Method and System for Imaging and Analysis of a Biological Specimen

Patent metadata

Field	Value
Publication number	US-2020096425-A1
Application number	US-201916660348-A
Country	US
Kind code	A1
Filing date	Oct 22, 2019
Priority date	May 25, 2016
Publication date	Mar 26, 2020
Grant date	—

Abstract

The present disclosure provides methods of preparing a biological specimen for imaging analysis, comprising fixing and clearing the biological specimen and subsequently analyzing the cleared biological specimen using microscopy. Also included are methods of quantifying cells, for example, active populations of cells in response to a stimulant. The present disclosure also provides devices for practicing the described methods. A flow-assisted clearing device provides rapid clearing of hydrogel-embedded biological specimens without the need of specialized equipment such as electrophoresis or perfusion devices.

First claim

1 - 31 . (canceled)

32 . A method for quantifying an active neuronal population of a subject animal exposed to a stimulant, the method comprising:
i) delivering a stimulant to the subject animal; ii) isolating the brain of the subject animal; iii) preparing the brain of the animal to produce a cleared brain by:
a) fixing the brain with a plurality of hydrogel monomers to produce a hydrogel-fixed brain;
b) clearing the hydrogel-fixed brain using a flow-assisted clearing device to produce a cleared brain, wherein the clearing is performed by immersing the hydrogel-fixed brain in a buffer and flowing the buffer through the hydrogel-fixed brain, wherein the buffer flow is unidirectional; and
c) contacting the cleared brain with a refractive index matching solution;
iv) imaging the cleared brain; and v) identifying the active neuronal population.

33 . The method of claim 32 , further comprising incubating the cleared brain in a mounting medium prior to imaging.

34 . The method of claim 33 , wherein the mounting medium is RapidClear Mounting Gel.

35 . The method of claim 32 , wherein the active neuronal population of the animal is labeled.

36 . The method of claim 35 , wherein the active neuronal population of the animal is labeled by targeted recombination in active populations (TRAP).

37 . The method of claim 32 , further comprising administering a tamoxifen to the animal to induce targeted recombination in active populations.

38 . The method of claim 32 , wherein the tamoxifen is 4-hydroxytamoxifen.

39 . The method of claim 32 , wherein the step of identifying the active neuronal population comprises:
i) illuminating the cleared brain with two light sheets from a first side and a second side to produce a sample image volume, wherein the second side is opposite to the first side; ii) registering the sample image volume to a reference image volume; and iii) identifying the active neuronal population from the registered sample image volume.

40 . The method of claim 32 , wherein the native image volume is deconvolved.

41 . The method of claim 32 , wherein the registering step comprises nonlinearly registering the sample image volume to the reference image volume to produce a nonlinear registration.

42 . The method of claim 32 , wherein identifying the active neuronal population further comprises:
i) identifying an active cell location in the sample image volume; and ii) mapping the active cell location to the registered sample image.

43 . The method of claim 32 , wherein the mapping step further comprises:
i) generating a binary mask volume for a specified region in the nonlinear registration; and ii) counting the number of active cells in the active cell location.

44 . The method of claim 43 , wherein the specified region is manually specified.

45 . The method of claim 43 , wherein the specified region comprises a pre-specified region from an Allen Brain Atlas image volume.

46 . The method of claim 32 , wherein the reference image volume is generated by:
i) globally aligning a plurality of image volumes obtained from a plurality of cleared brains to an Allen Brain Atlas image volume; and ii) averaging the plurality of globally aligned image volumes.

47 . The method of claim 32 , wherein the step of globally aligning comprises affine registration and/or spline registration.

48 . The method of claim 32 , wherein the Allen Brain Atlas image volume is an Allen Brain Atlas Nissl-stained image volume.

49 . The method of claim 32 , wherein the stimulant is amphetamine, caffeine, ephedrine, 3,4-methylenedioxymethamphetamine (MDMA), methylenedioxypyrovalerone (MDPV), prolintane, mephedrone, methamphetamine, nicotine, phenylpropanolamine, propylhexedrine, dimethylamylamine, pseudoephedrine, cathinone, or cocaine.

50 . (canceled)

51 . The method of claim 32 , wherein the stimulant generates a rewarding or aversive experience in the subject animal.

52 . The method of claim 32 , wherein the stimulant is a physical stimulant.

53 - 64 . (canceled)

Assignees

• Univ Leland Stanford Junior

Inventors

• Deisseroth Karl A
• Allen William E
• Hsueh Brian
• Ye Li

Classifications

• G01N2001/305

  Fixative compositions · CPC title

• B01L2300/0609

  Holders integrated in container to position an object · CPC title

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• G01N1/34

  Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)} · CPC title

• B01L2300/0832

  cylindrical, tube shaped · CPC title