Quantitative dna-based imaging and super-resolution imaging

Abstract

The present disclosure provides, inter alia, methods and compositions (e.g., conjugates) for imaging, at high spatial resolution, targets of interest.

What is claimed is: 
     
         1 . A protein-nucleic acid conjugate, comprising a protein linked to a docking strand that is capable of transiently binding to a complementary labeled imager strand. 
     
     
         2 . The protein-nucleic acid conjugate of  claim 1 , wherein the docking strand is transiently bound to the complementary labeled imager strand. 
     
     
         3 . The protein-nucleic acid conjugate of  claim 1  or  2 , wherein the protein is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         4 . The protein-nucleic acid conjugate of any one of  claims 1 - 3 , wherein the protein is linked to the docking strand through an intermediate linker. 
     
     
         5 . The protein-nucleic acid conjugate of  claim 4 , wherein the intermediate linker comprises biotin and streptavidin. 
     
     
         6 . The protein-nucleic acid conjugate of any one of  claims 3 - 5 , wherein the antibody is a monoclonal antibody. 
     
     
         7 . The protein-nucleic acid conjugate of any one of  claims 1 - 6 , wherein the complementary labeled imager strand is a complementary fluorescently-labeled imager strand. 
     
     
         8 . The protein-nucleic acid conjugate of  claim 7 , wherein the complementary fluorescently-labeled imager strand comprises at least one fluorophore. 
     
     
         9 . The protein-nucleic acid conjugate of any one of  claims 1 - 8 , wherein the complementary labeled imager strand is about 4 to about 30 nucleotides in length. 
     
     
         10 . The protein-nucleic acid conjugate of  claim 9 , wherein the complementary labeled imager strand is about 8 to about 10 nucleotides in length. 
     
     
         11 . A target bound to at least one protein-nucleic acid conjugate of any one of  claims 1 - 10 . 
     
     
         12 . The target of  claim 11 , wherein the target is a protein. 
     
     
         13 . A plurality of the protein-nucleic acid conjugates of any one of  claims 1 - 10 . 
     
     
         14 . The plurality of  claim 13 , wherein the plurality comprises at least two subsets of the protein-nucleic acid conjugates, and the protein-nucleic acid conjugates of each subset bind to different targets. 
     
     
         15 . A composition comprising the plurality of protein-nucleic acid conjugates of  claim 13  or  14 , wherein at least one of the protein-nucleic acid conjugates is bound to at least one target. 
     
     
         16 . A composition comprising:
 at least one protein-nucleic acid conjugate that comprises a protein linked to a docking strand, wherein the at least one protein-nucleic acid conjugate is bound to a target; and   at least one complementary labeled imager strand that is transiently bound to the at least one protein-nucleic acid conjugate.   
     
     
         17 . The composition of  claim 16 , comprising at least two complementary labeled imager strands, wherein the at least two complementary labeled imager strands are identical. 
     
     
         18 . The composition of  claim 16 , comprising at least two complementary labeled imager strands, wherein the at least two complementary labeled imager strands are different. 
     
     
         19 . The composition of any one of  claims 16 - 18 , wherein the number of complementary labeled imager strands is less than, greater than or equal to the number of protein-nucleic acid conjugates. 
     
     
         20 . The composition of any one of  claims 13 - 19 , wherein the composition comprises at least 2 different complementary labeled imager strands. 
     
     
         21 . The composition of any one of  claims 13 - 20 , wherein the composition comprises at least 5 different complementary labeled imager strands. 
     
     
         22 . The composition of any one of  claims 13 - 21 , wherein the composition comprises at least 10 different complementary labeled imager strands. 
     
     
         23 . The composition of  claim 22 , wherein the composition comprises at least 100 different complementary labeled imager strands. 
     
     
         24 . The composition of any one of  claims 16 - 23 , wherein the complementary labeled imager strands are complementary fluorescently-labeled imager strands. 
     
     
         25 . An antibody-DNA conjugate, comprising a monoclonal antibody linked to a docking strand that is bound to a complementary labeled imager strand, wherein the antibody and the docking strand are each biotinylated and linked to each other through a biotin-streptavidin linker. 
     
     
         26 . The antibody-DNA conjugate of  claim 25 , wherein the complementary labeled imager strand is a complementary fluorescently-labeled imager strand. 
     
     
         27 . The antibody-DNA conjugate of any one of  claims 1 - 26 , wherein the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         28 . The antibody-DNA conjugate of  claim 27 , wherein the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         29 . The antibody-DNA conjugate of any one of  claims 21 - 28 , wherein the thermal stability of a docking strand transiently bound to a complementary labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         30 . An aptamer-nucleic acid conjugate, comprising a nucleic acid aptamer linked to a docking strand that is transiently bound to a complementary labeled imager strand. 
     
     
         31 . The aptamer-nucleic acid conjugate of  claim 30 , wherein the complementary labeled imager strand is a complementary fluorescently-labeled imager strand. 
     
     
         32 . The aptamer-nucleic acid conjugate of  claim 30  or  31 , wherein the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         33 . The aptamer-nucleic acid conjugate of  claim 32 , wherein the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         34 . The aptamer-nucleic acid conjugate of any one of  claims 30 - 33 , wherein the thermal stability of a docking strand transiently bound to a complementary labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         35 . A method of detecting a target in a sample, the method comprising:
 contacting a sample with (a) at least one protein-nucleic acid conjugate that comprises a protein linked to a docking strand and (b) at least one fluorescently-labeled imager strand that is complementary to and transiently binds to the docking strand of the at least one protein-nucleic acid conjugate; and   determining whether the at least one protein-nucleic acid conjugate binds to the target in the sample.   
     
     
         36 . The method of  claim 35 , wherein the determining step comprises imaging transient binding of the at least one fluorescently-labeled imager strand to the docking strand of the at least one protein-nucleic acid conjugate. 
     
     
         37 . The method of  claim 35  or  36 , wherein the protein of the protein-nucleic acid conjugate is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         38 . The method of  claim 36 , wherein the antibody is a monoclonal antibody. 
     
     
         39 . The method of any one of  claims 35 - 38 , wherein the protein of the protein-nucleic acid conjugate is linked to the docking strand through an intermediate linker. 
     
     
         40 . The method of  claim 39 , wherein the intermediate linker comprises biotin and/or streptavidin. 
     
     
         41 . The method of any one of  claims 35 - 40 , wherein the complementary fluorescently-labeled imager strand comprises at least one fluorophore. 
     
     
         42 . The method of any one of  claims 35 - 41 , wherein the complementary fluorescently-labeled imager strand is about 4 to about 30 nucleotides in length. 
     
     
         43 . The method of  claim 42 , wherein the complementary fluorescently-labeled imager strand is about 8 to about 10 nucleotides in length. 
     
     
         44 . The method of any one of  claims 35 - 43 , wherein the sample is a cell or cell lysate. 
     
     
         45 . The method of any one of  claims 35 - 44 , wherein the target is a protein 
     
     
         46 . The method of any one of  claims 35 - 45 , wherein the target is obtained from a cell or cell lysate. 
     
     
         47 . The method of any one of  claims 35 - 46 , wherein the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         48 . The method of  claim 47 , wherein the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         49 . The method of any one of  claims 35 - 48 , wherein the thermal stability of a docking strand transiently bound to a labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         50 . A method of detecting at least one target in a sample, the method comprising:
 contacting a sample with (a) at least two protein-nucleic acid conjugates, each comprising a protein linked to a docking strand, and (b) at least two labeled imager strands that are complementary to and transiently bind to respective docking strands of the at least two different protein-nucleic acid conjugates; and   determining whether the at least two protein-nucleic acid conjugates bind to at least one target in the sample.   
     
     
         51 . The method of  claim 50 , wherein the determining step comprises, in the following order,
 imaging transient binding of one of the at least two labeled imager strands to a docking strand of one of the at least two protein-nucleic acid conjugates to produce a first image of signal, and   imaging transient binding of another of the at least two labeled imager strands to a docking strand of another of the at least two protein-nucleic acid conjugates to produce at least one other image of signal.   
     
     
         52 . The method of  claim 51 , further comprising combining the first image and the at least one other image to produce a composite image of signal, wherein the signal of the composite image is representative of the at least one target. 
     
     
         53 . The method of any one of  claims 50 - 52 , wherein the protein of the protein-nucleic acid conjugate is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         54 . The method of  claims 53 , wherein the antibody is a monoclonal antibody. 
     
     
         55 . The method of any one of  claims 50 - 54  wherein the protein of the protein-nucleic acid conjugate is linked to the docking strand through an intermediate linker. 
     
     
         56 . The method of  claim 55 , wherein the intermediate linker comprises biotin and streptavidin. 
     
     
         57 . The method of any one of  claims 50 - 56 , wherein each of the at least two labeled imager strands are fluorescently-labeled imager strands. 
     
     
         58 . The method of  claim 57 , wherein the at least two fluorescently-labeled imager strands are spectrally distinct, fluorescently-labeled imager strands. 
     
     
         59 . The method of any  claim 57  or  58 , wherein each of the at least two labeled imager strands comprises at least one fluorophore. 
     
     
         60 . The method of any one of  claims 50 - 59 , wherein each of the at least two labeled imager strands is about 4 to about 30 nucleotides in length. 
     
     
         61 . The method of  claim 60 , wherein each of the at least two labeled imager strands is about 8 to about 10 nucleotides in length. 
     
     
         62 . The method of any one of  claims 50 - 61 , wherein the sample is a cell or cell lysate. 
     
     
         63 . The method of any one of  claims 50 - 62 , wherein the at least one target is a protein. 
     
     
         64 . The method of any one of  claims 50 - 63 , wherein the at least one target is obtained from a cell or cell lysate. 
     
     
         65 . The method of any one of  claims 50 - 64 , wherein step of determining whether the at least two protein-nucleic acid conjugates bind to at least one target in the sample comprises determining whether the at least two protein-nucleic acid conjugates bind to at least two targets in the sample. 
     
     
         66 . The method of any one of  claims 50 - 65 , wherein the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         67 . The method of  claim 66 , wherein the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         68 . The method of any one of  claims 50 - 67 , wherein the thermal stability of a docking strand transiently bound to a labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         69 . A method of detecting at least one protein target in a sample, comprising:
 (a) contacting a sample with at least two protein-nucleic acid conjugates, each comprising a protein linked to a docking strand and (b) sequentially contacting the sample with at least two labeled imager strands that are complementary to and transiently bind to respective docking strands of the at least two protein-nucleic acid conjugates; and   determining whether the at least two protein-nucleic acid conjugates bind to at least one protein target in the sample.   
     
     
         70 . The method of  claim 69 , comprising, in the following ordered steps:
 contacting the sample with a first protein-nucleic acid conjugate and at least one other protein-nucleic acid conjugate;   contacting the sample with a first labeled imager strand that is complementary to and transiently binds to the docking strand of the first protein-nucleic acid conjugate;   imaging the sample to obtain a first image, optionally using time-lapsed imaging;   removing the first labeled imager strand;   contacting the sample with at least one other labeled imager strand that is complementary to and transiently binds to the docking strand of the at least one other protein-nucleic acid conjugate; and   imaging the sample to obtain at least one other image, optionally using time-lapsed imaging.   
     
     
         71 . The method of  claim 69 , comprising, in the following ordered steps:
 contacting the sample with a first protein-nucleic acid conjugate;   contacting the sample with a first labeled imager strand that is complementary to and transiently binds to the docking strand of the first protein-nucleic acid conjugate;   imaging the sample to obtain a first image, optionally using time-lapsed imaging;   removing the first labeled imager strand;   contacting the sample with at least one other protein-nucleic acid conjugate;   contacting the sample with at least one other labeled imager strand that is complementary to and transiently binds to the docking strand of the at least one other protein-nucleic acid conjugate; and   imaging the sample to obtain at least one other image, optionally using time-lapsed imaging.   
     
     
         72 . The method of  claim 70  or  71 , further comprising determining whether the first protein-DNA conjugate binds to a first target and/or whether the at least one other protein-DNA conjugate binds to at least one other target. 
     
     
         73 . The method of  claim 72 , further comprising assigning a pseudo-color to the signal in the first image, and assigning at least one other pseudo-color to the signal in the at least one other image. 
     
     
         74 . The method of  claim 73 , further comprising combining the first image and the at least one other image to produce a composite image of the pseudo-colored signals, wherein the pseudo-colored signals of the composite image are representative of the at least two targets. 
     
     
         75 . The method of any one of  claims 69 - 74 , wherein the protein of the protein-nucleic acid conjugate(s) is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         76 . The method of  claim 75 , wherein the antibody is a monoclonal antibody. 
     
     
         77 . The method of any one of  claims 69 - 76 , wherein the protein of the protein-nucleic acid conjugate(s) is linked to the docking strand through an intermediate linker. 
     
     
         78 . The method of  claim 77 , wherein the intermediate linker comprises biotin and/or streptavidin. 
     
     
         79 . The method of any one of  claims 69 - 78 , wherein each of the labeled imager strands is a fluorescently-labeled imager strand. 
     
     
         80 . The method of  claim 69 , wherein each of the labeled imager strands is a spectrally distinct, fluorescently-labeled imager strand. 
     
     
         81 . The method of  claim 69  or  70 , wherein each of the labeled imager strands comprises at least one fluorophore. 
     
     
         82 . The method of any one of  claims 69 - 81 , wherein each of the labeled imager strands is about 4 to about 30 nucleotides in length. 
     
     
         83 . The method of  claim 82 , wherein each of the labeled imager strands is about 8 to about 10 nucleotides in length. 
     
     
         84 . The method of any one of  claims 69 - 83 , wherein the sample is a cell or cell lysate. 
     
     
         85 . The method of any one of  claims 69 - 84 , wherein the target(s) is a protein. 
     
     
         86 . The method of any one of  claims 69 - 85 , wherein the target(s) is obtained from a cell or cell lysate. 
     
     
         87 . The method of any one of  claims 69 - 86 , wherein the step of determining whether the at least two protein-nucleic acid conjugates bind to at least one protein target in the sample comprises determining whether the at least two protein-nucleic acid conjugates bind to at least two protein targets in the sample. 
     
     
         88 . The method of any one of  claims 69 - 87 , wherein each of the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         89 . The method of any one of  claims 69 - 88 , wherein each of the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         90 . The method of any one of  claims 69 - 89 , wherein the thermal stability of a docking strand transiently bound to a labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         91 . A method of detecting a molecule, comprising
 contacting a sample containing at least one target with (a) at least one BP-NA conjugate, each BP-NA conjugate comprising a binding partner linked to a docking strand and (b) at least one labeled imager strand that is complementary to and transiently binds the docking strand of the at least one BP-NA conjugate; and   determining whether the at least one BP-NA conjugate binds to at least one target in the sample.   
     
     
         92 . The method of  claim 91 , wherein the determining step comprises imaging transient binding the at least one labeled imager strand to the docking strand of the at least one BP-NA conjugate. 
     
     
         93 . The method of  claim 91  or  92 , wherein the sample is a cell or cell lysate. 
     
     
         94 . The method of any one of  claims 91 - 93 , wherein the at least one target is obtained from a cell or cell lysate. 
     
     
         95 . The method of any one of  claims 91 - 94 , wherein the target is a naturally-occurring biomolecule. 
     
     
         96 . The method of  claim 95 , wherein the naturally-occurring biomolecule is a protein. 
     
     
         97 . The method of  claim 96 , wherein the protein is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         98 . The method of  claim 97 , wherein the antibody is a monoclonal antibody. 
     
     
         99 . The method of any one of  claims 91 - 98 , wherein the target is linked to the docking strand through an intermediate linker. 
     
     
         100 . The method of  claim 99 , wherein the intermediate linker comprises biotin and/or streptavidin. 
     
     
         101 . The method of  claim 95 , wherein the naturally-occurring biomolecule is a nucleic acid. 
     
     
         102 . The method of  claim 101 , wherein the nucleic acid is a nucleic acid aptamer. 
     
     
         103 . The method of any one of  claims 91 - 103 , wherein the labeled imager strand is a fluorescently-labeled imager strand. 
     
     
         104 . The method of  claim 103 , wherein the fluorescently-labeled imager strand comprises at least one fluorophore. 
     
     
         105 . The method of any one of  claims 91 - 104 , wherein the fluorescently-labeled imager strand is about 4 to about 30 nucleotides in length. 
     
     
         106 . The method of  claim 105 , wherein the fluorescently-labeled imager strand is about 8 to about 10 nucleotides in length. 
     
     
         107 . The method of any one of  claims 91 - 106 , wherein the binding partner is a protein or nucleic acid. 
     
     
         108 . The method of any one of  claims 91 - 107 , wherein the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         109 . The method of  claim 108 , wherein the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         110 . The method of any one of  claims 91 - 109 , wherein the thermal stability of a docking strand transiently bound to a labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         111 . A method of detecting a naturally-occurring biomolecule, comprising contacting a sample containing at least one target with (a) at least two different BP-NA conjugates, each BP-NA conjugate comprising a binding partner linked to a docking strand and (b) at least two labeled imager strands that are complementary to and transiently bind to respective docking strands of the at least two BP-NA conjugates; and
 determining whether the at least two BP-NA conjugates bind to at least one target in the sample.   
     
     
         112 . The method of  claim 111 , comprising, in the following ordered steps:
 contacting the sample with a first BP-NA conjugate and at least one other BP-NA conjugate; and   contacting the sample with a first labeled imager strand that is complementary to and transiently binds to the docking strand of the first BP-NA conjugate;   imaging the sample to obtain a first image, optionally using time-lapsed imaging;   removing the first fluorescently-labeled imager strand;   contacting the sample with at least one other labeled imager strand that is complementary to and transiently binds to the docking strand of the at least one other BP-NA conjugate; and   imaging the sample to obtain at least one other image, optionally using time-lapsed imaging.   
     
     
         113 . The method of  claim 111 , comprising, in the following ordered steps:
 contacting the sample with a first BP-NA conjugate;   contacting the sample with a first labeled imager strand that is complementary to and transiently binds to the docking strand of the first BP-NA conjugate;   imaging the sample to obtain a first image, optionally using time-lapsed imaging;   removing the first labeled imager strand;   contacting the sample with at least one other BP-NA conjugate;   contacting the sample with at least one other labeled imager strand that is complementary to and transiently binds to the docking strand of the at least one other BP-NA conjugate; and   imaging the sample to obtain a at least one other image, optionally using time-lapsed imaging.   
     
     
         114 . The method of  claim 112  or  113 , further comprising determining whether the first protein DNA conjugate binds to a first target and/or whether the at least one other protein-DNA conjugate binds to at least one other target. 
     
     
         115 . The method of  claim 114 , further comprising assigning a pseudo-color to the signal in the first image, and assigning at least one other pseudo-color to the signal in the at least one other image. 
     
     
         116 . The method of  claim 115 , further comprising combining the first image and the at least one other image to produce a composite image of the pseudo-colored signals, wherein the pseudo-colored signals of the composite image are representative of the at least one target. 
     
     
         117 . The method of any one of  claims 111 - 116 , wherein the sample is a cell or cell lysate. 
     
     
         118 . The method of any one of  claims 111 - 117 , wherein the at least one target is obtained from a cell or cell lysate. 
     
     
         119 . The method of any one of  claims 111 - 118 , wherein the target is a naturally-occurring biomolecule. 
     
     
         120 . The method of  claim 119 , wherein the naturally-occurring biomolecule is a protein. 
     
     
         121 . The method of  claim 120 , wherein the protein is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         122 . The method of  claim 121 , wherein the antibody is a monoclonal antibody. 
     
     
         123 . The method of any one of  claims 111 - 122 , wherein the target is linked to the docking strand through an intermediate linker. 
     
     
         124 . The method of  claim 123 , wherein the intermediate linker comprises biotin and/or streptavidin. 
     
     
         125 . The method of  claim 119 , wherein the naturally-occurring biomolecule is a nucleic acid. 
     
     
         126 . The method of  claim 125 , wherein the nucleic acid is a nucleic acid aptamer. 
     
     
         127 . The method of any one of  claims 111 - 126 , wherein the first labeled imager strand is a fluorescently-labeled imager strand. 
     
     
         128 . The method of any one of  claims 111 - 126 , wherein the at least one other labeled imager strand is a fluorescently-labeled imager strand. 
     
     
         129 . The method of  claim 127  or  128 , wherein the first labeled imager strand and/or the at least one other labeled imager strand comprises at least one fluorophore. 
     
     
         130 . The method of any one of  claims 111 - 129 , wherein at least one other labeled imager strand about 4 to about 30 nucleotides in length. 
     
     
         131 . The method of  claim 130 , at least one other labeled imager strand is about 8 to about 10 nucleotides in length. 
     
     
         132 . The method of any one of  claims 111 - 131 , wherein the binding partner is a protein or nucleic acid. 
     
     
         133 . The method of any one of  claims 111 - 132 , wherein the docking strand is a DNA docking strand. 
     
     
         134 . The method of any one of  claims 111 - 133 , wherein the docking strand comprises at least two domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         135 . The method of  claim 134 , wherein the docking strand comprises at least three domains, wherein each domain binds to a respectively complementary labeled imager strand. 
     
     
         136 . The method of any one of  claims 111 - 135 , wherein the thermal stability of a docking strand transiently bound to a labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands. 
     
     
         137 . A method of determining the number of targets in a test sample, comprising:
 obtaining a sample that comprises targets transiently bound directly or indirectly to labeled imager strands;   obtaining a time-lapsed image of the sample;   performing spot detection and localization on the image to obtain a high-resolution image of the sample;   calibrating k on ·c imager , wherein k on  is a second order association constant, and c imager  is concentration of labeled imager strands in the test sample;   determining variable τ d ; and   determining the number of test targets in the sample based on the equation, number of test targets=(k on ·C imager ·τ d ) −1 .   
     
     
         138 . The method of  claim 137 , wherein the test targets are protein targets. 
     
     
         139 . The method of  claim 138 , wherein the protein targets are bound to protein-nucleic acid conjugates that comprise a protein linked to a docking strand, and the labeled imager strands are complementary to and transiently bind to respective docking strands of the protein-nucleic acid conjugates. 
     
     
         140 . The method of  claim 139 , wherein the protein of the protein-nucleic acid conjugate is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         141 . The method of  claim 137 , wherein the test targets are single-stranded nucleic acids. 
     
     
         142 . The method of  claim 141 , wherein the single-stranded nucleic acids are DNA or RNA. 
     
     
         143 . The method of any one of  claims 137 - 142 , wherein each of the labeled imager strands is a fluorescently-labeled imager strand. 
     
     
         144 . The method of any one of  claims 137 - 143 , wherein each of the fluorescently-labeled imager strands comprises at least one fluorophore. 
     
     
         145 . The method of any one of  claims 137 - 144 , wherein each of the fluorescently-labeled imager strands is about 3 to about 30 nucleotides in length. 
     
     
         146 . The method of  claim 144 , wherein each of the fluorescently-labeled imager strands is about 8 to about 10 nucleotides in length. 
     
     
         147 . The method of any one of  claims 137 - 146 , wherein the time-lapsed image is obtained over a period of about 24 minutes. 
     
     
         148 . The method of any one of  claims 137 - 147 , wherein the time-lapsed image is a time-lapsed diffraction-limited fluorescence image. 
     
     
         149 . The method of any one of  claims 137 - 148 , wherein the step of performing localization on the image comprises performing Gaussian fitting on the image. 
     
     
         150 . The method of any one of  claims 137 - 149 , wherein the step of calibrating k on ·c imager  comprising calibrating k on ·c imager  with a control sample with a known number of targets. 
     
     
         151 . The method of any one of  claims 137 - 150 , wherein the step of determining variable τ d  comprises determining variable τ d  by fitting the signal OFF-time distribution to a cumulative distribution function. 
     
     
         152 . The method of any one of  claims 137 - 151 , wherein the number of test targets is determined with an accuracy of greater than 90%. 
     
     
         153 . A method of determining a relative amount of targets in a test sample, comprising:
 obtaining a sample that comprises targets transiently bound directly or indirectly to labeled imager strands;   obtaining a time-lapsed image of the sample;   performing spot detection and localization on the image to obtain a high-resolution image of the sample;   determining variable τ d ; and   determining the relative amount of two or more test targets in the sample based on τ d .   
     
     
         154 . The method of  claim 153 , wherein the test targets are protein targets. 
     
     
         155 . The method of  claim 154 , wherein the protein targets are bound to protein-nucleic acid conjugates that comprise a protein linked to a docking strand, and the labeled imager strands are complementary to and transiently bind to respective docking strands of the protein-nucleic acid conjugates. 
     
     
         156 . The method of  claim 155 , wherein the protein of the protein-nucleic acid conjugate is an antibody, an antigen-binding antibody fragment, or a peptide aptamer. 
     
     
         157 . The method of  claim 153 , wherein the test targets are single-stranded nucleic acids. 
     
     
         158 . The method of  claim 157 , wherein the single-stranded nucleic acids are DNA or RNA. 
     
     
         159 . The method of any one of  claims 153 - 158 , wherein each of the labeled imager strands is a fluorescently-labeled imager strand. 
     
     
         160 . The method of  claim 159 , wherein wherein each of the labeled imager strands comprises at least one fluorophore. 
     
     
         161 . The method of any one of  claims 153 - 160 , wherein each of the labeled imager strands is about 4 to about 30 nucleotides in length. 
     
     
         162 . The method of  claim 161 , wherein each of the labeled imager strands is about 8 to about 10 nucleotides in length. 
     
     
         163 . The method of any one of  claims 153 - 162 , wherein the time-lapsed image is obtained over a period of about 25 minutes. 
     
     
         164 . The method of any one of  claims 153 - 163 , wherein the time-lapsed image is a time-lapsed diffraction-limited fluorescence image. 
     
     
         165 . The method of any one of  claims 153 - 164 , wherein the step of performing localization on the image comprises performing Gaussian fitting on the image. 
     
     
         166 . The method of any one of  claims 153 - 165 , wherein the step of determining variable τ d  comprises determining variable τ d  by fitting the signal OFF-time distribution to a cumulative distribution function. 
     
     
         167 . The method of any one of  claims 153 - 166 , wherein the number of test targets is determined with an accuracy of greater than 90%. 
     
     
         168 . A single-stranded DNA probe comprising a target binding domain of about 20 nucleotides in length linked to a docking domain comprising at least one subdomain complementary to at least one labeled imager strand of about 4 to 30 nucleotides in length, and wherein the target binding domain is bound to a complementary domain of a single-stranded mRNA target strand. 
     
     
         169 . The single-stranded DNA probe of  claim 168 , wherein the at least one subdomain is transiently bound to the at least one labeled imager strand. 
     
     
         170 . The single-stranded DNA probe of  claim 169 , wherein the at least one labeled imager strand is fluorescently labeled. 
     
     
         171 . The single-stranded DNA probe of  claim 170 , wherein the at least one labeled imager strand comprises a fluorophore. 
     
     
         172 . The single-stranded DNA probe of  claim 168 , wherein the docking domain comprises at least two subdomains, wherein the at least two subdomains are respectively complementary to at least two labeled imager strands of about 4 to 30 nucleotides in length. 
     
     
         173 . The single-stranded DNA probe of  claim 172 , wherein the at least two subdomains are respectively complementary to at least two labeled imager strands of about 8 to 10 nucleotides in length. 
     
     
         174 . The single-stranded DNA probe of  claim 172  or  173 , wherein the at least two subdomains are transiently bound to respectively complementary labeled imager strands. 
     
     
         175 . The single-stranded DNA probe of any one of  claims 172 - 174 , wherein the respectively complementary labeled imager strands are distinctly labeled imager strands. 
     
     
         176 . The single-stranded DNA probe of any one of  claims 172 - 175 , wherein the respectively complementary labeled imager strands are respectively complementary fluorescently-labeled imager strands. 
     
     
         177 . The single-stranded DNA probe of  claim 176 , the respectively complementary labeled imager strands each comprise a fluorophore. 
     
     
         178 . The single-stranded DNA probe of  claim 168 , wherein the docking domain comprises at least three subdomains, wherein the at least three subdomains are respectively complementary to at least three labeled imager strands of about 4 to 30 nucleotides in length. 
     
     
         179 . The single-stranded DNA probe of  claim 178 , wherein the at least three subdomains are transiently bound to respectively complementary labeled imager strands. 
     
     
         180 . The single-stranded DNA probe of  claim 179 , wherein the respectively complementary labeled imager strands are distinctly labeled imager strands. 
     
     
         181 . The single-stranded DNA probe of  claim 179  or  180 , wherein the respectively complementary labeled imager strands are respectively complementary fluorescently-labeled imager strands. 
     
     
         182 . The single-stranded DNA probe of  claim 180  or  181 , wherein the respectively complementary labeled imager strands each comprise a fluorophore. 
     
     
         183 . The single-stranded DNA probe of any one of  claims 168 - 182 , wherein the target binding domain of about 20 nucleotides in length is linked at its 3′ end to a docking domain. 
     
     
         184 . The The single-stranded DNA probe of any one of  claims 168 - 182 , wherein the target binding domain of about 20 nucleotides in length is linked at its 5′ end to a docking domain. 
     
     
         185 . A method of performing drift correction for a plurality of images, wherein each of the plurality of images comprises a frame of a time sequence of images, wherein the time sequence of images captures a plurality of transient events, the method comprising:
 determining a time trace for each of a plurality of drift markers identified in the plurality of images, wherein a time trace for each drift marker corresponds to movement of an object in the image over the time sequence of images;   determining, with at least one computer processor, a first drift correction from at least one of the plurality of drift markers based, at least in part, on the time traces for the at least one of the plurality of drift markers;   determining a time trace for each of a plurality of geometrically-addressable marker sites from a plurality of drift templates identified from the plurality of images, wherein each drift template in the plurality of drift templates describes a geometrical relationship between the plurality of geometrically-addressable marker sites of transient events in the drift template;   determining a second drift correction based, at least in part, on the time traces for the plurality of geometrically-addressable marker sites from the plurality of drift templates;   correcting the plurality of images based, at least in part, on the first drift correction and the second drift correction; and   outputting a final image based on the corrected plurality of images.   
     
     
         186 . The method of  claim 185 , further comprising:
 identifying a plurality of localizations in each of the plurality of images;   creating a two-dimensional histogram of the plurality of localizations; and   identifying locations of the plurality of drift markers based, at least in part, on the two-dimensional histogram;   wherein determining the time traces for each of the plurality of drift markers comprises determining the time traces based, at least in part, on the locations of the plurality of drift markers.   
     
     
         187 . The method of  claim 186 , wherein identifying a plurality of localizations comprises:
 identifying a plurality of spots on each of the plurality of images; and   determining a fitted center position of each of the plurality of spots using a local Gaussian fitting algorithm;   wherein each of the plurality of localizations comprises the spot identified on an image and its associated fitted center position.   
     
     
         188 . The method of  claim 187 , wherein each of the plurality of localizations further comprises a detected photon count corresponding to the localization. 
     
     
         189 . The method of  claim 186 , wherein creating the two-dimensional histogram of the plurality of localizations comprises binning all localizations into a two-dimensional grid and using a total number of localizations in each bin as a histogram count. 
     
     
         190 . The method of  claim 186 , wherein creating the two-dimensional histogram of the plurality of localizations comprises binning all localizations into a two-dimensional grid and using a total number of photon count of the plurality of localizations in each bin as a histogram count. 
     
     
         191 . The method of  claim 186 , wherein identifying locations of the plurality of drift markers based, at least in part, on the two-dimensional histogram comprises at least one of the following:
 binarizing the two-dimensional histogram using one or more selection criteria, wherein the one or more selection criteria include a lower-bound threshold of a histogram value or a upper-bound threshold of a histogram value;   partitioning the binarized image into partitions and filtering the partitions based on one or more selection criteria, wherein the one or more selection criteria include one or more of a lower-bound threshold of an area of a partition area, an upper-bound threshold of the area, a lower-bound or an upper-bound of a longest or shortest linear dimension of a partition longest, and a lower-bound or an upper-bound of an eccentricity of a partition; and   expanding and shrinking the binarized image using one or more binary image operations, wherein the one or more binary image operations include one or more of the following: dilate, erode, bridge, close, open, fill, clean, top-hat, bottom-hat, thicken, thin, and more.   
     
     
         192 . The method of  claim 185 , wherein determining a first drift correction based, at least in part, on the time traces for the plurality of drift markers comprises:
 determining a relative time trace for each of the plurality of drift markers, wherein the relative time trace is determined by comparing the time trace for the drift marker with the average position of the same trace; and   determining a combined time trace based on the relative time traces for each of the plurality of drift markers;   wherein determining the first drift correction based, at least in part, on the time traces for the plurality of drift markers comprises determining the first drift correction based, at least in part, on the relative time traces for each of the plurality of drift markers.   
     
     
         193 . The method of  claim 192 , wherein determining the first drift correction based, at least in part, on the relative time traces for each of the plurality of drift markers comprises performing a weighted average of the relative time traces for each of the plurality of drift markers. 
     
     
         194 . The method of  claim 193 , wherein performing the weighted average comprises:
 determining a quality score for each of the relative time traces, wherein the quality score is determined based, at least in part, on a measure of variability over time associated with the time trace and/or a measure of localization uncertainty of individual localizations within the time trace.   
     
     
         195 . The method of  claim 194 , wherein the measure of variability over time comprises a standard deviation of the time trace over time. 
     
     
         196 . The method of  claim 194 , wherein the measure of localization uncertainty of individual localizations comprises, at least in part, an estimate of uncertainty from a Gaussian fitting or a comparison with other simultaneous localizations, wherein the other simultaneous localizations are from within a same image and from other time traces from the plurality of drift markers, wherein the comparison comprises a mean and standard deviation of all simultaneous localizations. 
     
     
         197 . The method of  claim 185 , further comprising:
 determining that a first drift marker of the plurality of drift markers is not present in at least one frame of the time sequence of images; and   linearly interpolating the time trace for the first drift marker for the at least one frame to produce a smoothed time trace for the first drift marker.   
     
     
         198 . The method of  claim 185 , wherein determining a time trace for each of a plurality of geometrically-addressable marker sites from a plurality of drift templates identified from the plurality of images comprises:
 identifying a plurality of localizations in each of the plurality of images;   creating a two-dimensional histogram of the plurality of localizations; and   identifying the plurality of drift templates based, at least in part, on the two-dimensional histogram;   wherein identifying the plurality of drift templates comprises evaluating the two-dimensional histogram using an lower-bound and/or an upper-bound threshold in a histogram count.   
     
     
         199 . The method of  claim 185 , wherein determining a time trace for each of a plurality of geometrically-addressable marker sites from a plurality of drift templates identified from the plurality of images comprises determining a time trace for each of a plurality of geometrically-addressable marker sites within each of the plurality of drift templates, and wherein determining the second drift correction comprises determining the second drift correction based, at least in part, on the time traces for each of the plurality of marker sites within each of the plurality of drift templates. 
     
     
         200 . The method of  claim 185 , wherein determining the second drift correction based, at least in part, on the time traces for each of the plurality of geometrically-addressable marker sites from each of the plurality of drift templates comprises:
 identifying a plurality of geometrically-addressable marker sites within each of the plurality of drift templates; and   determining a relative time trace for each of a plurality of geometrically-addressable drift markers for each of the plurality of drift templates;   wherein determining the second drift correction based, at least in part, on the time traces for the plurality of drift templates comprises determining the second drift correction based, at least in part, on the relative time traces for each of the plurality of drift markers within each of the plurality of drift templates.   
     
     
         201 . The method of  claim 200 , wherein identifying a plurality of geometrically addressable marker sites from each of the plurality of drift templates comprises determining a plurality of marker sites based on, at least in part, a two-dimensional histogram of the plurality of localizations in the corresponding drift template, and/or one or more selection criteria, wherein the one or more selection criteria include one or more of a total number of localizations, a surface density of localizations, and standard deviation of localizations. 
     
     
         202 . The method of  claim 200 , wherein determining the second drift correction based, at least in part, on the relative time traces for each of the plurality of drift markers within each of the plurality of drift templates comprises performing a weighted average of the relative time traces for each of the plurality of drift markers within each of the drift templates. 
     
     
         203 . The method of  claim 202 , wherein performing the weighted average comprises:
 determining a quality score for each of the relative time traces, wherein the quality score is determined based, at least in part, on a measure of variability over time associated with the time trace and/or a localization uncertainty within the time trace.   
     
     
         204 . The method of  claim 203 , wherein the measure of variability over time comprises a standard deviation of the time trace over time. 
     
     
         205 . The method of  claim 203 , wherein the measure of localization uncertainty of individual localizations comprises an estimate of uncertainty from a Gaussian fitting or a comparison with other simultaneous localizations, wherein the other simultaneous localizations are from within a same image and from the other time traces from the plurality of marker sites from the plurality of drift templates, wherein the comparison comprises a mean and standard deviation of all simultaneous localizations. 
     
     
         206 . The method of  claim 185 , wherein correcting the plurality of images based, at least in part, on the first drift correction and the second drift correction comprises correcting the plurality images using the first drift correction to produce a first corrected plurality of images, and wherein determining a time trace for each of a plurality of drift templates identified from the plurality of images comprises determining a time trace for each of the plurality of drift templates identified from the first corrected plurality of images. 
     
     
         207 . The method of  claim 185 , further comprising:
 smoothing the first drift correction prior to correcting the plurality of images using the first drift correction.   
     
     
         208 . The method of  claim 207 , wherein smoothing the first drift correction comprises processing the first drift correction using local regression with a window determined by a characteristic drift time scale of the first drift correction. 
     
     
         209 . The method of  claim 185 , further comprising smoothing the second drift correction prior to correcting the plurality of images using the second drift correction. 
     
     
         210 . The method of  claim 209 , wherein smoothing the second drift correction comprises processing the second drift correction using local regression with a window determined by a characteristic drift time scale of the second drift correction. 
     
     
         211 . The method of  claim 185 , further comprising:
 selecting a single drift marker of the plurality of drift markers; and   determining a third drift correction based, at least in part, on the selected single drift marker;   wherein correcting the plurality of images comprises correcting the plurality of images based, at least in part, on the third drift correction.   
     
     
         212 . The method of  claim 211 , wherein correcting the plurality of images based, at least in part, on the third drift correction is performed prior to correcting the plurality of images based, at least in part on the first drift correction and the second drift correction. 
     
     
         213 . The method of any of  claims 185  and  211 , further comprising:
 identifying locations of a first plurality of points in a first image of the plurality of frames; 
 identifying locations of a second plurality of points in a second image of the plurality of images, wherein the second image corresponds to a neighboring frame of the first image in the time sequence of images; and 
 determining a fourth drift correction based, at least in part, on differences between the locations of the first plurality of points and the second plurality of points; 
 wherein correcting the plurality of images comprises correcting the plurality of images based, at least in part, on the fourth drift correction. 
 
     
     
         214 . The method of  claim 213 , wherein the second image corresponds to a frame immediately following the frame corresponding to the first image in the time sequence of images. 
     
     
         215 . The method of  claim 213 , wherein determining the fourth drift correction based, at least in part, on differences between the locations of the first plurality of points and the second plurality of points comprises:
 creating a histogram of distances between the locations of the first plurality of points and the second plurality of points;   determining based, at least in part, on the histogram, pairs of points between the first image and the second image that correspond to the same transient event; and   determining a location offset between each of the determined pairs of points;   wherein determining the fourth drift correction is based on a vector average of the location offsets for each of the determined pairs of points.   
     
     
         216 . The method of  claim 185 , wherein the plurality of images correspond to DNA-based images and wherein the plurality of transient events are binding events between an imaging strand and a DNA docking strand. 
     
     
         217 . The method of  claim 216 , wherein the imaging strand is a fluorescent imaging probe configured to fluoresce when associated with the DNA docking strand. 
     
     
         218 . The method of  claim 185 , wherein at least one of the drift markers is a DNA based nanostructure. 
     
     
         219 . The method of  claim 218 , wherein the DNA based nanostructure is a DNA origami nanostructure with docking strands. 
     
     
         220 . The method of  claim 185 , wherein at least one of the drift templates is a DNA based nanostructure. 
     
     
         221 . The method of  claim 220 , wherein the DNA based nanostructure is a DNA origami nanostructure with docking strands. 
     
     
         222 . The method of  claim 185 , wherein at least one of the drift templates is a three-dimensional drift template. 
     
     
         223 . The method of  claim 222 , wherein the three-dimensional drift template is a tetrahedron. 
     
     
         224 . The method of  claim 185 , wherein at least one of the drift templates includes multiple colors corresponding to different types of transient events. 
     
     
         225 . The method of  claim 224 , wherein the different types of transient events include a first binding event of a first imaging strand with a first type of DNA docking strand and a second binding event of a second imaging strand with a second type of DNA docking strand. 
     
     
         226 . The method of  claim 185 , wherein outputting the final image comprises displaying the final image on a display. 
     
     
         227 . The method of  claim 185 , wherein outputting the final image comprises sending the final image to a computer via at least one network. 
     
     
         228 . The method of  claim 185 , wherein outputting the final image comprises storing the final image on at least one storage device. 
     
     
         229 . A non-transitory computer readable medium encoded with a plurality of instructions that, when executed by at least one computer processor, performs a method of performing drift correction for a plurality of images, wherein each of the plurality of images comprises a frame of a time sequence of images, wherein the time sequence of images captures a plurality of transient events, the method comprising:
 determining a time trace for each of a plurality of drift markers identified in the plurality of images, wherein a time trace for each drift marker corresponds to movement of an object in the image over the time sequence of images;   determining a first drift correction from at least one of the plurality of drift markers based, at least in part, on the time traces for the at least one of the plurality of drift markers;   determining a time trace for each of a plurality of geometrically-addressable marker sites from a plurality of drift templates identified from the plurality of images, wherein each drift template in the plurality of drift templates describes a geometrical relationship between the plurality of geometrically-addressable marker sites of transient events in the drift template;   determining a second drift correction based, at least in part, on the time traces for the plurality of geometrically-addressable marker sites from the plurality of drift templates;   correcting the plurality of images based, at least in part, on the first drift correction and the second drift correction; and   outputting a final image based on the corrected plurality of images.   
     
     
         230 . A computer, comprising:
 an input interface configured to receive a plurality of images, wherein each of the plurality of images comprises a frame of a time sequence of images, wherein the time sequence of images captures a plurality of transient events;   at least one processor programmed to:
 determine a time trace for each of a plurality of drift markers identified in the plurality of images, wherein a time trace for each drift marker corresponds to movement of an object in the image over the time sequence of images; 
 determine a first drift correction from at least one of the plurality of drift markers based, at least in part, on the time traces for the at least one of the plurality of drift markers; 
 determine a time trace for each of a plurality of geometrically-addressable marker sites from a plurality of drift templates identified from the plurality of images, wherein each drift template in the plurality of drift templates describes a geometrical relationship between the plurality of geometrically-addressable marker sites of transient events in the drift template; 
 determine a second drift correction based, at least in part, on the time traces for the plurality of geometrically-addressable marker sites from the plurality of drift templates; 
 correct the plurality of images based, at least in part, on the first drift correction and the second drift correction; and 
 determine a final image based on the corrected plurality of images; and an output interface configured to output the final image.