# US 11,994,451 B2 · Optical clearing and auto-fluorescence quenching solutions and method of use for enhanced microscopy imaging of biological tissues

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- Number: US 11,994,451 B2
- Document ID: US11994451B2
- Status: Granted
- Kind: B2
- Application number: 17/294,504
- Filed: 2019-11-20
- Published: 2021-12-30
- Granted: 2024-05-28
- Earliest claimed: 2018-11-21
- Assignees: Cornell University
- Applicants: Cornell University
- Inventors: Romulo Hurtado
- Web page: https://patentgrep.com/patent/US11994451B2

## CPC

- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G02B 21/0076: {arrangements using fluorescence or luminescence}
- 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 33/5058: {Neurological cells}
- G01N 33/5061: {Muscle cells}
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 2021/6432: {Quenching}
- 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 for rendering biological tissue sufficiently optically transparent for three-dimensional light microscopy imaging, comprising incubating biological tissue with an optical clearing solution, wherein the optical clearing solution comprises: (i) 20-50 wt % formamide, (ii) 10-90 wt % glycerol, and (iii) water as remainder. Also described herein is a method for ridding tissue of blood to make them amenable for optical clearing, comprising incubating biological tissue in a decolorizing solution, wherein the decolorizing solution comprises: (i) 0.5-3 wt % hydrogen peroxide, (ii) 0.05-1 wt % sodium azide, (iii) 5-20 wt % DMSO, and (iv) phosphate buffered saline as a remained. Also described herein is a method for reducing auto-fluorescence in biological tissue to permit imaging of the biological tissue in a fluorescence-based imaging technique with enhanced resolution, wherein the auto-fluorescence quenching solution comprises: 1-100 mM ammonium bicarbonate, (ii) 20-500 M copper sulfate, (iii) 5-20 wt % DMSO, and (iv) water as remainder.

## Claims

### Claim 1

A method for reducing auto-fluorescence in biological tissue to render the biological tissue imagable with enhanced resolution in a fluorescence-based imaging technique, the method comprising incubating biological tissue with an auto-fluorescence quenching solution for sufficient time for the auto-fluorescence quenching solution to impregnate the biological tissue and make the biological tissue sufficiently reduced in auto-fluorescence to permit imaging of the biological tissue in a fluorescence-based imaging technique with enhanced resolution, wherein said auto-fluorescence quenching solution comprises: (i) ammonium bicarbonate in a concentration of 1-100 mM, (ii) copper sulfate in a concentration of 20-500 M, (iii) 5-20 wt % dimethyl sulfoxide DMSO, and (iv) water as remainder.

### Claim 2 (depends on 1)

The method of claim 1, further comprising performing a fluorescence-based imaging technique on the biological tissue that has been reduced in auto-fluorescence.

### Claim 3 (depends on 1)

The method of claim 1, wherein said auto-fluorescence quenching solution consists only of components (i)-(iv).

### Claim 4 (depends on 1)

The method of claim 1, wherein said auto-fluorescence clearing solution comprises (i) ammonium bicarbonate in a concentration of 5-50 mM, (ii) copper sulfate in a concentration of 100-250 μM, (iii) 5-20% DMSO, and (iv) water as remainder.

### Claim 5 (depends on 1)

The method of claim 1, wherein said auto-fluorescence quenching solution has a pH of 7-8.

### Claim 6 (depends on 1)

The method of claim 1, wherein said auto-fluorescence quenching solution has a pH of about 7.5.

### Claim 7 (depends on 1)

The method of claim 1, further comprising rinsing the biological tissue at least twice with ultrapure water before impregnating the biological tissue with said auto-fluorescence quenching solution, wherein said rinsing is conducted for sufficient time to reduce the content of ionic species in the biological tissue.

### Claim 8 (depends on 1)

The method of claim 1, wherein said biological tissue is internal organ tissue.

### Claim 9 (depends on 8)

The method of claim 8, wherein said internal organ tissue is brain tissue.

### Claim 10 (depends on 1)

The method of claim 1, wherein said fluorescence-based imaging technique is immunofluorescence.