mediated binding to the P1 imager docking site sequence.

[0133] Figures 26A-26B : Measurement of off-rate of transient linkers using the Imager FP2 (Fluorescence Imager) according to some embodiments. Figures 19A-19I Figure 4. Workflow and dissociation rates of direct and transient linker-mediated binding of all 12 transient linkers to the FP2 imager docking site sequences.

[0134] Figures 27A-27B : Measurement of association rate of transient linkers using the Imager FP2 (fluorescence imager) according to some embodiments. Figures 19A-19I Figure 4. Workflow of the association rates of all 12 transient linkers for direct and transient linker-mediated binding to the FP2 imager docking site sequences.

[0135] Figure 28 :Representative field of view of the 4-way DNA origami alphabet experiment ( Figure 13EImaged DNA origami nanostructures: Round 1: "Y" with a 20 nm grid and a 10 nm grid (red); Round 2: "A" with a 20 nm grid and a 10 nm grid (green); Round 3: "L" with a 20 nm grid and a 10 nm grid (magenta); Round 4: "E" with a 20 nm grid and a 10 nm grid (cyan). The 20 nm and 10 nm grids were imaged in each round and used for drift correction and alignment of individual rounds. Scale bar 500 nm.

[0136] Figure 29 : According to some embodiments, a single DNA origami structure showing the letter "Y". Figure 13E The depicted image is an average of 246 individual structures. Scale bar 100 nm.

[0137] Figure 30 : According to some embodiments, a single DNA origami structure of the letter "A" is shown. Figure 13E The depicted image is an average of 224 individual structures. Scale bar 100 nm

[0138] Figure 31 : According to some embodiments, a single DNA origami structure showing the letter "L". Figure 13E The depicted image is an average of 121 individual structures. Scale bar 100 nm.

[0139] Figure 32 : According to some embodiments, a single DNA origami structure showing the letter "E". Figure 13E The depicted image is an average of 191 individual structures. Scale bar 100 nm.

[0140] Figures 33A-33B Direct comparison of imaging performance of a fluorescent imager, a high-speed imager, and a classical imager according to some embodiments. Mitochondria were immunolabeled with a primary antibody against Tom20. The secondary antibody was conjugated to the docking site of the A3 transient linker. In the first round of sequential imaging experiments, the fluorescent imager FP2 and the transient linker for FP2 were used. After data acquisition, both were washed away and replaced with the speed imager R2 and a transient linker with a 5xR2 motif. After the second round of data acquisition, the imager and transient linker were washed away again and replaced with the classical imager P1 and the corresponding transient linker. After the third round of data acquisition, another round of washing was performed, and the fluorescent imager FP2 and the transient linker for FP2 were reintroduced to verify negligible sample degradation. The comparison shows a significant improvement in signal-to-noise-to-background ratio using the fluorescent imager and the high-speed imager (SNR ≈ 40) compared to the classical imager P1 (SNR ≈ 8). Scale bar 2 μm.

[0141] Figures 34A-34B : Quantification of the erasure efficiency of all 12 transient linker sequences according to some embodiments. Figures 19A-19I Modified workflow: In the first round, only the imager was introduced without the transient linker. In the second round, the transient linker (20 nM) and the imager were used. In the final round of imaging, the solution was replaced with the corresponding erased strand (100 nM) and the same imager (no washing). After a 3-minute wait, the third round of imaging was performed. The analyzed individual docking sites were divided into ten random groups, from which the mean and standard deviation were calculated. All values ​​were normalized.

[0142] Figures 35A-35B : Observation of molecular target switching kinetics according to some embodiments. Figure 35A : U-2OS cells labeled with antibodies against the mitochondrial outer membrane protein Tom20 and α-tubulin. Before the time course shown, the sample was in a medium containing a transient linker that directed the imager R2 to the mitochondria. At the start of image acquisition, the corresponding eraser and a new transient linker that directed the imager to α-tubulin were added. As the α-tubulin signal mediated by the new transient linker increased (τ 1 / 2 ≈200 seconds), the eraser will quickly erase the mitochondrial signal (τ 1 / 2 ≈60 seconds). Figure 35B : Equivalent experiments using antibodies against NPM1 (nucleolus) and lamin B1 (nuclear envelope). Data were acquired using a spinning disk microscope and are diffraction-limited. Scale bar 5 μm.

[0143] Figures 36A-36D : Evaluation of non-specific binding of transient linkers and high-speed imaging systems according to some embodiments. Mitochondrial protein Tom20 in U-2OS cells was immunolabeled using primary and secondary antibodies with the A5 docking site. Figure 36A In the first round, the classical P1 imager and the transient junction directing P1 to A5 were introduced to identify the field of view. The histogram shows the 1D localization projection along the arrow at the highlighted region of interest and demonstrates a robust signal. Figure 36B : After two brief washes, the R2 imager was introduced. Since the corresponding transient linker was not present, no specific binding was expected and the corresponding histogram showed very few localization events. Figure 36C In the third round of imaging, all transient adapters except the correct adapter (A5-5xR2) were added to the R2 imager. Since none of the introduced transient adapters should be able to interact with the docking site, no specific interaction or downstream sampling is expected. The histogram again shows very few localization events, which confirms this. Figure 36D: In the fourth round of imaging, a matching transient linker (A5-5xR2) was introduced with imager R2. As expected, the histogram shows a robust signal comparable to (a). Scale bar 2 μm.

[0144] Figures 37A-37D : Evaluation of non-specific binding of transient linkers and classical imager systems according to some embodiments. Mitochondrial protein Tom20 in U-2OS cells was immunolabeled using primary and secondary antibodies with the A5 docking site. Figure 37A In the first round, the R2 high-speed imager and a transient joint directing R2 to A5 were introduced to identify the field of view. The histogram shows the 1D localization projection along the arrow at the highlighted region of interest and demonstrates a robust signal. Figure 37B : After two brief washes, the P1 imager was introduced. Since the corresponding transient linker was not present, no specific binding was expected and the corresponding histogram showed very few localization events. Figure 37C In the third round of imaging, all transient adapters except the correct adapter (A5-P1) were added to the P1 imager. Since none of the introduced transient adapters should be able to interact with the docking site, no specific interaction or downstream sampling is expected. The histogram again shows very few localization events, which confirms this. Figure 37D : In the fourth round of imaging, the matching transient joint (A5-P1) was introduced with imager P1. As expected, the histogram shows Figure 37A The signal is quite robust. Scale bar 2 μm.

[0145] Figures 38A-38D : Assessment of non-specific binding of transient linkers and fluorescence imager systems according to some embodiments. Mitochondrial protein Tom20 in U-2OS cells was immunolabeled using primary and secondary antibodies with the A5 docking site. Figure 38A In the first round, the classical P1 imager and the transient junction directing P1 to A5 were introduced to identify the field of view. The histogram shows the 1D localization projection along the arrow at the highlighted region of interest and demonstrates a robust signal. Figure 38B : After two brief washes, the FP2 imager was introduced. Since the corresponding transient linker was not present, no specific binding was expected and the corresponding histogram showed very few localization events. Figure 38C In the third round of imaging, all transient adapters except the correct adapter (A5-FP2) were added to the FP2 imager. Since none of the introduced transient adapters should be able to interact with the docking site, no specific interaction or downstream sampling is expected. The histogram again shows very few localization events, which confirms this. Figure 38D: In the fourth round of imaging, the matching transient connector (A5-FP2) was introduced with imager FP2. As expected, the histogram shows the same Figure 38A The signal is quite robust. Scale bar 2 μm.

[0146] Figure 39 According to some embodiments Figures 21A-21C Images of individual imaging rounds. In addition, for single-target imaging rounds, the sample is imaged again with all targets simultaneously for alignment purposes. Scale bar: 5 μm.

[0147] Figures 40A-40O : Cilia-targeting antibody photoactivatable site-specific conjugation (LASIC) according to some embodiments and Figures 22A-22B Images of each imaging wheel, and image segmentation. Figure 40A : Coomassie Brilliant Blue stained SDS-PAGE gel showing direct conjugation of seven different OyOlink adhesive probe sequences (AlphaThera) to cilium-specific antibodies. After incubation with OyOlink probes for 2 hours under 365 nm UV light, the conjugated heavy chain IgG shifted from 60 kDa (- lane) to 75 kDa (+ lane). Figures 40B-40J : Figures 22A-22B Images of the individual imaging wheels. Figure 40K : z-projection of the pH-Smo data, showing the entire 1.5-μm z-range. Figures 40I-40M : Magnified views of two analyzed cilia. Figures 40N-40O : Examples of surfaces, clusters, and filaments generated for pHSmo and ac-tub targets using Imaris software. Scale bar: 5 μm ( Figures 40B-40K ), 1μm( Figures 40L-40O ).

[0148] Figures 41A-41B According to some embodiments Figures 17A-17G Images of individual imaging wheels. In addition to the single-target imaging wheel, the sample is imaged again with all targets simultaneously for alignment purposes. Scale bar: 5 μm.

[0149] Figures 42A-42B According to some embodiments Figures 17I-17O Images of individual imaging wheels. In addition to the single-target imaging wheel, the sample is imaged again with all targets simultaneously for alignment purposes. Scale bar: 5 μm.

[0150] Figure 43 : According to some embodiments, Figures 17A-17PHeatmaps and log2 changes in distance for secretory pathway proteins in untreated and nocodazole-treated HeLa cells are shown. The heatmap on the right represents the log2 change in distance (distance in untreated cells / distance in nocodazole-treated cells).

[0151] Figure 44 According to some embodiments Figure 18B Individual optical sections of a super-resolution image of Tom20. The axial spacing between optical sections is 350 nm. The last panel shows a z-projection, with the color indicating the z position of the localization event. Scale bar: 5 μm.

[0152] Figure 45 According to some embodiments Figure 18A Individual optical sections from a super-resolution image of Sec61β. The axial spacing between optical sections is 350 nm. The last panel shows a z-projection, with the color indicating the z position of the localization event. Scale bar: 5 μm.

[0153] Figure 46 According to some embodiments Figure 18D Individual optical sections of a ManII super-resolution image. The axial spacing between optical sections is 350 nm. The last panel shows a z-projection, with the color indicating the z position of the localization event. Scale bar: 5 μm.

[0154] Figure 47 According to some embodiments Figure 18A Individual optical sections of a super-resolution image of Lamp1 in Figure 1. The axial spacing between optical sections is 350 nm. The last panel shows a z-projection, with the color indicating the z position of the localization event. Scale bar: 5 μm.

[0155] Figure 48 : According to some embodiments, contact sites between the ER (Sec61β), mitochondria (Tom20), lysosomes (Lamp1), and the Golgi complex (ManII-GFP). The first and third columns show the organelle pairs for which contact sites were calculated. Contact sites were calculated for the "blue" organelle relative to the "yellow" organelle. The second and fourth columns show the corresponding contact site maps for the organelles shown in the first or third column. The color map indicates distances >100 nm in blue, and distances <100 nm in white and red (see color bar). Scale bar: 1 μm.

[0156] definition

[0157] The present invention is best understood with reference to the following definitions.

[0158] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0159] Unless otherwise specified or obvious from the context, as used herein, the term "about" is understood to mean within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly dictates otherwise, all numerical values ​​provided herein are modified by the term "about."

[0160] As used in the specification and claims, the terms "comprising," "including," "containing," "having," and the like may have the meanings ascribed to them in U.S. patent law, and may mean "including," "comprising," and the like.

[0161] As used herein, the term "or" should be construed as being inclusive unless otherwise specified or obvious from the context.

[0162] A "transient" joint can be a molecule that can be separated from a target of interest without applying denaturants such as heat, formaldehyde, or stripping the invasive strand of a fluorescent reporter probe from a target of interest. In this way, a transient joint can be temporarily or non-permanently bound. Transient can be assessed according to an experiment or procedure, for example, if a certain portion (e.g., 100% or close to 100%, such as approximately 90%, 95%, 98%, 99%, etc.) will bind to and release the target during the experiment or procedure, then the joint can be said to be transient. Transient can also be assessed based on the binding duration (e.g., between approximately 1 second and 1 minute). Binding time is typically a statistical distribution over time, with the mean value being reported as "binding time." A transient joint can have a washing efficiency of 100% or close to 100%, such as approximately 90%, 95%, 98%, 99%, etc. Transient joints can be, but not necessarily only, effectively removed by rinsing (e.g., with water or a buffer solution). The transient linker does not need to be washed in order to dissociate from the target (although dissociation may also be transient before rebinding to the target).

[0163] 47,48,49, or 50.

[0164] According to some embodiments herein, the term "first / second target" refers to a molecule that specifically binds to and labels a point of interest in a sample, such as a molecule, complex, structure, organelle, wild-type, engineered, or mutant nucleic acid sequence, peptide or protein, or cell in the sample. The "first / second target" is indirectly bound to the "first / first imaging molecule" via a "first / second linker," which simultaneously binds to the "first / second target" and the "first / second imaging molecule." The indirect interaction between the "first / second target" and the "first / second imaging molecule" allows for detection of the molecule, complex, structure, organelle, or cell in the sample by detecting the "first / second imaging molecule."

[0165] In various embodiments, a first linker and its associated imaging molecule can be separated from a first target by contacting the sample with an eraser molecule, and a second target can be applied to label and enable detection of the same or a different point of interest. The first / second distinction refers to this removal and addition of targets, and thus multiple first targets may be applied simultaneously or sequentially before applying an eraser or other means of removing the first target or targets.

[0166] In some embodiments, the first target and the second target (and additional target(s), if any) are applied simultaneously. According to these embodiments, after applying the eraser to disrupt the first target-first linker-first imaging molecule association, the second target does not need to be applied; instead, the second linker and the second imaging molecule can be applied directly to the sample because the second target is already in the same location and marks the point of interest for which the second target is specific. DETAILED DESCRIPTION

[0167] Over the past few years, DNA-PAINT has emerged as one of the most promising super-resolution microscopy methods. Its most prominent advantages are spectrally unlimited multiplexing (Exchange-PAINT), high spatial resolution (sub-5 nm), and the ability to count targets of interest with very predictable binding kinetics. However, an undisputed issue is the relatively slow imaging speed, as the apparent blinking of DNA-PAINT is based on the diffusion of dye-labeled DNA oligonucleotides ("imagers") and the transient binding of these oligonucleotides to the targets of interest.

[0168] In the past two years, rational sequence design and fluorescent imagers have addressed this issue, increasing the speed of DNA-PAINT by up to two orders of magnitude. However, the problem with these approaches is that the sequence design of these imagers significantly reduces the sequence space, which ultimately limits the multiplexing capability (>6 targets of interest).

[0169] In other words, conventional DNA-PAINT is either very slow, susceptible to background artifacts, or very limited in multiplexing space, which is in stark contrast to the needs of spatial omics experiments.

[0170] To make DNA-PAINT the tool of choice for spatial omics experiments and in vitro binding analysis, achieving the fastest and most highly multiplexed DNA-PAINT is crucial.

[0171] Applicants have designed an "adapter" for DNA-PAINT that will enable the fastest and most highly multiplexed fluorescent coatings to address this problem.

[0172] Although certain embodiments of the present invention may be described in the context of DNA-PAINT, the present invention has broad applicability and offers advantages in confocal imaging, amplification microscopy, and other dye-based fluorescence imaging modalities.

[0173] In this concept, the binding of the imager chain is mediated by a linker chain that transiently binds to a docking site at the target of interest. Thus, the linker chain has at least two regions.

[0174] The first part is the binding site for the imager and can be any sequence, including established speed sequences and fluorescence imagers. This part can be the same in each round of the multiplexed experiment (i.e., the same imager can be used for each round).

[0175] The second part of joint is attached to the docking position at target of interest.The sequence can be any sequence (about 1,000,000 available sequences) under the transient binding scheme.Because the joint chain is not fluorescent, background will not be affected, so it can be used with high concentration (that is, it is possible to achieve rapid binding frequency).Therefore, this part of joint does not need to be a speed-optimized sequence.In this case, the trick is to achieve rapid imaging by high concentration of joint chain, so the sequence pool of multiplex will not be limited.

[0176] 1, left panel, a transient linker 102 can include a first end 104 having a target complementary sequence and a second end 106 having a fluorescent imaging molecule complementary sequence. The first end 104 and the second end 106 can be directly adjacent to each other or can be separated by additional nucleotides.

[0177] The right side of Figure 1 is a timeline depicting the sequential binding of the adapter 102 and the imager 108. Ideally, the time that a given adapter docking site 110 on the target of interest (T1) is unoccupied (denoted as y=0) is minimized. The binding time of the adapter can be configured using the number of nucleotides shown in Table 1 below.

[0178]

[0179] Table 1 shows a general relationship, and the binding time may be affected by one or more factors, such as salinity, temperature, pH, etc.

[0180] Because the linker is not fluorescent, the amount and / or concentration can be increased to decrease the time between binding events without increasing the risk of photobleaching or increasing the fluorescence background.

[0181] Reference Figure 9 and Figure 10 Other aspects of the present invention provide a method 900, system 1000, and kit 1000a for single-molecule imaging according to embodiments of the present invention. In step S902, a sample having multiple targets is exposed to multiple transient non-fluorescent single-stranded nucleic acid adaptor molecules 1002. In step S904, the sample is exposed to multiple fluorescent imaging molecules 1004. In step S906, the sample is exposed to an excitation source 1008 having a wavelength capable of exciting the multiple fluorescent imaging molecules.

[0182] In step S908, the sample is exposed to an eraser molecule 1012 adapted and configured to quench the transient non-fluorescent single-stranded nucleic acid linker molecule. In step S902, the sample can be exposed to a second plurality of transient non-fluorescent single-stranded nucleic acid linker molecules having a second, different target-complementary sequence. The sample can then be exposed to a plurality of fluorescent imaging molecules. These plurality of fluorescent imaging molecules can be reintroduced or can remain in the sample environment of step S904. The sample-linker-imager complex can then be imaged again.

[0183] Imaging can be performed using various imagers 1010, such as a microscope. The excitation source 1008 can be integrated into the imager 1010. The kit 1000a can include instructions, such as printed material, detailing the methods described herein (eg, 900).

[0184] Imaging methods

[0185] In some aspects, the invention relates to a method of imaging.

[0186] In some embodiments, the method includes making a first mark; and acquiring a first image.

[0187] In some embodiments, performing the first labeling includes applying one or more targets comprising a first target to the sample, wherein the first target comprises a first target single-stranded nucleic acid; applying a first linker comprising a first linker single-stranded nucleic acid to the sample; and applying a first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif to the sample.

[0188] In some embodiments, acquiring a first image comprises acquiring a first image of a first detection motif.

[0189] In some embodiments, the first linker ssN comprises: a first region having sufficient sequence complementarity to bind to the first target ssN; and a second region having sufficient sequence complementarity to bind to the first imaging molecule ssN.

[0190] In some embodiments, the first linker mediates the association between the first target and the first imaging molecule.

[0191] In some embodiments, the first target is used to label a molecule, complex, structure, organelle, etc. in a sample, such that the first imaging molecule can be localized to the molecule, complex, structure, or organelle via the first target and the first linker. In this way, the molecule, complex, structure, or organelle can be detected by the imaging method described herein by detecting the first detection motif.

[0192] The nature of the first target is not limited. It will be appreciated by those skilled in the art that various molecules / structures in the sample can be labeled in a variety of ways. For example, the first target can include antibodies, non-antibody proteins that specifically interact with molecules or structures, nucleic acids, etc. Antibodies or polypeptides that specifically bind to proteins, protein complexes, nucleic acids, cell structures, organelles, or cells are widely available in the art and can be easily selected by those skilled in the art. Similarly, nucleic acids that specifically bind to or are complementary to nucleic acids of interest are also widely available based on specific imaging experiments.

[0193] In some embodiments, the first target, first linker, first imaging molecule, etc. is not limited to one first target, one first linker, or one first imaging molecule. Based on this specification, one of ordinary skill in the art will understand that the imaging methods herein allow for the use of multiple first targets, first linkers, and first imaging molecules to simultaneously label and image multiple molecules, complexes, structures, organelles, etc. in a sample.

[0194] According to the present specification, a person skilled in the art will understand that the choice of the first detection motif is not limited. The detection motif can be selected according to the available imaging device.

[0195] For example, if the imaging device is a fluorescence microscope, the first detection motif can be a fluorescent motif, such as a fluorescent protein (GFP, RFP, YFP, CFP, etc.), a fluorescent small molecule (xanthene derivatives, cyanine derivatives, squaraine derivatives or ring-substituted squaraine, squaraine rotaxane derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, tetrapyrrole derivatives, dipyrromethene derivatives, etc.), quantum dots, etc.

[0196] For example, if the imaging device is an optical microscope or other type of microscope that can detect metal nanoparticles, the first detection motif can be metal nanoparticles, such as gold nanoparticles, tungsten nanoparticles, silica nanoparticles, iron nanoparticles, copper nanoparticles, selenium nanoparticles, molybdenum nanoparticles, silver nanoparticles, gadolinium nanoparticles, holmium nanoparticles, rhenium nanoparticles, platinum nanoparticles, etc.

[0197] For example, if the imaging device is adapted to perform Raman scattering microscopy or otherwise detect Raman scattering, the first detection motif may be a Raman scattering motif, such as a Raman dye, such as a Raman dye suitable for stimulated Raman scattering microscopy.

[0198] For example, if the imaging device is capable of detecting radioactivity or isotopes of an element, the first detection motif may be the isotope.

[0199] In some embodiments, the complementary base pairs K between the first target single-stranded nucleic acid and the first region of the first adapter, between the first imaging molecule single-stranded nucleic acid and the second region of the adapter, and / or between the first imaging molecule single-stranded nucleic acid and the second region of the adapter are selected based on a specific experiment. on , K off , K d The number of such molecules is such that the indirect association between the first target and the first imaging molecule is suitable for the imaging device to specifically detect the first imaging molecule labeled with the first target anchored to the molecule, complex, structure, organelle or cell, and such indirect association can be easily disrupted / competed by the eraser molecule (described elsewhere herein) to allow another round of labeling and detection.

[0200] In some embodiments, the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 1 to 30, such as from 5 to 20, or from 8 to 12. In some embodiments, the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or any range therebetween.

[0201] In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is on The range is about 1*10 3 1 / M*s and approximately 1*10 8 1 / M*s, for example, between about 1*10 4 1 / M*s and approximately 1*10 7 1 / M*s, or about 1*10 5 1 / M*s and approximately 1*10 6 In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is on About 1*10 3 1 / M*s, approximately 1*10 4 1 / M*s, 1*10 5 1 / M*s, 1*10 6 1 / M*s, 1*10 7 1 / M*s, 1*10 8 1 / M*s or any range therebetween.

[0202] In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is off In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is between about 10 1 / s and about 0.00001 1 / s, such as between about 1 1 / s and about 0.0001 1 / s, between about 0.1 1 / s and about 0.001 1 / s, or between about 0.03 1 / s and about 0.003 1 / s. off The range is about 10 1 / s, such as about 1 1 / s, 0.1 1 / s, 0.01 1 / s, 0.001 1 / s, 0.0001 1 / s, 0.00001 1 / s, or any range therebetween.

[0203] In some embodiments, the K between the first target single-stranded nucleic acid and the first region of the adapter is dThe K between the first target single-stranded nucleic acid and the first region of the adapter is in a range of 100 μM to 0.1 nM, such as 10 μM to 1 nM, 1 μM to 10 nM, or 300 nM to 30 nM. d About 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM, about 1 nM, or any range therebetween.

[0204] In some embodiments, the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the adapter ranges from 1 to 30, such as from 5 to 20, or from 8 to 12. In some embodiments, the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the adapter ranges from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or any range therebetween.

[0205] In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is on The range is about 1*10 3 1 / M*s and approximately 1*10 8 1 / M*s, for example, between about 1*10 4 1 / M*s and approximately 1*10 7 1 / M*s, or about 1*10 5 1 / M*s and approximately 1*10 6 In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is on About 1*10 3 1 / M*s, approximately 1*10 4 1 / M*s, 1*10 5 1 / M*s, 1*10 6 1 / M*s, 1*10 7 1 / M*s, 1*10 8 1 / M*s or any range therebetween.

[0206] In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is offThe K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is between about 1 / s and about 0.00001 / s, for example, between about 1000 1 / s and about 0.0001 1 / s, between about 100 1 / s and about 0.001 1 / s, between about 10 1 / s and about 0.01 1 / s, between about 1 1 / s and about 0.1 1 / s. In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is between about 1 / s and about 0.01 1 / s. off About 10000 1 / s, about 1000 1 / s, about 100 1 / s, about 10 1 / s, about 1 1 / s, 0.1 1 / s, 0.01 1 / s, 0.001 1 / s, 0.0001 1 / s, 0.00001 1 / s, or any range therebetween.

[0207] In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is d The range is between 100 μM and 0.1 nM, such as between 10 μM and 1 nM, between 1 μM and 10 nM, or between 300 nM and 30 nM. In some embodiments, the K between the first imaging molecule single-stranded nucleic acid and the second region of the linker is d About 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM, about 1 nM, or any range therebetween.

[0208] As described elsewhere herein, during the first labeling process, there can be more than one first target, first linker, first imaging molecule, etc., so that more than one point of interest in the sample (e.g., molecules, complexes, structures, organelles, cells, etc.) can be simultaneously labeled and detected without first removing the label.

[0209] Thus, in some embodiments, performing a first labeling comprises: applying a plurality of first targets to the sample, each target comprising a first target single-stranded nucleic acid; applying a plurality of first linkers to the sample, each first linker comprising a first linker single-stranded nucleic acid; and applying a plurality of first imaging molecules to the sample, each first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif. In some embodiments, acquiring a first image comprises acquiring an image of a plurality of first detection motifs of the plurality of first imaging molecules. In some embodiments, each first linker mediates the association of each of the plurality of first targets with each of the plurality of first imaging molecules in a sequence-specific manner. In some embodiments, the plurality of first detection motifs do not interfere with each other during acquisition of the first image.

[0210] In some embodiments, a detection motif can interfere with another detection motif if the detection signals given by the two motifs overlap or are identical. For example, the emission spectrum of an enhanced green fluorescent protein (EGFP) molecule is identical to that of another EGFP molecule, so the fluorescence signals of the two molecules can interfere with each other. As another example, the emission spectrum of an EGFP molecule overlaps with the emission spectrum of an enhanced cyan fluorescent protein (ECFP) molecule, so for some fluorescence microscopes, the two molecules may interfere with each other.

[0211] A feature of the imaging methods herein is that the methods allow for dissociation of the first imaging molecule from the first target without washing or stripping the sample (which typically results in sample disruption and / or reduced image quality after washing / stripping). Instead, according to the imaging methods herein, the first imaging molecule can be readily dissociated from the first target by nucleic acids that compete with one or more hybrids required to form the first target-first linker-first imaging molecule complex. Notably, the methods herein can include washing / stripping; these steps are not required nor incompatible.

[0212] Thus, in some embodiments, the imaging method further comprises applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker; performing a second labeling; and acquiring a second image.

[0213] In some embodiments, performing the second labeling comprises: applying a second target comprising a second target single-stranded nucleic acid to the sample; applying a second linker comprising a second linker single-stranded nucleic acid to the sample; and applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample.

[0214] In some embodiments, performing the second labeling does not include applying a second target to the sample. According to some embodiments, in the first labeling, the one or more targets applied to the sample already include both the first target and the second target. According to these embodiments, the targets only need to be applied to the sample once.

[0215] In some embodiments, acquiring a second image comprises acquiring a second image of a second detection motif.

[0216] In some embodiments, the description of the first target, the first linker, and / or the first imaging molecule also applies to the second target, the second linker, and / or the second imaging molecule.

[0217] In some embodiments, the first detection motif and the second detection motif interfere with each other, eg, produce overlapping or identical signals.

[0218] In some embodiments, the second linker single-stranded nucleic acid comprises a third region having sufficient complementarity to bind to the second target single-stranded nucleic acid; and a fourth region having sufficient complementarity to bind to the second imaging molecule single-stranded nucleic acid. In some embodiments, the second linker mediates the association between the second target and the second imaging molecule.

[0219] In some embodiments, the eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first adapter. In some embodiments, the eraser molecule prevents hybridization between the first target single-stranded nucleic acid and the first region of the adapter. In some embodiments, the eraser molecule prevents hybridization between the first imaging molecule single-stranded nucleic acid and the second region of the adapter.

[0220] In some embodiments, the first target, first linker, first imaging molecule, and eraser molecule are not washed from the sample prior to applying the second target, second linker, and second imaging molecule.

[0221] In some embodiments, the signal of the first detection motif and the signal of the second detection motif overlap or are the same.

[0222] In some embodiments, in any one or each of the first label and the second label, 2 or more, such as 3 or more, 4 or more, 5 or more, 6 or more or 7 or more different detection motifs with different signals are used. In some embodiments, in any one or each of the first label and the second label, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 different detection motifs with different signals are used.

[0223] In some embodiments, the sample is expanded according to expansion microscopy techniques. For example, M'Saad et al. describe expansion microscopy techniques (Nature Communications volume 11, Article number: 3850 (2020)).

[0224] Device

[0225] In some aspects, the present invention relates to an apparatus, such as an apparatus for acquiring imaging, such as an apparatus for performing the imaging methods described herein.

[0226] In some embodiments, the apparatus comprises: a sample holder for holding a sample; a computer-operated liquid applicator for applying liquid to the sample; a computer-operated microscope; and a computer.

[0227] In some embodiments, the computer is programmed to: (a) operate a liquid applicator to perform a first application of: one or more targets comprising a first target for specifically binding to a first component in a sample; a first imaging molecule comprising a first detection motif detectable by a microscope; and a first linker for mediating association between the first target and the first imaging molecule.

[0228] In some embodiments, the computer is programmed to: (b) operate the microscope to record a first signal of the first detection motif.

[0229] In some embodiments, the computer is programmed to: (c) operate the liquid applicator to perform a second application of: an eraser molecule for interrupting the first linker-mediated interaction between the first target and the first linker; a second target for specifically binding to a second component in the sample; a second imaging molecule comprising a second detection motif detectable by a microscope; and a second linker for mediating association between the second target and the second imaging molecule. In some embodiments, the second target is one of the one or more targets applied in (a) (along with the first target), and thus (c) does not include application of the second target.

[0230] In some embodiments, the computer is programmed to: (d) operate the microscope to record a second signal from a second detection motif,

[0231] In some embodiments, the computer is programmed to perform operations (a), (b), (c), and (d) sequentially in the order (a), (b), (c), and (d).

[0232] In some embodiments, the first / second targets, first / second linkers, first / second imaging molecules, and eraser molecules are the same as or similar to those described elsewhere herein (eg, in the "Imaging Methods" section).

[0233] In some embodiments, the computer is programmed so that the device does not remove the liquid applied in operation (a) before performing operations (c) and (d).

[0234] In some embodiments, the first signal and the second signal overlap or are identical to each other.

[0235] In some embodiments, the device further comprises a reservoir for storing one or more of the target, the first linker, the first imaging molecule, the eraser, the second linker, and the second imaging molecule.

[0236] In some embodiments, the device further comprises at least one selected from the group consisting of a first target, a first linker, a first imaging molecule, an eraser, a second target, a second linker, and a second imaging molecule.

[0237] In some embodiments, the device comprises a first target, a first linker, a first imaging molecule, an eraser, a second target, a second linker, and a second imaging molecule.

[0238] Example 1: Prophetic Example

[0239] Embodiments of the present invention open the door to a wide range of potential applications, including the following.

[0240] Example 1-1: Spatial Omics

[0241] Nature Methods The journal highlighted spatial omics as a highly multiplexed, spatially resolved technique as the method of the year 2021 ( https: / / www.nature.com / articles / s41592-020-01033-y ). Spatial omics methods (MERFISH, SeqFISH+) are at the forefront of imaging. Xiaowei Zhuang's (Harvard University) laboratory invented multiplexed error-resistant fluorescence in situ hybridization (MERFISH), which allows the observation of hundreds of transcribed RNA molecules or hundreds of genomic sites ( https: / / www.science.org / doi / 10.1126 / science.aaa6090 Long Cai (from Caltech) pioneered SeqFISH+, which allows visualization of tens of thousands of different RNA species within cells. Both methods rely primarily on diffraction-limited readout ( https: / / www.nature.com / articles / s41586-020-03126-2 ). They can also be used in combination with STORM or DNA-PAINT.

[0242] Embodiments of the present invention provide at least two advantages. First, because the linker and imager are instantaneously bound, this should reduce wash times (significantly, the time between images). Second, because the readout is no longer diffraction-limited, it can be used for high-density targets within cells. For example, this can enable spatial proteomics using conventional fluorescence microscopy.

[0243] Example 1-2: In vitro transcription assay

[0244] Highly multiplexed fluorescent PAINT can also be used in the context of in vitro transcription assays. Because the probes are fluorescent, the complexity of illumination and downstream device design can be greatly reduced. Furthermore, super-resolution readout can improve throughput by increasing surface density. Combined with fast imaging probes and rapid washes (due to the instantaneous binding of linkers to imagers), this will significantly increase the speed and throughput of such assays.

[0245] Reduce costs

[0246] A simple DNA strand within the length of an IDT (DNA synthesis company) linker or imager costs about $10. A fluorescently labeled DNA strand costs about $200-500. A fluorescence imager with a fluorescent molecule on one end and a quencher molecule on the other costs about $1000. By using a multiplexing linker strategy, embodiments of the present invention can reduce the cost of experiments / assays by 20-100 times.

[0247] Example 2: Resolving cellular complexity through infinitely multiplexed super-resolution imaging

[0248] Mapping the complex spatial relationships among many different molecules within cells is crucial for understanding the complexity of cellular function. Super-resolution fluorescence microscopy provides the required spatial resolution but struggles to visualize more than four distinct targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging expands this number but is limited by its low throughput. Here, we present a new imaging approach that enables rapid multiplexed super-resolution microscopy of a virtually unlimited number of molecular targets by utilizing fluorescent labels combined with transient linker-mediated high-throughput DNA-PAINT (FLASH-PAINT) switching. We demonstrate the cell biology versatility of FLASH-PAINT in mammalian cells with four applications: i) mapping nine proteins in single mammalian cells, ii) elucidating the functional organization of primary cilia by imaging nine targets, iii) revealing changes in the vicinity of twelve distinct targets in intact and dissociated Golgi stacks, and iv) studying interorganelle contacts at 3D super-resolution.

[0249] Example 2-1:

[0250] Understanding cellular function is closely linked to the ability to visualize how organelles and the molecules that make up them respond to different physiological and disease states. However, meaningful, information-rich visualization is a challenge because it depends on the ability to identify molecules (especially proteins) and their many interacting partners, and to resolve their spatial organization. Fluorescence microscopy has long been key here, revealing specific proteins with resolutions of hundreds of nanometers, or with the advent of optical super-resolution microscopy, reaching tens of nanometers or even sub-ten nanometer resolution. Single-molecule localization microscopy (SMLM) is the preferred choice for cell biology studies among different super-resolution microscopy modes because of its high 3D resolution (typically around 20-70 nm), sensitivity (single molecule), and relatively low instrumentation requirements. In SMLM, single molecules spontaneously switch between "ON" (bright) and "OFF" (dark) states, and super-resolution images are built by computationally localizing individual ON molecules over thousands of camera frames. In contrast to SMLM techniques such as (F)PALM and (D)STORM, which rely on photophysical switching between bright and dim fluorescent states, DNA-PAINT exploits the transient, reversible binding of a fluorescently labeled short oligonucleotide strand, termed the “imager” (or “imager probe”), to a complementary “docking strand” attached to a target of interest, such as a protein typically tagged via an antibody. Figure 13A , left half). In traditional DNA-PAINT, there is no true dark fluorescent state; instead, the "OFF" state relies on the rapid diffusion of unbound imagers into a uniform background blur, preventing their localization; only when the imager binds to a docking strand is the transiently immobilized imager observed as a discrete fluorescent spot that can be localized ("OFF"). Unconstrained by photophysical switching, the dye and buffer in DNA-PAINT can be selected to maximize brightness. Furthermore, due to the large reservoir of imagers in solution, bound imager probes can be replaced even if they bleach. This results in a higher density of localization events in the final image, which is an otherwise limiting factor, especially when imaging thick volumes such as cells. The combination of these advantages allows for resolutions of <5 nm.

[0251] While DNA-PAINT and other super-resolution techniques offer a tenfold or greater improvement in resolution compared to traditional fluorescence microscopy, their impact on biomedical research has been limited by the lack of multicolor imaging techniques, which are essential for decoding the complex organization of cells at the molecular level. For example, the mammalian Golgi complex is organized as stacks of multiple cisternae arranged in an endo-trans arrangement. These stacks are often linked laterally to form a highly convoluted "ribbon." The complex role and structure of the Golgi apparatus and its interactions with the trans-Golgi network (TGN), endoplasmic reticulum (ER) exit sites (ERES), the ER-Golgi intermediate compartment (ERGIC), and many other organelles are mediated by over a thousand different proteins that interact in a selective, carefully orchestrated manner, as governed by their specific spatial distribution. While there is a typical "textbook" Golgi apparatus, the shape and orientation of the Golgi ribbon vary significantly between cells. This variability makes it impossible to combine separate, independently recorded super-resolution images of different subsets of two or three different proteins into a comprehensive image of ten or more colors, which would cover only a small part of the Golgi's role in cell biology.

[0252] Multicolor SMLM has traditionally been limited by the limited availability of bright, spectrally distinguishable probes. Consequently, two-color imaging has been the standard in SMLM, with the exception of three or four colors. Multiplexing approaches for sequential imaging of different markers offer a way to overcome this limitation and, for example, have been shown to extend diffraction-limited multicolor fluorescence imaging to approximately 100 markers. In super-resolution microscopy, multiplexing has been achieved by a variant of DNA-PAINT called exchange-PAINT. Here, different targets are labeled with orthogonal ssDNA docking strands and then imaged sequentially using different imager probes. However, the imager probes used to date have slow binding kinetics, resulting in data acquisition times of an hour or more per color channel. Adding wash times between sequential imaging cycles often increases the total data acquisition time for a single cell to several days.

[0253] Recently developed speed-optimized and fluorescent imager probes allow for up to approximately 100-fold faster imaging in DNA-PAINT, which, at first glance, appears to be a solution to this severe throughput limitation. However, due to limitations in the DNA sequence design of these specialized probes, only six speed-optimized probes and two fluorescent probes have been discovered to date. This limits the promise of Rapid Exchange-PAINT to scenarios with only a few targets. Furthermore, requiring a specific imager probe for each target does not scale well to tens, hundreds, or even thousands of probes, as dye-conjugated oligonucleotides are expensive, probe exchange with extensive washing after each imaging cycle is time-consuming, and cumulatively damages the sample.

[0254] Here, we introduce fluorescent labeling combined with transient linker-mediated high-throughput DNA-PAINT (FLASH-PAINT) switching, a method that allows rapid, essentially unlimited multiplexing in super-resolution imaging. Orthogonal ssDNA-based linkers are used, which direct any imager probe (e.g., speed-optimized probe or fluorescent probe) to a specific target ( Figure 13A ), eliminating the color limitations of super-resolution microscopy. Key to the success of FLASH-PAINT is that linkers only transiently bind to docking strands. This allows for rapid, efficient, and gentle linker replacement between imaging cycles. Furthermore, it enables the introduction of "erasers," oligonucleotides complementary to individual transient linkers. These erasers hybridize to any selected transient linker, efficiently neutralizing it and eliminating the need for any wash steps.

[0255] This study demonstrates the broad utility of FLASH-PAINT by mapping the spatial distribution of nine different proteins in U-2OS cells and revealing the complex spatial arrangement of nine proteins on individual primary cilia and twelve Golgi-associated proteins in single cells. Furthermore, this study characterized the number and size of contacts between the ER, mitochondria, lysosomes, and the Golgi complex at 3D super-resolution.

[0256] Example 2-2: Joint Design

[0257] Minimal crosstalk between targets is a key requirement for successful multiplexed imaging. Therefore, multiplexing methods have traditionally emphasized the efficient erasure of previous rounds of fluorescent labeling (e.g., by photobleaching, UV cleavage, or chemical stripping) before imaging the next round of labeling. This difficult process of label removal stands in stark contrast to the nearly 100% dissociation efficiency of the imager from the docking site in DNA-PAINT, which is precisely facilitated by the transient nature of imager docking site association (~1 s). It is hypothesized that the same principles of this transient binding can be applied to linkers that only transiently bind to their targets. The conceptual challenge of such an approach is that such linkers will inevitably be bound to the target only a fraction of the time, thereby reducing the overall binding frequency of the imager probe to the docking site, compared to traditional linker-free DNA-PAINT methods. Importantly, however, the transient linker itself is not fluorescent and can therefore be used at concentrations several orders of magnitude higher than the imager strands in traditional DNA-PAINT experiments (e.g., c TA =10 nM–100 nM): For example, at a transient linker concentration of 50 nM, the average binding time is 100 s and the association rate is 2 x 10 6 M -1 s-1 , about 91% of the docking sites are occupied by linkers.

[0258] In this study, a set of transient linkers was designed, each consisting of two binding motifs, one for the imager probe and the other for the docking sequence, separated by a short 2-nucleotide (nt) spacer. As imaging probe motifs, three previously published sequences were selected: a conventional DNA-PAINT imager, a speed-optimized imager, and a fluorescent imager (Table 1). -1 To increase the target dissociation rate by orders of magnitude, this study designed 12 orthogonal 10-nt motifs (Table 2-3) with a GC content of 40%-50%.

[0259] Table 1

[0260]

[0261]

[0262] Table 2

[0263]

[0264]

[0265] Table 3

[0266]

[0267]

[0268] Example 2-3: Transient linkers are highly specific and can bind efficiently and reversibly

[0269] To initially demonstrate the concept of FLASH-PAINT, this study used DNA origami nanostructures. To directly compare linker-mediated binding with direct binding, this study used an SMLM instrument to image a mixture of two different DNA origami species. One species had binding sites for the imager probe arranged in a rectangular frame, while the other species had linker docking sites arranged in a 3x4 grid with a 20nm pitch ( Figures 13A-13B). In the first round of imaging, only the imager strand was introduced. As expected, only the first species could be observed because the imager should not bind to the linker docking site on the second origami species. In the second round of studies, the linker and imager were introduced, so that both DNA origami species were visible. For the third round of imaging, to test the linker dissociation efficiency, the mixture of linker and imager strands was washed away and then only the imager was reintroduced. The resulting images were similar to the first images, confirming excellent dissociation efficiency. Counting the number of imager probe binding events recorded in the three images confirmed that the nonspecific binding of the imager probe to the linker docking site was less than 1% ( Figure 13B ; 0-3 vs. 316-457 events), the efficiency of linker dissociation exceeded 99% (0-2 events after washing).

[0270] Next, we designed an experiment to compare the association rate of linker-mediated binding with that of direct binding ( Figure 13C ). This study again mixed two different DNA origami species, one with a single docking site for linker-mediated binding and the other with a direct binding site. To distinguish the two DNA origami from each other, this study used orthogonal docking sites arranged in rectangles, framed the single docking site, and sequentially imaged these frames in two additional rounds ( Figures 19A-19I and 20). We then used a high-speed imager to measure the association rates of the imager with the two types of DNA origami at a constant imager concentration (10 nM) but different linker concentrations ( Figure 13D and 21A -21C). In the low linker concentration regime, the association rate of the imager binding to the DNA origami through the linker increases when the linker concentration is increased. This can be explained by an increase in the occupancy of docking sites by the linker chain. However, when the linker chain concentration exceeds about 20nM, the association rate decreases. A similar decrease can be simultaneously observed in the association rate of the imager binding directly to the second DNA origami species, which is consistent with the decrease in the available imager concentration. This can be explained by the high concentration of linker chains in solution competing for the imager, thereby depleting the pool of imagers available for binding to the DNA origami. Analytical descriptions of the direct and linker-mediated association rates are provided in the Supplementary Information and match the experimental data points very well ( Figure 13D Importantly, the linker-mediated association rate of the imager with the DNA origami reached that of direct binding (c TA =0 nM), a level comparable to DNA-PAINT, which relies on direct, linker-free binding of the imager. This demonstrates that it is possible to introduce transient linkers without substantially impairing imager-target association.

[0271] Next, we measured the association and dissociation rates of 36 designed linkers (Table 4), 12 for each linker for high speed (linker concentration 20 nM) ( Figures 22A-22B and 23A-23B), Classic ( Figures 24A-24B and 24A-24B) and fluorescence ( Figures 26A-26B and 27A-27B) imagers. In all cases measured, this study found that the association rate of linker-mediated binding was in a similar range as that of direct binding. This confirms that transient linkers can generally be used without substantially impairing the association of the imager with the target.

[0272] Table 4

[0273]

[0274]

[0275] Examples 2-4: High-resolution multiplexed quantitative super-resolution microscopy

[0276] To test multiplexed imaging via transient linkers, we designed DNA origami structures with four different orthogonal docking sites arranged in the shapes of the letters "Y," "A," "L," and "E" ( Figure 13E and 20 The localization accuracy of all four rounds of imaging was approximately 2 nm, which allowed us to clearly distinguish adjacent binding sites of the letter pattern ( Figure 13E and 28 -32) and demonstrated that the use of transient linkers did not compromise resolution. Using a speed-optimized imager, each imaging run took approximately 25 minutes, for a total imaging time of approximately 100 minutes.

[0277] Another unique feature of transient linkers is the ability to image the same target of interest using different imagers. This allows us to compare the imaging performance of different imagers using the same sample and imaging conditions. The outer membrane protein Tom20 in COS-7 cells was immunolabeled with an antibody with ssDNA docking sites and imaged using high-speed, fluorescence, and classical imagers under epi illumination through the linker ( Figures 33A-33B While the signal-to-noise ratio (SNR) of the fluorescence imager (SNR ~ 30) and speed imager (SNR ~ 40) is high enough to easily separate the bound imager from the diffuse background, the low SNR in the case of the classical imager (SNR ~ 8) prevents artifact-free localization of targets in thick samples. This demonstrates the clear advantages of high-speed and fluorescence imager probes over the classical DNA-PAINT imager.

[0278] Example 2-5: Eraser allows for quick and efficient switching between joints without washing

[0279] In classic exchange-PAINT, switching between targets is achieved by thoroughly washing one imager and subsequently introducing the next, but this is time-consuming (typically about 10 minutes). It is inferred that by introducing an eraser chain ( Figure 14A and Table 5), the wash step can be eliminated in FLASH-PAINT. The higher affinity eraser binds to the transient linker (effectively permanently) and neutralizes it by preventing it from binding to the corresponding docking site.

[0280] We characterized the erasure efficiency of all twelve transient linker sequences using DNA origami structures and found it to be greater than 98% in all cases ( Figure 14B and 44 As a test in biological samples, this study monitored the reorientation of the imager probe from the docking site on mitochondria (immunolabeling of the mitochondrial outer membrane protein Tom20) to the microtubule docking site (immunolabeling of α-tubulin) by simultaneously introducing the eraser of the transient linker into the mitochondrial docking site and the new transient linker into the microtubule docking site. Figure 14C Even without any flow or active perfusion, mitochondrial signaling appears along with microtubule signaling (τ 1 / 2 ≈200s) and disappears quickly (τ 1 / 2 ≈60s)( Figure 35A By switching the imager signal from the nucleolar protein NPM1 to lamin B1 at the nuclear lamina, similarly efficient switching between the two targets could be observed in the denser environment of the cell nucleus ( Figure 15B ).

[0281] To evaluate the nonspecific binding of transient linkers (and imagers) for classical, high-speed, and fluorescent DNA-PAINT imaging in the context of cell imaging, we imaged anti-Tom20 immunolabeled cells using matched and unmatched transient linker-imager combinations ( Figures 36A-36D , 37A-37D, and 38A-38D). It was found that in all three cases, non-specific binding was negligible (<2%) compared to specific binding, even with the addition of 11 mismatched linkers.

[0282] Table 5

[0283]

[0284]

[0285]

[0286] Example 2-6: FLASH-PAINT achieves spectrally unlimited multiplexed super-resolution microscopy in cells

[0287] To test the capabilities of FLASH-PAINT for rapid, efficient, and spectrally unlimited multiplexed super-resolution microscopy, nine immunolabeled targets were imaged in U2OS cells, including three Golgi proteins (GM130, GRASP55, GRASP65), three mitochondrial-associated targets (OMP25, HADHA, dsDNA), two nucleolar-localized targets (NPM1, RPA40), and the nuclear envelope (lamin-B1) ( Figures 15A-15B and 39). This study then imaged each target individually in nine rounds, followed by a tenth round in which all targets were imaged together for spatial alignment. The imaging experiment was completed in only approximately three hours, including the time spent switching between targets. This study achieved an average localization accuracy of approximately 11.2 nm. To broadly demonstrate the cell biology utility of FLASH-PAINT, this study tested the new method in three additional applications.

[0288] Example 2-7: 9-channel FLASH-PAINT resolution of molecular organization in primary cilia

[0289] Primary cilia function as cellular antennas, not only receiving signals but also potentially transmitting them by releasing vesicles from their tips. Their characteristic architecture consists of a core microtubule axoneme surrounded by a specialized membrane enriched in GPCRs such as Smo, and a transition zone (TZ) structure near the cilium base that controls access to this privileged domain. To obtain a comprehensive view of primary cilia, it is necessary to incorporate the spatial distribution of individual proteins organized and enriched in these sub-diffraction (<200 nm) compartments. However, this task is hampered by the different states that primary cilia can exist in (e.g., in response to stimuli, assembly, and disassembly), which makes it difficult to combine data from different datasets.

[0290] This study tested whether FLASH-PAINT can visualize ciliary nanostructures in 3D and reveal the characteristic protein assemblies of individual ciliary compartments. To directly conjugate FLASH-PAINT docking sites to antibodies against different ciliary targets, this study employed a photoactivatable site-specific conjugation (LASIC) protocol that conjugates oligonucleotides directly to primary antibodies. Alternatively, LASIC can be used to conjugate oligonucleotides to secondary antibodies ( Figure 40A This study used 9-channel FLASH-PAINT to perform 3D imaging of multiple ciliated cells ( Figures 16A-16G and 40B-40C). In this study, a single cilium was analyzed and, as expected, ciliary membrane proteins (pHSmo, INPP5E, and Arl13b) were observed as tubes surrounding glutamylated and acetylated microtubules (Glu-tub and Ac-tub; Figures 16A-16BThe basal body distal appendage protein CEP164 is ring-shaped around the base of the cilium, with the TZ protein Rpgrip1l at its distal end (blue box, arrow). Figures 16A-16B The distribution of other ciliary proteins, Sept2 and the cargo transport protein Ift88, is more variable. This 9-color super-resolution image is compared with another cilium with a large bulbous tip (yellow box, asterisk, Figure 16B ) images, reveal striking differences: the latter cilium exhibits thinning of the axoneme just before the bulbous tip, indicated by pH Smo, and additional varicosities (arrows, Figure 16B Interestingly, this is not evident in microtubule reports, perhaps because the axoneme-microtubule complex thins and becomes singlet as it approaches the tip.

[0291] We next analyzed the spatial distribution along the ciliary axis. We localized the single-molecule clusters relative to the pH-Smo signal and the central filament generated by the Ac-tub signal. Figure 40D Furthermore, because the TZ is close to the basal body, which has recently been shown to differentially accumulate Ac / Glu tubulin, we divided the cluster data into quartiles based on their distance from CEP164 (proximal, middle 1, middle 2, and distal squares; Figures 16A-16B Quantifying the distribution of clusters within these quartiles revealed differences between normal and globular cilia, with Ift88 and Sept2 enriched at the proximal and two mid-regions of normal cilia but recruited along the entire length of cilia with globular tips, the latter indicating enhanced activation ( Figures 16C-16D When the median distance from the central filament was measured in this study, Ac-tub and Glu-tub showed the lowest values, as expected ( Figure 16E Compared with normal cilia, bulbous tip cilia showed shorter distances in the proximal and middle regions for all targets ( Figure 16E Finally, when we analyzed the median distances between different protein clusters (see Example 2-11), we observed that the median distances between Sept2 and all other proteins in globular tip cilia were greater than those in normal cilia in both the proximal and distal regions (black dashed rectangle, Figures 16F-16G In contrast, for Ift88, this was only the case in the distal region (purple dashed rectangle). These findings highlight the power of multiplexed super-resolution microscopy to identify distinct nanoscale and long-range states in primary cilia, including rare / transient stages that would be missed in ensemble-averaged studies of the cilium.

[0292] Example 2-8: 12-way FLASH-PAINT maps the spatial organization of the secretory pathway

[0293] We next tested FLASH-PAINT to better visualize the complex 3D structure of the Golgi apparatus. We used 12-way super-resolution imaging to investigate the spatial organization of the secretory pathway by highlighting components of the ER exit sites (ERES), the ER-Golgi intermediate compartment (ERGIC), the cis-, intermediate-, and trans-Golgi cisternae, the trans-Golgi network (TGN), and COPI and COPII vesicles in the same cell. As expected, the Golgi ribbon appears as a highly coiled 3D structure near the nuclear lamina in interphase HeLa cells ( Figure 17A and 41A -41B). ERES (TANGO1), ERGIC (ERGIC-53), and COPI (β′-COP) vesicles are distributed throughout the cytoplasm ( Figure 42B Cross-sections of Golgi stacks reveal a hierarchical organization of cis (GRASP65, GM130), intermediate (ManII-GFP), and trans cisternae (Golgin97, p230), as well as the trans-Golgi network (TGN46). Figure 17E and 41A -41B). Frontal view of the Golgi stack shows megalin localized to the edge of the Golgi cisternae ( Figure 17C and 17G ), which is consistent with the EM data. COPI (β'-COP) vesicles were mainly observed at the periphery of the Golgi ribbon ( Figure 17F ), close to its budding site. Most COPII envelope (Sec31A) spots are significantly larger than TANGO1 (ERES) spots, and usually one or more TANGO1 spots decorate each Sec31A spot ( Figure 17F ), which supports the idea that multiple TANGO1 proteins surround the budding site of ERES.

[0294] To visualize the 3D organization of the Golgi ribbon, we used recently developed methods to generate surfaces from single-molecule localization data of GM130, ManII-GFP, Golgin97, and lamin B1. Figure 17D The median distance between the localization of each marker and the localization of all other markers within 500 nm was plotted as a heat map ( Figure 17H Consistent with the expected organization of Golgi stacks, this quantification showed that stack-associated proteins (GM130, GRASP65, ManII-GFP, p230, Golgin97) were closer to each other than proteins outside this group.

[0295] Interphase nocodazole-treated cells were imaged using the same markers ( Figures 42A-42B ), revealing a Golgi micro-stack with a cis-trans structure ( Figure 17Iand 17M ) and marginal localization of megalin ( Figure 17K and 17O ) are largely intact, supporting the long-held hypothesis that nocodazole-induced mini-stacks represent a valid morphological model of the native Golgi apparatus. Visual comparison of this data with Golgi data from untreated cells shows that the mini-stacks are closer to the ERES, as marked by Sec31A and TANGO1 ( Figure 17J and 17N Quantification of the median distance of Golgi stack proteins to Sec31A under both conditions revealed a significant decrease from >100 nm to the 50 nm range ( Figure 17P and 43 This observation is consistent with a model in which ER export is crucial for Golgi regeneration. COPI vesicles are located at the periphery of Golgi mini-stacks ( Figure 17J and 17N ), as they did in untreated cells, indicating that vesicle budding was unaffected by nocodazole. Collectively, these data provide the first visualization and quantification of the complex organization of secretory pathway proteins in the same cell, providing strong morphological context and molecular specificity for future studies.

[0296] Example 2-9: Whole-cell FLASH-PAINT maps the number and size of inter-organelle contact sites

[0297] In recent years, contacts between organelles have been recognized to play a key role in coordinating cellular functions, and dysfunction of such contacts may be associated with neurodegenerative diseases. Inspired by earlier work using diffraction-limited microscopy, this study imaged four different organelles, mitochondria (Tom20), ER (Sec61β), Golgi apparatus (ManII), and lysosomes (Lamp1), at 3D super-resolution in HeLa cells in a volume approximately 2.5 μm thick ( Figures 18A-18I For these experiments, this study used fluorescent imagers, which enable high-quality imaging of thick volumes deep within cells in two ways: First, the fluorescent nature of the unbound state reduces the background of unbound probes in solution, which is crucial for the large excitation volumes used in highly inclined and laminated optical sheets (HILO) or epi-illumination. Second, the fluorescent nature protects the unbound imagers from bleaching in solution. This is particularly important for large excitation volumes, as bleaching of the imagers in solution reduces the effective concentration of functional imagers, thereby reducing the blinking frequency and data acquisition speed in DNA-PAINT experiments.

[0298] This study collected 41 million localizations in 173 minutes with an average localization accuracy of 16.6nm. Using surface reconstruction of the local point cloud, this study generated a 3D representation of the imaged organelles. Using the organelle surface, this study quantified the number of contact sites between different organelles ( Figure 18F ), defined as a spatial proximity of <100 nm between two membranes. The number of contact sites obtained is consistent with that extracted from diffraction-limited microscopy data by Valm et al. Super-resolution imaging allowed us to additionally quantify the average area of ​​contact sites. The median size of all contact sites found in this study was within 0.1 μm 2 to 0.2μm 2 ER-mitochondria contact sites were the most abundant contact points and also showed the largest median and largest size variance, with approximately 20% of contact sites larger than 1 μm. 2 ( Figure 18H and 18I ).

[0299] Example 2-10

[0300] Through transient linkers and erasers, this study introduces a new concept in FLASH-PAINT that can rapidly switch fluorescent probes from one target to another. Based on DNA technology, theoretically up to 4 10 The 10-nt long transient adaptors represent over 1 million sequences, far exceeding the approximately 20,000 different proteins expressed in cells. While not all 1 million sequences will be suitable due to off-target binding, crosstalk, unwanted secondary structure formation, and other effects, this concept provides effectively unlimited multiplexing capacity for any currently practical proteomics study.

[0301] Importantly, the same fluorescent imager probes can be reused (or a small number if one wants to image multiple targets simultaneously in different colors). FLASH-PAINT can therefore take advantage of the latest generation of DNA-PAINT probes, which are optimized for speed and fluorescence but are severely limited in sequence design and therefore not directly suitable for highly multiplexed imaging. As shown here, this combination enables the generation of super-resolution images of complex subcellular structures such as cilia or the Golgi complex with high quality, deep into the cell, and within minutes rather than hours per imaged target.

[0302] Linkers that bind stably, i.e., non-transiently, have been successfully used in diffraction-limited and super-resolution microscopy. While both types of linkers enable the sequential labeling of many targets with only a few fluorescent probes, stable linkers suffer from the same problem faced by linker-free sequential multiplexing methods: the previously imaged target needs to be eliminated before the next target can be imaged. This is typically achieved by: (i) removing the linkers (using dissociation buffers or toehold-mediated displacement), (ii) permanently blocking them (using blocking chains that saturate the binding sites where the imager probes normally bind), or (iii) destroying them (using enzymes). However, all of these methods require long incubation and washing periods, which slow data acquisition, can be inefficient, leading to crosstalk or background, and / or can damage the sample, especially if applied repeatedly over many imaging cycles.

[0303] Compared to static linkers, the designed transient linkers readily dissociate from their targets without the need for toehold-mediated displacement or dissociation buffers. This rapid and simple dissociation makes the sequence of the transient linker that specifically binds to its target docking site easily accessible to the complementary eraser strand. As shown in this study ( Figure 14B 、 34A -34B, 39, 40A-40O, 41A-41B, 42A-42B), which results in highly efficient (99% to 99.8%) neutralization of transient linkers. Since the erasers are each specific to a particular docking site, they do not quench the signal of other targets (in contrast to the closed strands that bind to the universal imager probe binding site described above). Therefore, there is no need to wash the previous transient linker and its eraser before the next imaging round. In fact, the eraser can be introduced simultaneously with the next linker ( Figure 14C and 35A -35B), which minimizes the transition time between imaging rounds.

[0304] In the experiments, the complete transition from one target to the next took approximately 1-10 minutes ( Figure 14C and 35A-35B). Although significantly faster than alternative sequential multiplexing methods, this study did not utilize any flow chambers in its proof-of-concept experiments and was therefore limited by diffusion. The introduction of flow, combined with further optimization of the adapter dissociation rate constants, could reduce the transition time between targets to less than a minute. Here, we demonstrate imaging of up to 12 channels, demonstrating the broad cell biological utility of FLASH-PAINT. In this study, the main obstacles to expanding FLASH-PAINT to more targets were limited access to validated, high-quality antibodies and the lack of automated microfluidics—neither of which is fundamental. While the focus here was on immunolabeling, FLASH-PAINT is equally useful for spatial transcriptomics studies and for tracking DNA in the nucleus using fluorescence in situ hybridization. As demonstrated by MERFISH and SeqFISH+, barcode multiplexing schemes with only tens of adapters allow for 1000-fold or even higher multiplexing. The low crosstalk of transient adapters has the potential to minimize error rates in barcode multiplexing. This in turn should enable researchers to use more barcodes in the codebook (i.e., those with smaller Hamming distances), thereby accessing a wider variety of targets with fewer imaging rounds.

[0305] It is expected that transient linkers will find widespread application in diffraction-limited spatial omics approaches. Localization of single-scintillation molecules is only required for super-resolution—if not, the concentration of imager probes can be increased to provide diffraction-limited images, e.g. Figure 14C and 35A -35B. Compared to many established technologies in the field, transient linkers and erasers allow for rapid label exchange without requiring harsh, time-consuming processing steps between imaging rounds, such as stripping the probe from the sample or photocleaving or bleaching it. Furthermore, transient linkers and erasers are inexpensive: the unlabeled oligonucleotides used here cost a fraction of the cost of dye-labeled oligonucleotides.

[0306] Importantly, FLASH-PAINT is not conceptually limited to imaging a single color at a time. It is anticipated that it can be readily combined with imager probes of multiple fluorescent colors. Furthermore, our technique synergizes with innovative simultaneous multicolor approaches such as supermultiplexed vibrational imaging. With these synergistic effects and a wide range of potential applications extending to transcriptomics and chromatin tracking, FLASH-PAINT will become an enabling technology for a wide range of biological applications.

[0307] Examples 2-11: Materials and Methods

[0308] Material

[0309] Unmodified, Cy3b-modified, and biotinylated DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). The M13mp18 scaffold (cat: N4040S) was obtained from New England BioLabs. Tris 1M pH 8.0 (cat: AM9856), EDTA 0.5M pH 8.0 (cat: AM9261), magnesium 1M (cat: AM9530G), and sodium chloride 5M (cat: AM8759) were obtained from Ambion. Ultrapure water (cat: 10977015) was purchased from Gibco. 200 μL PCR tubes (cat: AB-0620) were obtained from Thermo Scientific. Polyethylene glycol (PEG)-8000 (cat: 89510-250G-F) was purchased from Sigma. Streptavidin (cat: S-888) was purchased from Thermo Fisher. BSA biotin (cat: A8549) was obtained from Sigma-Aldrich. Tween 20 (cat: P9416-50mL), glycerol (cat: 65516-500mL), methanol (cat: 32213-2.5L), protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa (PCD) (cat: P8279), 3,4-dihydroxybenzoic acid (PCA) (cat: 37580-25G-F), and (+-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (cat: 238813-5G) were purchased from Sigma. Sodium hydroxide (cat: P3911-1kg) was purchased from Sigma-Aldrich. Potassium chloride (cat: 3624-01) was purchased from Baker Analyzed ACS Reagent. 30mL syringes (cat: 302832) were obtained from BD. Biocompatible silicone tubing (cat: 10831), flow chamber 6-well μ-slide VI 0.58-well glass-bottom μ-slides (cat: 80607) and glass-bottom 8-well μ-slides (cat: 80827) were obtained from ibidi. 8-well 1.5H glass-bottom chambers (cat: C8-1.5HN) were purchased from Cellvis. 15 mL (cat: 352096) and 50 mL (cat: 352070) polypropylene conical tubes and tissue culture flasks (cat: 353136) were purchased from FALCON. Dulbecco's modified Eagle's medium (DMEM) (cat: 21063-929), McCoy's 5A medium (cat: 16600-082), Opti-MEM (cat: 31985-070), 0.05% trypsin-EDTA (cat: 25300-054), fetal bovine serum (FBS) (cat: 16000-044), and 1× phosphate-buffered saline (PBS) pH 7.2 (cat: 10010-023) were ordered from Gibco. 10% formalin (cat: HT501128-4L), heat-inactivated FBS (cat: F4135-500ML), and 1 mg / mL fibronectin (cat: F0895-2MG) were purchased from Sigma-Aldrich. HeLa CRM-CCL-2 cells (cat: CRM-CCL-2), U-2OS cells (cat: HTB-96), COS-7 cells (cat: CRL-1651), and hTERT-RPE cells (cat. CRL-4000) were obtained from ATCC. Paraformaldehyde (cat: 15710) and glutaraldehyde (cat: 16219) were obtained from Electron Microscopy Sciences. Bovine serum albumin (cat: 001-000-162) was purchased from Jackson ImmunoResearch. Triton X-100 (cat: T8787-60ML) was purchased from Sigma. Antibodies against GM130 (cat: 610822), Sec31A (COPII) (cat: 612350), and p230 (cat: 611280) were obtained from BD Biosciences. Antibodies against lamin B1 (cat: ab16048), HADHA (cat: ab 110302), GRASP65 (cat: ab174834), dsDNA (cat: ab3519), and Septin2 (ab187654) were obtained from abcam.Antibodies against GRASP55 (cat: 10598-1-AP), GM130 (cat: 11308-1-AP), TGN46 (cat: 10598-1-AP), Inpp5e (17797-1-AP), Arl13b (17711-1-AP), Ift88 (13967-1-AP), CEP164 (22227-1-AP), and RPGRIP1L (55160-1-AP) were purchased from Proteintech. Antibodies against Tom20 (cat: sc-11415) and RPA40 (cat: sc-374443) were ordered from Santa Cruz. Antibodies against NPM1 (cat: NB600-1030) were obtained from Novus Bio. Antibodies against GOLGB1 (megalin) (cat: HPA011555), MIA3 (Tango1) (cat. HPA055922), acetylated tubulin (T6793), and α-tubulin (cat: T5168) were ordered from Sigma. Antibodies against GOLGA1_1 Golgin-97 (cat: HPA044329) were purchased from Atlas Antibodies. Antibodies against LMAN1 ERGIC-53 (cat: MA5-25345) were ordered from Invitrogen. Antibodies against glutamylated tubulin (AB3201) were ordered from Millipore. Antibodies against Lamp1 (9091) were purchased from Cell Signaling Technology. Anti-mCherry antibodies (GT844 and GT857) were obtained from GeneTex. Anti-COPI antibody (CMIA10) was custom-made at the Rothman laboratory. DNA-labeled secondary anti-rabbit antibody, DNA-labeled secondary anti-mouse antibody, and DNA-labeled GFP nanobody were custom ordered from Massive Photonics. Oligonucleotides conjugated to OyOlink probes were purchased from AlphaThera.

[0310] buffer

[0311] Three buffers were used for sample preparation and imaging: buffer A (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% Tween 20, pH 7.5); buffer B (10 mM MgCl2, 5 mM Tris-HCl pH 8, 1 mM EDTA, 0.05% Tween 20, pH 7.5) and buffer C (1× PBS, 500 mM NaCl). Figure 13B 、 13E, 15A-15B, 17A-17P, 18A-18I, 28-32, 33A-33B, 36A-36D, 37A-37D, 38A-38D, 39, 41A-41B, 42A-42B, and 44-47, imaging buffer was supplemented with: 1× Trolox, 1× PCA, and 1× PCD.

[0312] Trolox, PCA, and PCD

[0313] 100× Trolox: 100 mg Trolox, 430 μL 100% methanol, 345 μL 1 M NaOH in 3.2 mL H₂O. 40× PCA: Combine 154 mg PCA, 10 mL water, and NaOH, and adjust the pH to 9.0. 100× PCD: 9.3 mg PCD, 13.3 mL buffer (100 mM Tris-HCl pH 8, 50 mM KCl, 1 mM EDTA, 50% glycerol).

[0314] DNA origami self-assembly

[0315] All DNA origami structures were designed using the Picasso design tool (see Figure 20 The self-assembly of DNA origami was completed in a one-pot reaction with a total volume of 50 μL, consisting of 10 nM scaffold chain (sequences are shown in Table 6 ), 100 nM folding staple, 10 nM (or 1 μM ( Figure 13E and 28 A mixture of 1 μM DNA-PAINT or FLASH-PAINT handles (Table 2) and a biotinylated staple (Table 7) was prepared in folding buffer (1× TE buffer (10 mM Tris and 1 mM EDTA) containing 12.5 mM MgCl). The reaction mixture was then subjected to a thermal annealing ramp using a thermal cycler. The reaction mixture was first incubated at 80°C for 5 minutes, then cooled from 60°C to 4°C in steps of 1°C every 3.21 minutes, and then held at 4°C.

[0316] Table 6

[0317]

[0318]

[0319]

[0320]

[0321] Table 7

[0322]

[0323] DNA origami PEG purification

[0324] DNA origami structures with letters, 10 nm, and 20 nm grids were purified by three rounds of PEG precipitation by adding equal volumes of PEG buffer (15% PEG-8000, 500 mM NaCl, 1× TE buffer, pH 8.0), centrifuging at 14,000 g for 30 min at 4°C, removing the supernatant, and resuspending in folding buffer ( Figure 13E and 28 -32).

[0325] DNA origami sample preparation

[0326] For DNA origami sample preparation, use μ-slide VI 0.5 (ibidi) was used as the sample chamber. First, 100 μL of biotinylated bovine albumin (1 mg / mL, dissolved in buffer A) was flushed into the chamber and incubated for 5 minutes. The chamber was then washed with 500 μL of buffer A. Then a volume of 100 μL of streptavidin (0.5 mg / mL, dissolved in buffer A) was flushed into the chamber and allowed to bind for 5 minutes. After washing with 500 μL of buffer A and then with 500 μL of buffer B, 100 μL of biotinylated DNA structure (about 200 pM) in buffer B was flushed into the chamber and incubated for 8 minutes. The chamber was then washed with 500 μL of buffer B. Finally, 100 μL of imager solution in the corresponding imaging buffer was flushed into the chamber.

[0327] plasmids

[0328] To label the mitochondrial outer membrane ( Figures 15A-15B ), this study expressed GFP-OMP25 from a plasmid. In order to mark the intermediate Golgi vesicles ( Figures 17A-17P and 18A-I). In this study, GFP-ManII was expressed from plasmids. mCherry-Sec61β was obtained from Addgene (plasmid 49155).

[0329] Cell culture

[0330] HeLa cells and COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). U-2OS cells were cultured in McCoy 5A medium supplemented with 10% FBS. The night before immunolabeling, cells were seeded on ibidi 8-well glass coverslips at a rate of approximately 30,000 cells / well. RPE-pHSmo cells were maintained in DMEM / F12 supplemented with 10% FBS, 1× Pen / Strep, 1× non-essential amino acids, and 1 mM sodium pyruvate. For cilia formation, 250 μL of a 50,000 cell / mL RPE-pHSmo cell suspension was plated into 4 wells of an 8-well cell observation chamber coated with 10 μg / mL fibronectin for 1 hour. The cells were incubated at 37°C for two days to reach confluence. On the third day, the culture medium was replaced with a culture medium supplemented with 0.5% FBS, and a starvation period of another two days was initiated.

[0331] transient transfection

[0332] Transfection was performed using a super electroporator NEPA21 II (Nepa Gene). In an electroporation cuvette (Bulldog Bio; 12358-346), cells were concentrated to approximately 1 million cells in 90 μL, to which 10 μL of approximately 1 μg / μL plasmid DNA was added. The cells were electroporated using the following program: 125V perforation pulse, 3ms pulse width, 50ms pulse interval, two pulses, a decay rate of 10% and + polarity, followed by a 25V transfer pulse, 50ms pulse width, 50ms pulse interval, five pulses, a decay rate of 40% and ± polarity.

[0333] Golgi mini-stack induction

[0334] HeLa cells were treated with 5 μg / mL nocodazole in culture medium for 4 h at 37°C and then fixed to induce Golgi mini-stacking.

[0335] Figures 14A-14C and 35A-35B cell fixation and labeling

[0336] Cells were fixed with 3% PFA and 0.1% GA for 15 minutes. After four washes (30 seconds, 60 seconds, 2 × 5 minutes), cells were blocked and infiltrated with 3% BSA and 0.25% Triton X-100 for 1 hour at room temperature. Next, cells were incubated overnight with the primary antibody (Table 8) in 3% BSA and 0.1% Triton X-100 at 4 ° C. The next day, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), cells were incubated with the secondary antibody for about 2 hours at room temperature. Next, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), samples were post-fixed for 10 minutes with 3% PFA and 0.1% GA. Finally, before adding imaging solution, samples were washed three times with 1 × PBS for 5 minutes each time.

[0337] Table 8

[0338]

[0339]

[0340]

[0341] Figures 15A-15B and 39 cell fixation and labeling

[0342] The cells were fixed with 4% PFA for 1 hour. After four washes (30 seconds, 60 seconds, 2×5 minutes), the cells were blocked and permeabilized with 3% BSA and 0.25% Triton X-100 for 1 hour at room temperature. Next, the cells were incubated with anti-GM-130 and lamin B1 primary antibodies (Table 8) in 3% BSA and 0.1% TritonX-100 at 4°C overnight. The other primary antibodies were pre-incubated with the corresponding nanobodies (Table 8) at 4°C overnight. The next day, after four washes (30 seconds, 60 seconds, 2×5 minutes), the cells were incubated with nanobodies corresponding to anti-GM130 (host: mouse) antibodies and anti-lamin B1 (host: rabbit) antibodies at room temperature for about 2 hours. Next, in order to block unlabeled epitopes, an excess of unlabeled secondary nanobodies was added to the pre-incubated antibody and nanobody mixture at room temperature for 5 minutes. Next, the cells were incubated with the pooled antibody and nanobody mixture at room temperature for approximately 2.5 hours. After four washes (30 seconds, 60 seconds, 2×5 minutes), the samples were post-fixed with 3% PFA and 0.1% GA for 10 minutes. Finally, the samples were washed three times with 1× PBS for 5 minutes each before adding the imaging solution. Cell fixation preserves cilia ( Figure 16A -16H and 40A-40O)

[0343] After induction of cilia formation, RPE-pHSmo cells were washed with 1×PBS and fixed with 10% formalin for 15 minutes. Next, the cells were washed three times with 1×PBS and permeabilized with PBS / 0.1% Triton X-100 (PBST) for 10 minutes. After permeabilization, the cells were washed with PBST and blocked with 3% BSA / PBST solution for 1 hour. To conjugate the cilia-targeted primary antibody (Table 8) with the adhesive oligonucleotide-OyOlink molecule, 1 μg of purified antibody and 1 μg of OyOlink (1:3 molar ratio) were mixed with PBS in a 100 μL transparent PCR tube for a total of 10 μL. The tube was then incubated for 2 hours on a UV transilluminator equipped with a 365 nm excitation light source. After light-induced cross-linking, the volumes were mixed and 200 μL of 3% BSA / PBST were added. 1 μL of 2.5 μM nano-GFP A3 and 0.5 μL of mouse anti-acetylated tubulin were added to the mixture. 150 μL of this solution was then added to one of the wells containing ciliated pHSmo cells and incubated overnight at 4°C. The following day, the cells were washed three times with PBST for 5 minutes each and incubated with anti-mouse A19 secondary antibody diluted 1:500 in blocking buffer for 2 hours. The samples were then washed three times with PBST for 5 minutes each, washed twice with 1× PBS, and incubated with 10% PFA and 0.1% GA for 10 minutes. After post-fixation, the samples were washed three times with 1× PBS each and stored at 4°C until imaging.

[0344] Cell fixation preserves the Golgi complex ( Figures 17A-17P , 41A-41B and 42A-42B)

[0345] The cells were fixed with 4% PFA for 30 minutes. After four washes (30 seconds, 60 seconds, 2×5 minutes), the cells were blocked and permeabilized with 3% BSA and 0.25% Triton X-100 for 1 hour at room temperature. Next, the cells were incubated with anti-MIA3 antibodies, anti-p230 antibodies and GFP nano antibodies in 3% BSA and 0.1% TritonX-100 overnight at 4°C. In addition, all other primary antibodies were pre-incubated with the corresponding nano antibodies (Table 8) at 4°C overnight. The next day, after four washes (30 seconds, 60 seconds, 2×5 minutes), the cells were incubated with the nano antibodies corresponding to anti-MIA3 antibodies and anti-p230 antibodies at room temperature for about 2 hours. Next, unlabeled excess secondary nano antibodies (for blocking unlabeled epitopes) were added to the pre-incubated antibody-nano antibody mixture for 5 minutes at room temperature. Next, the cells were incubated with the pooled antibody-nano antibody mixture for about 2.5 hours at room temperature. After four washes (30 s, 60 s, 2 x 5 min), samples were post-fixed with 3% PFA and 0.1% GA for 10 min. Finally, samples were washed three times with 1 x PBS for 5 min each before adding imaging solution.

[0346] Cell fixation preserves ER, Golgi complex, lysosomes and mitochondria ( Figures 18A-18I and 44-47)

[0347] Cells were fixed with 3% PFA and 0.1% GA for 15 minutes. After four washes (30 seconds, 60 seconds, 2 × 5 minutes), cells were blocked and permeabilized with 3% BSA and 0.25% Triton X-100 for 1 hour at room temperature. Next, cells were incubated with primary antibodies and nano antibodies (Table 8) in 3% BSA and 0.1% Triton X-100 at 4 ° C overnight. The next day, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), cells were incubated with secondary antibodies at room temperature for about 2 hours. Next, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), samples were post-fixed for 10 minutes with 3% PFA and 0.1% GA. Finally, before adding imaging solution, samples were washed three times with 1 × PBS for 5 minutes each time.

[0348] Cell fixation preserves the nuclear lamina and nucleolus ( Figure 35A )

[0349] Cells were fixed with 2.4% PFA for 30 minutes. After four washes (30 seconds, 60 seconds, 2 × 5 minutes), cells were blocked and infiltrated with 3% BSA and 0.25% Triton X-100 for 1 hour at room temperature. Next, cells were incubated overnight with the primary antibody (Table 8) in 3% BSA and 0.1% Triton X-100 at 4 ° C. The next day, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), cells were incubated with the secondary antibody for 2 hours at room temperature. Next, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), samples were post-fixed for 10 minutes with 3% PFA and 0.1% GA. Finally, before adding imaging solution, samples were washed three times with 1 × PBS for 5 minutes each time.

[0350] Cell fixation preserves mitochondria ( Figures 33A-33B , 35A-35B, 36A-36D, and 37A-37D)

[0351] The cells were fixed with 3% PFA and 0.1% GA for 15 minutes. After four washes (30 seconds, 60 seconds, 2 × 5 minutes), the cells were blocked and permeabilized with 3% BSA and 0.25% Triton X-100 for 1 hour at room temperature. Next, the cells were incubated overnight with an anti-Tom20 primary antibody (Table 8) in 3% BSA and 0.1% Triton X-100 at 4 ° C. The next day, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), the cells were incubated with a secondary antibody at room temperature for 2 hours. Next, after four washes (30 seconds, 60 seconds, 2 × 5 minutes), the samples were post-fixed for 10 minutes with 3% PFA and 0.1% GA. Finally, before adding the imaging solution, the samples were washed three times with 1 × PBS for 5 minutes each time.

[0352] Super-resolution microscopy setup

[0353] Fluorescence imaging was performed on an inverted Nikon Eclipse Ti2 microscope (Nikon Instruments) equipped with a perfect focus system of an Andor Dragonfly unit. The Dragonfly was used in BTIRF mode, applying an objective type TIRF or HiLo configuration with an oil immersion objective (Nikon Instruments, Apo SR TIRF 60×, NA 1.49, oil). For excitation, a 561 nm laser (1 W nominal laser power) was used. The beam was coupled into a multimode fiber passing through the Andor Borealis unit, shaping the beam from a Gaussian profile to a uniform flat top. A dichroic mirror CR-DFLY-DMQD-01 was used. Fluorescence was spectrally filtered with an emission filter (TR-DFLY-F600-050) and imaged with a scientific complementary metal oxide semiconductor (sCMOS) camera (Sona4BV6X, Andor Technologies) without further amplification, resulting in an effective pixel size of 108 nm. Three-dimensional super-resolution imaging was performed by introducing astigmatism through a cylindrical lens in front of the camera.

[0354] Imaging conditions

[0355] A high-level summary of all experiments is described in Table 9.

[0356] Table 9

[0357]

[0358]

[0359] Image analysis

[0360] Spotting and subsequent super-resolution reconstruction, drift correction, filtering, and alignment of raw fluorescence microscopy images were performed using the "Picasso" software package. X, Y, and Z drift correction was performed using redundant cross-correlation integrated in the same software package. PYMEVisualize was used. 42 Surfaces were reconstructed from the localization data and subsequently analyzed for contact sites. To identify and quantify clusters and distances on the 9-way ciliary dataset, individual Picasso-reconstructed ciliary datasets were loaded into Imaris (Oxford Instruments, version 10.0) to generate surfaces that were used to mask the localization data for each target at the cilium. After applying the surface mask, localizations were processed using a Gaussian filter equivalent to one pixel. The filtered data were then used to generate spots using the Imaris spot detection algorithm to represent the size of localization clusters. These spots were used to quantify the number of clusters and the distances between targets. Additionally, Actub clusters were used to generate filaments, which represent the position of the axoneme along the length of the cilium.

[0361] Example 2-12: Derivation of the effective association rate of imager probes bound to DNA origami in the presence of transient linkers

[0362] like Figures 13C-13D As shown, for a given DNA origami, the number of transient binding events observed per time unit can be described by the average dark time, i.e., the time during which no imager probe is bound to the DNA origami (depending on the design of the DNA origami, whether through a transient linker or directly). These times T off瞬时接头介导 and T off直接 can be described as the effective association rate k by introducing the transient linker-mediated and direct binding cases, respectively a有效瞬时接头介导 and k a有效直接 The imager probe concentration x added to the imaging buffer 成像器探针 The function (definitions are summarized at the end of this derivation):

[0363]

[0364] The effective association rate k of the imager probe bound to the DNA origami docking site via a transient linker a有效瞬时接头介导 The binding affinity of transient linkers to imager probes is affected by: (i) the occupancy of the docking site by the transient linker, and (ii) the affinity between the transient linker and the imager probe. The latter not only affects the efficiency of imager probe recruitment into the DNA origami, but also transient linkers in solution compete for these imager probes, thereby reducing the pool of imager probes available for transient linker binding to the DNA origami target. The latter phenomenon also affects k a有效直接 , because both DNA origami species were imaged in the same sample.

[0365] In order to derive ka有效瞬时接头介导 , two assumptions are made: the imager probe can only bind to the docking site when a transient linker chain is present. Second, only the unbound portion of the imager probe present in solution can bind to the docking site. a有效瞬时接头介导 , which can be described as the product of the duty cycle D, the fraction of time that the transient linker occupies the docking site, and the association rate constant for the imager probe to bind to the transient linker, k a成像器探针 :

[0366] k a有效瞬时接头介导 =D*k a,成像器探针 (3)

[0367] The duty cycle can be expressed as:

[0368]

[0369] The average time τ during which no transient linker is bound to the docking site off瞬时接头 Depends on the concentration of transient linker c 瞬时接头 , and the association rate constant k for the transient linker binding to the docking site a瞬时接头 :

[0370]

[0371] For a transient linker concentration of 50 nM, an average binding time of 100 s, and 2 x 10 6 M -1 s -1 The association rate is, for example, 91%.

[0372] As mentioned above, a high concentration of transient linkers in solution, i.e., not bound to any docking site, will result in a non-negligible fraction of the imager probes being bound to these transient linkers, without generating a localizable signal. Only the free fraction f of the unbound imager probes is available for binding to transient linkers bound to docking sites, thereby reducing the imager probe concentration in solution to below the concentration initially added to the imaging buffer:

[0373] c 游离成像器探针 =f*c 成像器探针 (6)

[0374] According to Jarmoskaite et al. 1 , the portion f that is not bound to the imager probe can be expressed as:

[0375]

[0376] Here, K D is the equilibrium dissociation constant between the transient linker and the imager probe:

[0377]

[0378] K D can be estimated by measuring the average ON and OFF times of the imager probe binding to the DNA origami with the same 瞬时接头 = 0. It is assumed here that these times depend only on the oligonucleotide sequence and that possible effects of the surrounding environment (DNA origami vs. transient linker) are negligible.

[0379] Combining Equations 3-8, the effective association rate constant for the imager probe binding to the docking site via the transient linker is given as c 瞬时接头 The function can be expressed as:

[0380]

[0381] For comparison: with k a成像器探针 In contrast, the effective association rate k of the imager probe directly binding to the complementary docking site on the DNA origami in the presence of a transient linker in solution is a有效直接 As c 瞬时接头 The function of will also decrease by a factor of f (i.e., Equation 6 applies), but is independent of the duty cycle D. It can be expressed as:

[0382]

[0383] Figure 13D The solid curve in is calculated using equations 9 and 10 and the following values:

[0384] c 成像器探针 =10nM

[0385] τ on成像器探针 =0.25s

[0386] τ on瞬时接头 =100s

[0387] k a成像器探针 =45*10 6 M -1 s -1

[0388] k a瞬时接头 =3*10 6 M -1 s -1

[0389] The following definitions were used:

[0390] T off瞬时接头介导 : Average time that no imager probe binds to the docking site of a DNA origami designed to bind the imager probe via a transient linker

[0391] T off直接 : Average time without imager probe binding to the docking site of the DNA origami, which is designed to directly bind the imager probe

[0392] T off瞬时接头 : Average time without transient linker binding to the docking site of the corresponding DNA origami

[0393] T on瞬时接头 : Average time for transient linker binding to the docking site of the corresponding DNA origami

[0394] T off成像器探针 : The average time that no imager probe binds to a specific docking site in the absence of a transient linker

[0395] T on成像器探针 : the average time for the imager probe to bind to its complementary sequence, either as part of a transient linker or as a direct docking site on the corresponding DNA origami

[0396] k a有效瞬时接头介导 : Effective association rate constants for imager probe binding to DNA origami docking sites via transient linkers, including corrections for duty cycle and competition with transient linkers in solution

[0397] k a有效直接 : Effective association rate constants for direct binding of imager probes to DNA origami with suitable docking sites in the presence of transient linkers, including competition for binding with transient linkers in solution

[0398] k a成像器探针 : Association rate constant of the imager probe binding to its complementary sequence

[0399] k a瞬时接头 : Association rate constant for transient linker binding to the docking site

[0400] D: Duty cycle, which is the fraction of time that the transient linker occupies the docking site

[0401] C 成像器探针 : the molar concentration of the imager probe initially added to the imaging buffer

[0402] C 游离成像器探针 : the molar concentration of imager probe in solution that is not bound to the transient linker

[0403] C 瞬时接头 : Molar concentration of transient linker

[0404] f: unbound fraction of imager probe

[0405] K D: The equilibrium dissociation constant between the imaging probe and the transient linker

[0406] Examples of implementation methods

[0407] In some aspects, the present invention relates to the following non-limiting embodiments:

[0408] Embodiment 1: A microscope imaging method, comprising:

[0409] exposing a sample having a plurality of targets to a plurality of transient single-stranded nucleic acid adaptor molecules; and

[0410] exposing the sample to a plurality of single-stranded nucleic acid imaging molecules; and

[0411] exposing the sample to an illumination source having a wavelength capable of interacting with the plurality of single-stranded nucleic acid imaging molecules;

[0412] The transient single-stranded nucleic acid linker molecule comprises:

[0413] a first region having a target-complementary sequence; and

[0414] The second region has a complementary sequence to the single-stranded nucleic acid imaging molecule.

[0415] Embodiment 2: The method according to embodiment 1, wherein the number of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the estimated or actual number of targets.

[0416] Embodiment 3: The method according to embodiment 1, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than that of the plurality of single-stranded nucleic acid imaging molecules.

[0417] Embodiment 4: The method of embodiment 1, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules by a ratio selected from the group consisting of: at least about 1, at least about 10, and at least about 100.

[0418] Embodiment 5: The method according to embodiment 1, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than about 500 times the ratio of the plurality of single-stranded nucleic acid imaging molecules.

[0419] Embodiment 6: The method according to embodiment 1, wherein the target complementary sequence is less than 11 nucleotides.

[0420] Embodiment 7: The method according to embodiment 1, wherein the target complementary sequence is selected from: 6 to 10 nucleotides and 8 to 10 nucleotides.

[0421] Embodiment 8: The method according to embodiment 1, further comprising:

[0422] exposing the sample to an eraser molecule adapted and configured to quench the transient single-stranded nucleic acid adaptor molecule;

[0423] exposing the sample to a second plurality of transient single-stranded nucleic acid adaptor molecules having a second, different, target-complementary sequence; and

[0424] exposing the sample to the plurality of single-stranded nucleic acid imaging molecules; and

[0425] The sample is exposed to an illumination source having a wavelength capable of interacting with the plurality of single-stranded nucleic acid imaging molecules.

[0426] Embodiment 9: The method of embodiment 8, wherein the method is performed without washing the plurality of transient single-stranded nucleic acid adaptor molecules from the sample.

[0427] Embodiment 10: The method of embodiment 8, wherein the eraser molecule and the second plurality of transient single-stranded nucleic acid adaptor molecules are introduced simultaneously.

[0428] Embodiment 11: The method of embodiment 8, wherein the eraser molecule and the second plurality of transient single-stranded nucleic acid adaptor molecules are introduced sequentially.

[0429] Embodiment 12: The method of embodiment 1, wherein the plurality of single-stranded nucleic acid imaging molecules comprises a speed-optimized sequence.

[0430] Embodiment 13: The method of embodiment 1, wherein the plurality of single-stranded nucleic acid imaging molecules are fluorescent.

[0431] Embodiment 14: The method of embodiment 1, wherein:

[0432] The plurality of single-stranded nucleic acid imaging molecules are fluorescent; and

[0433] The detected light change is the fluorescence emitted by the single-stranded nucleic acid imaging molecule.

[0434] Embodiment 15: The method according to embodiment 1, wherein the single-stranded nucleic acid imaging molecules are detected individually to generate single-molecule localization super-resolution microscopy images.

[0435] Embodiment 16: The method according to embodiment 1, wherein the sample is a biological tissue section.

[0436] Embodiment 17: The method of embodiment 1, wherein:

[0437] The plurality of targets are antibodies or binding ligands that bind to a plurality of specific proteins in the sample; and

[0438] Each type of antibody or binding ligand is conjugated to a different single-stranded nucleic acid.

[0439] Embodiment 18: The method of embodiment 1, wherein the single-stranded nucleic acid is an RNA or DNA molecule.

[0440] Embodiment 19: The method of embodiment 1, wherein the single-stranded nucleic acid imaging molecule comprises a single-stranded nucleic acid coupled to a molecule exhibiting a Raman signature detectable by a Raman microscope.

[0441] Embodiment 20: The method of embodiment 1, wherein the single-stranded nucleic acid imaging molecule comprises a single-stranded nucleic acid coupled to a nanoparticle.

[0442] Embodiment 21: The method of Embodiment 20, wherein the nanoparticles are gold nanoparticles.

[0443] Embodiment 22: The method of embodiment 20, wherein the interaction is scattering.

[0444] Embodiment 23: A kit comprising:

[0445] a plurality of transient single-stranded nucleic acid adaptor molecules; and

[0446] a plurality of single-stranded nucleic acid imaging molecules; and

[0447] The transient single-stranded nucleic acid linker molecule comprises:

[0448] a first region having a target-complementary sequence; and

[0449] The second region has a complementary sequence to the single-stranded nucleic acid imaging molecule.

[0450] Embodiment 24: The kit according to embodiment 23, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than that of the plurality of single-stranded nucleic acid imaging molecules.

[0451] Embodiment 25: The kit of embodiment 23, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules by a ratio selected from the group consisting of: at least about 1, at least about 10, and at least about 100.

[0452] Embodiment 26: The kit according to embodiment 23, wherein the number or concentration of the plurality of transient non-fluorescent single-stranded nucleic acid adaptor molecules is greater than the ratio of about 500 times of the plurality of fluorescent imaging molecules.

[0453] Embodiment 27: An imaging method, comprising:

[0454] Perform the first marking, which includes:

[0455] applying one or more targets including a first target comprising a first target single-stranded nucleic acid to the sample;

[0456] applying a first adapter comprising a first adapter single-stranded nucleic acid to the sample; and

[0457] applying a first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif to the sample; and

[0458] acquiring a first image of the first detection motif,

[0459] Wherein the first linker single-stranded nucleic acid comprises:

[0460] a first region having sufficient sequence complementarity to bind to the target single-stranded nucleic acid; and

[0461] a second region having sufficient sequence complementarity to bind to the first imaging molecule single-stranded nucleic acid, and

[0462] wherein the first linker binds the target and the first imaging molecule.

[0463] Embodiment 28: The method of embodiment 27, wherein at least one of the following applies:

[0464] (a) the first target comprises the first target single-stranded nucleic acid attached to an antibody or polypeptide, the antibody or polypeptide specifically binding to a site of interest in the sample, optionally a protein, protein complex, nucleic acid, cellular structure, organelle or cell;

[0465] (b) The first target comprises the first target single-stranded nucleic acid attached to a targeting nucleic acid that specifically binds to or is complementary to a point of interest in the sample, optionally a nucleic acid.

[0466] Embodiment 29: The method of embodiment 27, wherein at least one of the following applies:

[0467] (a) the first detection motif is a fluorescent motif, optionally a fluorescent protein, a fluorescent small molecule or a quantum dot,

[0468] (b) the first detection motif is a metal nanoparticle, optionally a gold nanoparticle,

[0469] (c) said first detection motif is a Raman scattering motif, optionally a Raman dye, optionally a Raman dye suitable for stimulated Raman scattering microscopy,

[0470] (d) The first detection motif is an isotope.

[0471] Embodiment 30: The method of embodiment 27, wherein at least one of the following applies:

[0472] (a) the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 1 to 30,

[0473] (b) the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 5 to 20,

[0474] (c) The number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 8 to 12.

[0475] Embodiment 31: The method of embodiment 27, wherein at least one of the following applies:

[0476] (a) K between the first target single-stranded nucleic acid and the first region of the linker on The range is 1*10 4 1 / M*s and 1*10 7 1 / M*s,

[0477] (b) K between the first target single-stranded nucleic acid and the first region of the linker off The range is between 1 1 / s and 0.0001 1 / s,

[0478] (c) K between the first target single-stranded nucleic acid and the first region of the linker d The range is between 10 μM and 1 nM.

[0479] Embodiment 32: The method of embodiment 27, wherein at least one of the following applies:

[0480] (a) the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 1 to 30,

[0481] (b) the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 5 to 20,

[0482] (c) The number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 8 to 12.

[0483] Embodiment 33: The method of embodiment 27, wherein at least one of the following applies:

[0484] (a) K between the first imaging molecule single-stranded nucleic acid and the second region of the linkeron Range is 1*10 4 1 / M*s and 1*10 7 1 / M*s,

[0485] (b) K between the first imaging molecule single-stranded nucleic acid and the second region of the linker off The range is between 1000 1 / s and 0.0001 1 / s,

[0486] (a) K between the first imaging molecule single-stranded nucleic acid and the second region of the linker d The range is between 10 μM and 1 nM.

[0487] Embodiment 34: The method of embodiment 27, comprising:

[0488] Performing the first marking comprises:

[0489] applying a plurality of first targets to the sample, each first target comprising a first target single-stranded nucleic acid;

[0490] applying a plurality of first adapters to the sample, each first adapter comprising a first adapter single-stranded nucleic acid; and

[0491] applying a plurality of first imaging molecules to the sample, each first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif; and

[0492] acquiring a first image of said plurality of first detection motifs of said plurality of first imaging molecules,

[0493] wherein each of said first linkers mediates the association of each of said plurality of first targets with each of said plurality of first imaging molecules in a sequence-specific manner, and

[0494] Wherein the plurality of first detection motifs do not interfere with each other during acquisition of the first image.

[0495] Embodiment 35: The method of Embodiment 27, wherein at least one of the following applies:

[0496] (a) The method further comprises:

[0497] applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker;

[0498] Perform a second marking, which includes:

[0499] applying a second target comprising a second target single-stranded nucleic acid to the sample;

[0500] applying a second adaptor comprising a second adaptor single-stranded nucleic acid to the sample; and

[0501] applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample; and

[0502] acquiring a second image of the second detection motif,

[0503] (b) the one or more targets applied in the first labeling further include a second target comprising a second single-stranded nucleic acid, and the method further comprises:

[0504] applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker;

[0505] Perform a second marking, which includes:

[0506] applying a second adaptor comprising a second adaptor single-stranded nucleic acid to the sample; and

[0507] applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample; and

[0508] acquiring a second image of the second detection motif,

[0509] Wherein, for (a) and (b), the second linker single-stranded nucleic acid comprises:

[0510] a third region having sufficient complementarity to bind to the second target single-stranded nucleic acid; and

[0511] a fourth region having sufficient complementarity to bind to the second imaging molecule single-stranded nucleic acid, and

[0512] Wherein, for (a) and (b), the second linker mediates the association between the second target and the second imaging molecule.

[0513] Embodiment 36: The method of embodiment 35, wherein

[0514] The eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first linker, and

[0515] The eraser molecule

[0516] preventing hybridization between the first target single-stranded nucleic acid and the first region of the adapter, or

[0517] Hybridization between the first imaging molecule single-stranded nucleic acid and the second region of the linker is prevented.

[0518] Embodiment 37: The method of embodiment 35, wherein at least one of the following applies:

[0519] (a) the first target, the first linker, the first imaging molecule and the eraser molecule are not washed off the sample before applying the second target, the second linker and the second imaging molecule,

[0520] (b) the one or more targets, the first linker, the first imaging molecule and the eraser molecule are not washed from the sample prior to applying the second linker and the second imaging molecule.

[0521] Embodiment 38: The method of embodiment 35, wherein the signal of the first detection motif and the signal of the second detection motif overlap or are the same.

[0522] Embodiment 39: The method according to embodiment 35, wherein in each of the first label and the second label, 4 or more different detection motifs with different signals are used.

[0523] Embodiment 40: The method of embodiment 35, wherein the sample is expanded according to expansion microscopy.

[0524] Embodiment 41: A device comprising:

[0525] a sample holder for holding a sample;

[0526] a computer-operated liquid applicator for applying the liquid to the sample;

[0527] Computer-operated microscopes; and

[0528] computer,

[0529] The computer is programmed to:

[0530] (a) operating the liquid applicator to perform a first application of:

[0531] one or more targets, including a first target for specifically binding to a first component in the sample;

[0532] a first imaging molecule comprising a first detection motif detectable by said microscope; and

[0533] a first linker for mediating the association between the first target and the first imaging molecule,

[0534] (b) operating the microscope to record a first signal from the first detection motif,

[0535] (c) operating the liquid applicator to perform a second application of (c1) or (c2);

[0536] (c1) The second applying includes applying the following:

[0537] an eraser molecule for interrupting the interaction between the first target and the first linker mediated by the first linker;

[0538] a second target for specifically binding to a second component in the sample;

[0539] a second imaging molecule comprising a second detection motif detectable by said microscope; and

[0540] a second linker for mediating the association between the second target and the second imaging molecule,

[0541] (c2) The one or more targets applied in (a) further include a second target for specifically binding to a second component in the sample, and the second applying includes applying the following:

[0542] an eraser molecule for interrupting the interaction between the first target and the first linker mediated by the first linker;

[0543] a second imaging molecule comprising a second detection motif detectable by said microscope; and

[0544] a second linker for mediating the association between the second target and the second imaging molecule,

[0545] (d) operating said microscope to record a second signal from said second detection motif,

[0546] The computer is programmed to perform operations (a), (b), (c) and (d) sequentially in the order of (a), (b), (c) and (d).

[0547] Embodiment 42: The device of embodiment 41, wherein the device does not remove the liquid applied in operation (a) before performing operations (c) and (d).

[0548] Embodiment 43: The apparatus of embodiment 41, wherein the first signal and the second signal overlap or are identical to each other.

[0549] Embodiment 44: The device of embodiment 41, wherein the first detection motif and the second detection motif are

[0550] (a) the first detection motif or the second detection motif is a fluorescent motif, optionally a fluorescent protein, a fluorescent small molecule or a quantum dot,

[0551] (b) the first detection motif or the second detection motif is a metal nanoparticle, optionally a gold nanoparticle,

[0552] (c) the first detection motif or the second detection motif is a Raman scattering motif, optionally a Raman dye, optionally a Raman dye suitable for stimulated Raman scattering microscopy,

[0553] (d) The first detection motif or the second detection motif is an isotope.

[0554] Embodiment 45: The device of embodiment 41 further comprises a reservoir for storing the one or more targets, the first linker, the first imaging molecule, the eraser, the second target, the second linker, and the second imaging molecule.

[0555] Embodiment 46: The device of embodiment 41, further comprising at least one selected from the group consisting of the first target, the first linker, the first imaging molecule, the eraser, the second target, the second linker, and the second imaging molecule, wherein

[0556] The first target comprises a first target single-stranded nucleic acid;

[0557] The first linker comprises a first linker single-stranded nucleic acid;

[0558] The first imaging molecule comprises a first imaging molecule single-stranded nucleic acid attached to the first detection motif;

[0559] The eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first linker;

[0560] The second target includes a second target single-stranded nucleic acid;

[0561] The second linker comprises a second linker single-stranded nucleic acid;

[0562] The second imaging molecule comprises a second imaging molecule single-stranded nucleic acid attached to the second detection motif;

[0563] Wherein the first linker single-stranded nucleic acid comprises:

[0564] a first region having sufficient sequence complementarity to bind to the first target single-stranded nucleic acid; and

[0565] a second region having sufficient sequence complementarity to bind to the first imaging molecule single-stranded nucleic acid, and

[0566] Wherein the second linker single-stranded nucleic acid comprises:

[0567] a third region having sufficient sequence complementarity to bind to the second target single-stranded nucleic acid; and

[0568] The fourth region has sufficient sequence complementarity to bind to the second imaging molecule single-stranded nucleic acid.

[0569] Embodiment 47: The device of embodiment 46, comprising the first target, the first linker, the first imaging molecule, the eraser, the second target, the second linker, and the second imaging molecule.

[0570] equivalent

[0571] While preferred embodiments of the present invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims.

[0572] Incorporated by Reference

[0573] All patents, published patent applications, and other references cited herein are expressly incorporated by reference in their entirety.

Claims

1. A microscope imaging method, comprising: exposing a sample having multiple targets to a plurality of transient single-stranded nucleic acid adaptor molecules; and exposing the sample to a plurality of single-stranded nucleic acid imaging molecules; and exposing the sample to an illumination source having a wavelength capable of interacting with the plurality of single-stranded nucleic acid imaging molecules; The transient single-stranded nucleic acid linker molecule comprises: a first region having a target-complementary sequence; and The second region has a complementary sequence to the single-stranded nucleic acid imaging molecule. 2 . The method of claim 1 , wherein the number of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the estimated or actual number of targets. 3 . The method of claim 1 , wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than that of the plurality of single-stranded nucleic acid imaging molecules.

4. The method of claim 1, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules by a ratio selected from the group consisting of: at least about 1, at least about 10, and at least about 100. 5 . The method of claim 1 , wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules by a factor of about 500. The method of claim 1 , wherein the target-complementary sequence is less than 11 nucleotides. The method according to claim 1 , wherein the target-complementary sequence is selected from the group consisting of: 6 to 10 nucleotides and 8 to 10 nucleotides.

8. The method according to claim 1, further comprising: exposing the sample to an eraser molecule adapted and configured to quench the transient single-stranded nucleic acid adaptor molecule; exposing the sample to a second plurality of transient single-stranded nucleic acid adaptor molecules having a second, different, target-complementary sequence; and exposing the sample to the plurality of single-stranded nucleic acid imaging molecules; and The sample is exposed to an illumination source having a wavelength capable of interacting with the plurality of single-stranded nucleic acid imaging molecules.

9. The method of claim 8, wherein the method is performed without washing the plurality of transient single-stranded nucleic acid adaptor molecules from the sample.

10. The method of claim 8, wherein the eraser molecule and the second plurality of transient single-stranded nucleic acid adaptor molecules are introduced simultaneously. The method of claim 8 , wherein the eraser molecule and the second plurality of transient single-stranded nucleic acid adaptor molecules are introduced sequentially.

12. The method of claim 1, wherein the plurality of single-stranded nucleic acid imaging molecules comprises a speed-optimized sequence.

13. The method of claim 1, wherein the plurality of single-stranded nucleic acid imaging molecules are fluorescent.

14. The method of claim 1, wherein: The plurality of single-stranded nucleic acid imaging molecules are fluorescent; and The detected light change is the fluorescence emitted by the single-stranded nucleic acid imaging molecule.

15. The method of claim 1, wherein the single-stranded nucleic acid imaging molecules are detected individually to generate single-molecule localization super-resolution microscopy images. The method according to claim 1 , wherein the sample is a biological tissue section.

17. The method of claim 1, wherein: The plurality of targets are antibodies or binding ligands that bind to a plurality of specific proteins in the sample; and Each type of antibody or binding ligand is conjugated to a different single-stranded nucleic acid.

18. The method according to claim 1, wherein the single-stranded nucleic acid is an RNA or DNA molecule.

19. The method of claim 1, wherein the single-stranded nucleic acid imaging molecule comprises a single-stranded nucleic acid coupled to a molecule exhibiting a Raman signature detectable by a Raman microscope.

20. The method of claim 1, wherein the single-stranded nucleic acid imaging molecule comprises a single-stranded nucleic acid coupled to a nanoparticle.

21. The method of claim 20, wherein the nanoparticles are gold nanoparticles.

22. The method of claim 20, wherein the interaction is scattering.

23. A kit comprising: multiple transient single-stranded nucleic acid adaptor molecules; and multiple single-stranded nucleic acid imaging molecules; and The transient single-stranded nucleic acid linker molecule comprises: a first region having a target-complementary sequence; and The second region has a complementary sequence to the single-stranded nucleic acid imaging molecule.

24. The kit of claim 23, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than that of the plurality of single-stranded nucleic acid imaging molecules.

25. The kit of claim 23, wherein the number or concentration of the plurality of transient single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of single-stranded nucleic acid imaging molecules by a ratio selected from the group consisting of: at least about 1, at least about 10, and at least about 100.

26. The kit of claim 23, wherein the number or concentration of the plurality of transient non-fluorescent single-stranded nucleic acid adaptor molecules is greater than the number or concentration of the plurality of fluorescent imaging molecules by a factor of about 500.

27. A method of imaging, comprising: Perform the first marking, which includes: applying one or more targets including a first target comprising a first target single-stranded nucleic acid to the sample; applying a first adapter comprising a first adapter single-stranded nucleic acid to the sample; and applying a first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif to the sample; and acquiring a first image of the first detection motif, Wherein the first linker single-stranded nucleic acid comprises: a first region having sufficient sequence complementarity to bind to the target single-stranded nucleic acid; and a second region having sufficient sequence complementarity to bind to the first imaging molecule single-stranded nucleic acid, and wherein the first linker binds the target and the first imaging molecule.

28. The method of claim 27, wherein at least one of the following applies: (a) the first target comprises the first target single-stranded nucleic acid attached to an antibody or polypeptide, the antibody or polypeptide specifically binding to a site of interest in the sample, optionally a protein, protein complex, nucleic acid, cellular structure, organelle or cell; (b) The first target comprises the first target single-stranded nucleic acid attached to a targeting nucleic acid that specifically binds to or is complementary to a point of interest in the sample, optionally a nucleic acid.

29. The method of claim 27, wherein at least one of the following applies: (a) the first detection motif is a fluorescent motif, optionally a fluorescent protein, a fluorescent small molecule or a quantum dot, (b) the first detection motif is a metal nanoparticle, optionally a gold nanoparticle, (c) said first detection motif is a Raman scattering motif, optionally a Raman dye, optionally a Raman dye suitable for stimulated Raman scattering microscopy, (d) The first detection motif is an isotope.

30. The method of claim 27, wherein at least one of the following applies: (a) the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter ranges from 1 to 30, (b) the number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first adapter is in the range of 5-20, (c) The number of complementary base pairs between the first target single-stranded nucleic acid and the first region of the first linker is in the range of 8-12.

31. The method of claim 27, wherein at least one of the following applies: (a) K between the first target single-stranded nucleic acid and the first region of the adapter on The range is 1*10 4 1 / M*s and 1*10 7 1 / M*s, (b) K between the first target single-stranded nucleic acid and the first region of the linker off The range is between 1 1 / s and 0.00011 / s, (c) K between the first target single-stranded nucleic acid and the first region of the adapter d The range is between 10 μM and 1 nM.

32. The method of claim 27, wherein at least one of the following applies: (a) the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 1 to 30, (b) the number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 5 to 20, (c) The number of complementary base pairs between the first imaging molecule single-stranded nucleic acid and the second region of the linker ranges from 8 to 12.

33. The method of claim 27, wherein at least one of the following applies: (a) K between the first imaging molecule single-stranded nucleic acid and the second region of the linker on The range is 1*10 4 1 / M*s and 1*10 7 1 / M*s, (b) K between the first imaging molecule single-stranded nucleic acid and the second region of the linker off The range is between 1000 1 / s and 0.0001 1 / s, (a) K between the first imaging molecule single-stranded nucleic acid and the second region of the linker d The range is between 10 μM and 1 nM.

34. The method of claim 27, comprising: Performing the first marking comprises: applying a plurality of first targets to the sample, each first target comprising a first target single-stranded nucleic acid; applying a plurality of first adapters to the sample, each first adapter comprising a first adapter single-stranded nucleic acid; and applying a plurality of first imaging molecules to the sample, each first imaging molecule comprising a first imaging molecule single-stranded nucleic acid and a first detection motif; and acquiring a first image of said plurality of first detection motifs of said plurality of first imaging molecules, wherein each of said first linkers mediates the association of each of said plurality of first targets with each of said plurality of first imaging molecules in a sequence-specific manner, and Wherein the plurality of first detection motifs do not interfere with each other during acquisition of the first image.

35. The method of claim 27, wherein at least one of the following applies: (a) The method further comprises: applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker; Perform a second marking, which includes: applying a second target comprising a second target single-stranded nucleic acid to the sample; applying a second adaptor comprising a second adaptor single-stranded nucleic acid to the sample; and applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample; and acquiring a second image of the second detection motif, (b) the one or more targets applied in the first labeling further include a second target comprising a second single-stranded nucleic acid, and the method further comprises: applying an eraser molecule to the sample to disrupt the association between the first target and the first imaging molecule mediated by the first linker; Perform a second marking, which includes: applying a second adaptor comprising a second adaptor single-stranded nucleic acid to the sample; and applying a second imaging molecule comprising a second imaging molecule single-stranded nucleic acid and a second detection motif to the sample; and acquiring a second image of the second detection motif, Wherein, for (a) and (b), the second linker single-stranded nucleic acid comprises: a third region having sufficient complementarity to bind to the second target single-stranded nucleic acid; and a fourth region having sufficient complementarity to bind to the second imaging molecule single-stranded nucleic acid, and Wherein, for (a) and (b), the second linker mediates the association between the second target and the second imaging molecule.

36. The method of claim 35, wherein The eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first linker, and The eraser molecule preventing hybridization between the first target single-stranded nucleic acid and the first region of the adapter, or Hybridization between the first imaging molecule single-stranded nucleic acid and the second region of the linker is prevented.

37. The method of claim 35, wherein at least one of the following applies: (a) the first target, the first linker, the first imaging molecule and the eraser molecule are not washed off the sample before applying the second target, the second linker and the second imaging molecule, (b) the one or more targets, the first linker, the first imaging molecule and the eraser molecule are not washed from the sample prior to applying the second linker and the second imaging molecule.

38. The method of claim 35, wherein the signal of the first detection motif and the signal of the second detection motif overlap or are identical.

39. The method of claim 35, wherein in each of the first label and the second label, four or more different detection motifs with different signals are used.

40. The method of claim 35, wherein the sample is expanded according to expansion microscopy.

41. An apparatus comprising: a sample holder for holding a sample; a computer-operated liquid applicator for applying a liquid to the sample; computer-operated microscopes; and computer, The computer is programmed to: (a) operating the liquid applicator to perform a first application of: one or more targets, including a first target for specifically binding to a first component in the sample; a first imaging molecule comprising a first detection motif detectable by said microscope; and a first linker for mediating the association between the first target and the first imaging molecule, (b) operating the microscope to record a first signal from the first detection motif, (c) operating the liquid applicator to perform a second application of (c1) or (c2); (c1) The second applying includes applying the following: an eraser molecule for interrupting the interaction between the first target and the first linker mediated by the first linker; a second target for specifically binding to a second component in the sample; a second imaging molecule comprising a second detection motif detectable by said microscope; and a second linker for mediating the association between the second target and the second imaging molecule, (c2) The one or more targets applied in (a) further include a second target for specifically binding to a second component in the sample, and the second applying includes applying the following: an eraser molecule for interrupting the interaction between the first target and the first linker mediated by the first linker; a second imaging molecule comprising a second detection motif detectable by said microscope; and a second linker for mediating the association between the second target and the second imaging molecule, (d) operating said microscope to record a second signal from said second detection motif, The computer is programmed to perform operations (a), (b), (c) and (d) sequentially in the order of (a), (b), (c) and (d).

42. The device of claim 41, wherein the device does not remove the liquid applied in operation (a) before performing operations (c) and (d).

43. The apparatus of claim 41, wherein the first signal and the second signal overlap or are identical to each other.

44. The device of claim 41, wherein the first detection motif and the second detection motif are (a) the first detection motif or the second detection motif is a fluorescent motif, optionally a fluorescent protein, a fluorescent small molecule or a quantum dot, (b) the first detection motif or the second detection motif is a metal nanoparticle, optionally a gold nanoparticle, (c) the first detection motif or the second detection motif is a Raman scattering motif, optionally a Raman dye, optionally a Raman dye suitable for stimulated Raman scattering microscopy, (d) The first detection motif or the second detection motif is an isotope.

45. The device of claim 41, further comprising a reservoir for storing the one or more targets, the first linker, the first imaging molecule, the eraser, the second target, the second linker, and the second imaging molecule.

46. ​​The device of claim 41 , further comprising at least one selected from the group consisting of the first target, the first linker, the first imaging molecule, the eraser, the second target, the second linker, and the second imaging molecule, wherein the first target comprises a first target single-stranded nucleic acid; The first linker comprises a first linker single-stranded nucleic acid; The first imaging molecule comprises a first imaging molecule single-stranded nucleic acid attached to the first detection motif; The eraser molecule comprises an eraser molecule single-stranded nucleic acid having sufficient sequence complementarity to bind to the first region or the second region of the first linker; The second target includes a second target single-stranded nucleic acid; The second linker comprises a second linker single-stranded nucleic acid; The second imaging molecule comprises a second imaging molecule single-stranded nucleic acid attached to the second detection motif; Wherein the first linker single-stranded nucleic acid comprises: a first region having sufficient sequence complementarity to bind to the first target single-stranded nucleic acid; and a second region having sufficient sequence complementarity to bind to the first imaging molecule single-stranded nucleic acid, and wherein the second linker single-stranded nucleic acid comprises: a third region having sufficient sequence complementarity to bind to the second target single-stranded nucleic acid; and The fourth region has sufficient sequence complementarity to bind to the second imaging molecule single-stranded nucleic acid.

47. The device of claim 46, comprising the first target, the first linker, the first imaging molecule, the eraser, the second target, the second linker, and the second imaging molecule.

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