SEEING THROUGH THE BRAIN

Chinmaya SadangiDepartment of Experimental Epileptology

Philipps University, Marburg

csadangi@gmail.comTwitter: @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. csadangi@gmail.com

@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

www.theaddictivebrain.wordpress.com