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SEEING THROUGH THE BRAIN
Chinmaya SadangiDepartment of Experimental Epileptology
Philipps University, Marburg
[email protected]: @addictivebrain; Web: www.theaddictivebrain.wordpress.com
AGENDA
• Clearing brains
• 3DISCO
• uDISCO
• CLARITY
INTRODUCTION
• Brain is made up 86 billion neurons.
• Constitute of at least 12% lipids.
• Lipids make imaging the whole brain difficult.
• Optical imaging methods are not suitable.
THE TRADITIONAL METHOD
Time consuming and error prone process.
Loss of information – only the surface is imaged.
CLEARING THE BRAIN
• New technique – clear the lipids.
• Image 3D structural and molecular information across the mm-cm scale at sub-micron resolution.
Seo et al. (2016), Molecules and cells
• German anatomist, Walter Spalteholz, cleared first tissue in 1910.
• Organic solution – Benzyl alcohol and methyl salicylate.
• Critical step – Refractive index (RI) matching.
Aqueous based clearing
Urea, Sorbitol Days-weeks-months
Immunolabeling varies
Expands or no change
Solvent basedclearing
BAAB, DBE Hours - days Yes, but limited Shrinkage
Electrophoresis Based clearing
SDS, FocusClear Days - Weeks Yes Slight expansion
3DISCO
• 3D imaging of solvent-cleared organs.
• Alcohol and BAAB – embryonic hippocampi.
• THF (Tetrahdyrofuran) and BAAB – adult spinal cord.
• New method: DBE (Dibenzyl ether) and THF.
• Fast to perform; few hours for small organs (spinal cord, lymph nodes) and two day for large organs such as the brain
• Sequential solution incubation steps.
• Few minutes of work but long incubation type.
• Diverse labeling methods including transgenic expression of fluorophores (CFP, GFP, YFP)
PRE-DISCO
• Perfuse animals 5-10 min with 0.9% saline (Nacl).
• 15-20 min with 4% PFA.
• Store the sample in PFA at 4ºC O/N.
• Fluorescent samples – Short time in PFA.
• PBS wash: 3-4 times.
SEQUENTIAL INCUBATION
• 50, 70, 80, 100 % THF.
• Samples in DCM.
• Samples in DBE until clear.
Erturk et al. (2012), Nature protocols
CHEMICAL HANDLING
Small tissues – lungs
50% THF 30 min
70% THF -
80% THF 30 min
100% THF 3 x 30 min
DCM 20 min
DBE > 30 min
Brain
2 h
2 h
2 h
2 h, ON, 2 h
-
2 x 2 h
Whole brain
6 h
6 h
12 h
2 x 6 h
-
2 x 2 h
Erturk et al. (2012), Nature protocols
IMAGING AND DATA ANALYSIS
• Imaging possible with Confocal microscopy, light sheet microscopes, and 2-photon microscopy.
• Large data set.
LIMITATIONS
• Fixed tissues.
• Cannot be used with electron microscopy.
• Cannot be used with lipophilic dyes.
• Cannot be stored in final solution.
• Cannot clear whole animals.
Important information:
• Dissect the region of interest.
• Better penetration of antibodies and faster clearing.
• Use of strong fluorophores.
CONCLUDING REMARKS
• Special lens for the microscope.
Dodt et al. (2015), Neurophotonics
uDISCO
• Ultimate DISCO for volumetric imaging.
• Preserve fluorescent proteins.
• Whole body shrinks up to 65%.
• Determine long-distance neuronal and vascular projections and spatial information on stem cell transplants.
• Uses Diphenyl ether (DPE), RI = 1.579.
• Mixture of BAAB and DPE = BAAB-D.
• Use of alpha-tocopherol and tert—butanol instead of THF.
• Internal organs and hard tissues like bones could be cleared.
WHOLE BODY CLEARING
CLARITY
• Invented by Kwang Chung and Karl Deisseroth.
• Fixation of molecules of interest within a solid polymer network using formaldehyde and hydrogel monomers.
• Lipid removal by using SDS.
• Tissue amenable to immunohistochemistry or in-situ hybridization (ISH).
• Sample is submerged in a refractive index (RI) matched fluid for imaging.
Advantages:
• Can be applied to any organ or tissue.
• Does not quench endogenous fluorescent probes.
• Multiple rounds of immunolabeling or ISH.
• Can be applied to previous fixed samples.
• Cleared tissue, can be stored for long periods of time.
CLEARING PROCESS
• Replace 50 mL clearing solution after 1 day and incubate at 37˚C with shaking.
• Samples in PBS-T at RT or 37˚C with shaking to wash out SDS micelles.
• Replace PBS-T after 1 day and continue incubation.
ELECTROPHORESIS CHAMBER
IMMUNOSTAINING
• Incubate samples in primary antibody solution (2 days).
• Wash samples with buffer (1day).
• Incubate samples in secondary antibody (2 days).
• Wash samples with buffer (1 day).
Sample Imaging
• Incubate samples in imaging solution – FocusClear, RapiClear, Glycerol.
• Visual transparent samples, mount with imaging solution in a sealed chamber.
Multiple staining
• Place samples in 50 mL PBST, incubate at RT or 37˚C O/N with shaking.
• Samples in 50 mL clearing solution, incubate at 60˚ O/N to wash first round of antibodies.
• Samples in 50 mL PBST, incubate at RT or 37˚C O/N with shaking.
THE WILCO DISH
Anna Beyler, Kay M. Tye lab, MIT & Chinmaya, Unpublished data.
Interstellate, Vol. 1, Sung Yong Kim, Chung lab, MIT
LIMITATIONS
• Slight tissue expansion, but returns to normal after RI matching.
• ~ 8 % protein loss.
• Doesn’t preserve lipids or other molecules lacking functional group.
• Can CLARITY samples be stored long enough for further analysis?
CONCLUSION
• Choose a method depending on your sample size.
• Brain mapping.
• Neural circuits. [email protected]
@addictivebrain
3DISCO CLARITY
Solvent based Electrophoresis basedLimited immunolabeling Multipe rounds of labelingTissue shrinkage Slight expansion$ - $$ $$ - $$$
Whole animal clearing possible Whole animal clearing hasn’t been tried
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