Method and System for Imaging and Analysis of a Biological Specimen

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.

What is claimed is: 
     
         1 . A method of preparing a biological specimen for imaging analysis, the method comprising:
 i) fixing the biological specimen with a plurality of hydrogel monomers to produce a hydrogel-fixed specimen;   ii) clearing the hydrogel-fixed specimen using a flow-assisted clearing device to produce a cleared specimen, wherein the clearing is performed by:
 a) immersing the hydrogel-fixed specimen in a buffer; 
 b) flowing the buffer through the hydrogel-fixed specimen, wherein the buffer flow is unidirectional; and 
   iii) contacting the cleared specimen with a refractive index matching solution prior to imaging analysis.   
     
     
         2 . The method of  claim 1 , wherein the flow-assisted clearing device comprises:
 a sample chamber comprising an inlet and an outlet, wherein the inlet delivers the buffer into the sample chamber and the outlet draws the buffer out of the sample chamber;   a sample holder, wherein the sample holder can be removably placed inside the sample chamber, and wherein the sample holder allows the buffer to flow through; and   a buffer circulator.   
     
     
         3 . The method of  claim 2 , wherein the buffer delivered by the inlet is fresh. 
     
     
         4 . The method of  claim 2 , wherein the buffer delivered by the inlet is reused. 
     
     
         5 . The method of  claim 2 , wherein the buffer circulator is a temperature-controlled circulator. 
     
     
         6 . The method of  claim 1 , wherein the flow-assisted clearing device comprises:
 a container;   a sample holder, wherein the sample holder allows the buffer to flow through, and wherein the sample holder can be removably placed inside the container; and   a buffer circulator.   
     
     
         7 . The method of  claim 6 , wherein the buffer circulator comprises a rotating rod. 
     
     
         8 . The method of  claim 7 , wherein the rotating rod is a magnetic rotating rod and is controlled by an external magnetic field. 
     
     
         9 . The method of  claims 1-8 , wherein a plurality of flow-assisted clearing devices are arranged in parallel to the buffer circulator. 
     
     
         10 . The method of  claim 9 , wherein each of the plurality of flow-assisted clearing devices clears a plurality of hydrogel-fixed specimens. 
     
     
         11 . The method of  claims 1-10 , wherein the flow-assisted clearing device is not an electrophoresis chamber. 
     
     
         12 . The method of  claims 1-11 , wherein the flow-assisted clearing device further comprises a buffer filter component. 
     
     
         13 . The method of  claims 1-12 , wherein the buffer circulator generates a unidirectional flow of the buffer through the sample holder. 
     
     
         14 . The method of  claims 1-13 , wherein fixing the biological specimen comprises contacting the biological specimen with paraformaldehyde. 
     
     
         15 . The method of  claim 14 , wherein contacting the biological specimen with paraformaldehyde comprises transcardial perfusion. 
     
     
         16 . The method of  claims 1-15 , wherein the plurality of hydrogel monomers comprise acrylamide. 
     
     
         17 . The method of  claim 16 , wherein the plurality of hydrogel monomers comprise 1% acrylamide. 
     
     
         18 . The method of  claims 1-17 , wherein clearing the hydrogel-fixed specimen further comprises substantially removing a plurality of cellular components from the hydrogel-fixed specimen. 
     
     
         19 . The method of  claim 18 , wherein the plurality of cellular components comprise lipids. 
     
     
         20 . The method of  claims 1-19 , wherein clearing the hydrogel-fixed specimen is performed at about 40° C. 
     
     
         21 . The method of  claims 1-20 , wherein the buffer comprises a Tris buffer and an ionic surfactant. 
     
     
         22 . The method of  claim 21 , wherein the Tris buffer is Tris-Boric buffer. 
     
     
         23 . The method of  claim 22 , wherein the ionic surfactant is sodium dodecyl sulfate. 
     
     
         24 . The method of  claims 1-23 , wherein the buffer is at about pH 8.5. 
     
     
         25 . The method of  claims 1-24 , wherein the refractive index matching solution has a refractive index that matches that of the cleared specimen. 
     
     
         26 . The method of  claim 25 , wherein the refractive index matching solution has a refractive index of 1.45. 
     
     
         27 . The method of  claim 25 , wherein the refractive index matching solution is RapidClear. 
     
     
         28 . The method of  claim 25 , wherein the refractive index matching solution is FocusClear. 
     
     
         29 . The method of  claims 1-28 , wherein the biological specimen is a central nervous system tissue. 
     
     
         30 . The method of  claim 29 , wherein the central nervous system tissue is a whole brain. 
     
     
         31 . The method of  claims 1-28 , wherein the biological specimen is a whole spinal cord. 
     
     
         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 according to the method of  claims 1-30  to produce a cleared brain;   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  claims 32-34 , 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  claims 32-36 , further comprising administering a tamoxifen to the animal to induce targeted recombination in active populations. 
     
     
         38 . The method of  claims 32-37 , wherein the tamoxifen is 4-hydroxytamoxifen. 
     
     
         39 . The method of  claims 32-38 , 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  claims 32-39 , wherein the native image volume is deconvolved. 
     
     
         41 . The method of  claims 32-40 , 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  claims 32-41 , 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  claims 32-42 , 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  claims 32-45 , 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  claims 32-46 , wherein the step of globally aligning comprises affine registration and/or spline registration. 
     
     
         48 . The method of  claims 32-47 , wherein the Allen Brain Atlas image volume is an Allen Brain Atlas Nissl-stained image volume. 
     
     
         49 . The method of  claims 32-48 , 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 . The method of  claims 32-49 , wherein the stimulant is cocaine. 
     
     
         51 . The method of  claims 32-50 , wherein the stimulant generates a rewarding or aversive experience in the subject animal. 
     
     
         52 . The method of  claims 32-48 , wherein the stimulant is a physical stimulant. 
     
     
         53 . The method of  claim 52 , wherein the physical stimulant is a foot shock. 
     
     
         54 . A flow-assisted clearing device, the device comprising:
 a sample chamber comprising an inlet and an outlet, wherein the inlet delivers the buffer into the sample chamber and the outlet draws the buffer out of the sample chamber;   a sample holder, wherein the sample holder can be removably placed inside the sample chamber, and wherein the sample holder allows the buffer to flow through; and   a buffer circulator.   
     
     
         55 . The device of  claim 54 , wherein the sample holder can hold a plurality of specimens. 
     
     
         56 . The device of  claims 54-55 , wherein the buffer circulator is temperature controlled. 
     
     
         57 . The device of  claims 54-56 , wherein the buffer circulator is configured to provide unidirectional flow of the buffer into the sample chamber. 
     
     
         58 . The device of  claims 54-57 , wherein the buffer circulator comprises a circulator inlet and a circulator outlet that is operably connected respectively to an outlet and an inlet of a sample chamber, and wherein the buffer circulator is configured to provide buffer flow entering the inlet and exiting the outlet of the sample chamber. 
     
     
         59 . The device of  claims 54-58 , wherein the buffer circulator further comprises a conduit operably connected to a receptacle. 
     
     
         60 . The device of  claim 59 , wherein the receptacle is a buffer reservoir. 
     
     
         61 . The device of  claims 54-60 , wherein the buffer circulator comprises a centrifugal pump, a peristaltic pump, an oscillatory pump, or a diaphragm pump. 
     
     
         62 . The device of  claims 54-61 , further comprising a buffer filter component. 
     
     
         63 . The device of  claims 54-62 , wherein the device is not an electrophoresis chamber. 
     
     
         64 . The device of  claims 54-63 , wherein the device comprises a plurality of sample chambers operably connected in parallel to the buffer circulator.