# US 2021/0172003 A1 · Molecular imaging and related methods > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0172003 A1 - Document ID: US20210172003A1 - Status: Published application - Kind: A1 - Application number: 17/086,016 - Filed: 2020-10-30 - Published: 2021-06-10 - Earliest claimed: 2013-03-06 - Assignees: Optical Biosystems, Inc. - Applicants: Optical Biosystems, Inc. - Inventors: Marc BEAL; Ronald M. COOK; Peter Jay COASSIN; Arturo ORJALO; Jekwan RYU - Web page: https://patentgrep.com/patent/US20210172003A1 ## CPC - C12Q 1/6841: In situ hybridisation - C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53) - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging} - G02B 21/0004: {specially adapted for specific applications} - G02B 21/0096: {with photometer devices (photometers per se G01J)} - G02B 21/082: {for incident illumination only} - G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} - G02B 21/18: Arrangements with more than one light path, e.g. for comparing two specimens - C12Q 2600/158: Expression markers - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS ## Abstract A method of imaging single molecules includes exposing a test sample to a probe. The probe includes a first portion that specifically binds to a target molecule and a second portion that is detectable as the result of one or more chemical groups that interact with light at one or more wavelengths. The probe binds to a target molecule to provide a complex. The method also includes exposing the complex to one or more wavelengths of light that interact with the one or more chemical groups; and detecting a result from the interaction of the light and the one or more chemical groups to provide an image of the one or more single molecules. The image possesses a resolution better than 450 nm over a view field area of at least 1×105 μm2, and the image is obtained in a single detection step without variation of any detection settings. ## Claims ### Claim 1 A method of imaging single molecules, the method comprising: obtaining a sample that includes a plurality of complexes, a respective complex of the plurality of complexes including a target molecule bound to a probe, the probe including one or more fluorescent groups that interact with light at one or more wavelengths; exposing an area of the sample to one or more wavelengths of light that interact with the one or more fluorescent groups, wherein an interference pattern is generated on the area of the sample in a region of overlap between two or more coherent laser beams; and detecting a result from the interacting of the one or more wavelengths of light that interact with the one or more fluorescent groups using 20× objective magnification to provide an image of one or more single molecules without variation of any detection settings so that the image possesses a resolution better than 450 nm over the imaged area of at least 1×105 μm2. ### Claim 2 (depends on 1) The method of claim 1, wherein: the interference pattern is generated on the area of the sample in the region of overlap of at least four paired coherent laser beams. ### Claim 3 (depends on 1) The method of claim 1, wherein: the two or more coherent laser beams are focused toward the area of the sample using the 20× objective magnification. ### Claim 4 (depends on 1) The method of claim 1, further comprising: quantifying the single molecules from the image having the imaged area of at least 1×105 μm2. ### Claim 5 (depends on 1) The method of claim 1, wherein: the targeted molecule is selected from a group consisting of mRNAs, lnc RNAs, snRNAs, a chromosome, a DNA strand comprising BrdU, a DNA strand comprising EdU, a protein, and a small molecule. ### Claim 6 (depends on 1) The method of claim 1, wherein: the one or more fluorescent groups include a fluorescent compound selected from a group consisting of fluorescent organic dyes, quantum dots, intercalator fluorescent dyes and expressible fluorescent proteins. ### Claim 7 (depends on 1) The method of claim 1, wherein: the density of the one or more fluorescent groups within the field of view is less than 1000 molecules per m2. ### Claim 8 (depends on 1) The method of claim 1, wherein: the imaged area is at least 1×106 μm2. ### Claim 9 (depends on 1) The method of claim 1, wherein: the sample contains a plurality of cells including a plurality of target molecules. ### Claim 10 (depends on 9) The method of claim 9, wherein: exposing live cells in the sample to a plurality of probes to form the plurality of complexes. ### Claim 11 (depends on 10) The method of claim 10, wherein: a respective probe of the plurality of probes includes a first portion that specifically binds to a target molecule and a second portion that is modifiable to include the one or more fluorescent groups that interact with light at one or more wavelengths. ### Claim 12 (depends on 11) The method of claim 11, further comprising: after the respective probe specifically binds to a target molecule, modifying the second portion of the respective probe to include the one or more fluorescent groups that interact with light at one or more wavelengths. ### Claim 13 (depends on 11) The method of claim 11, wherein: the second portion of the probe is modified using a type of chemical reaction selected from a group of chemical reactions consisting of: Click chemistry; a Diels-Alder reaction; Staudinger ligation; hydrazine ligation; oxime ligation; native chemical ligation; tetrazine ligation; maleimide-thiol ligation; active ester-amine ligation; carbodiimide phosphate conjugation; and, carboxy conjugation. ### Claim 14 (depends on 9) The method of claim 9, wherein: the plurality of target molecules includes an mRNA molecule; and probes in the sample include a plurality of oligonucleotides that are capable of hybridizing to the mRNA molecules, each oligonucleotide including a single fluorescent label, providing a set of singly-labeled oligonucleotides to afford a set of oligonucleotide-mRNA hybridized products. ### Claim 15 (depends on 9) The method of claim 9, wherein: the plurality of target molecules includes an lnc RNA molecule; and probes in the sample include a plurality of oligonucleotides that are capable of hybridizing to the lnc RNA molecules, each oligonucleotide including a single fluorescent label, providing a set of singly-labeled oligonucleotides to afford a set of oligonucleotide-lnc RNA hybridized products. ### Claim 16 (depends on 9) The method of claim 9, wherein: the plurality of target molecules includes an snRNA molecule; and probes in the sample include a plurality of oligonucleotides that are capable of hybridizing to the snRNA molecules, each oligonucleotide including a single fluorescent label, providing a set of singly-labeled oligonucleotides to afford a set of oligonucleotide-snRNA hybridized products. ### Claim 17 (depends on 9) The method of claim 9, wherein: the plurality of target molecules includes a chromosome or a portion of a chromosome; and probes in the sample include a plurality of oligonucleotides that are capable of hybridizing to the chromosome or the portion of the chromosome, each oligonucleotide including a single fluorescent label, providing a set of singly-labeled oligonucleotides to afford a set of oligonucleotide-chromosome hybridized products. ### Claim 18 (depends on 9) The method of claim 9, wherein: the target molecule is an mRNA selected from a group consisting of: CCNB1 mRNA, CENPE mRNA, AURKB mRNA, PLK1 mRNA, PLK4 mRNA, TAGLN mRNA, ACTG2 mRNA, TPM1 mRNA, MYH111 mRNA, DES mRNA, EF1AX mRNA, AR mRNA, HSPD1 mRNA, HSPCA mRNA, K-ALPHA1 mRNA, MLL5 mRNA, UGT2B15 mRNA, WNT5B5 mRNA, ANXA11 mRNA, FOS mRNA, SFRP1 mRNA, FN1 mRNA, ITGB8 mRNA, THBS2 mRNA, HNT mRNA, CDH10 mRNA, BMP4 mRNA, ANKH mRNA, SEP4 mRNA, SEP7 mRNA, PTN mRNA, VEGF mRNA, SRY mRNA, EGR3 mRNA, FoxP1 mRNA, FoxM1 mRNA, TGCT1 mRNA, ITPKB mRNA, RGS4 mRNA, and BACE1 mRNA. ### Claim 19 (depends on 9) The method of claim 9, wherein: the target molecule is BrdU incorporated into a replicating DNA strand of a cell; the probes in the sample include an anti-BrdU antibody comprising one or more fluorescent groups; and the method includes: providing an amount of BrdU to the plurality of live cells; incubating the provided BrdU with the plurality of live cells for a time period that allows for a significant amount of the BrdU to be incorporated into proliferating cells; providing an amount of the anti-BrdU antibody to the plurality of cells incorporating the BrdU; and incubating the provided antibody with the plurality of live cells incorporating the BrdU for a time period that allows for binding of a significant amount of the anti-BrdU antibody to the BrdU incorporated into the plurality of cells. ### Claim 20 (depends on 9) The method of claim 9, wherein: the target molecule is EdU incorporated into a replicating DNA strand of a cell; the probes in the sample include an amount of a fluorescently labeled, azide-based Click reagent; and the method includes: providing an amount of EdU to the plurality of live cells; incubating the provided EdU with the plurality of live cells for a time period that allows for a significant amount of the EdU to be incorporated into proliferating cells; and providing an amount of the fluorescently labeled, azide-based Click reagent under conditions that allow reaction between the incorporated EdU and the Click reagent.