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Fundamentals of clearing, imaging and volume rendering thick or whole mount tissues

Judy Cathcart, AOMFjudy@aomf.ca

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Fundamentals of clearing, imaging and volume rendering thick or whole mount tissues

• Cleared tissue• Clearing techniques• Antibody labeling• Imaging considerations• Data processing requirements

http://logosbio.com/x_clarity/x_clarity/features.php

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Tissue is opaque because its heterogeneous components refract light in a way that inhibits its propagation in the direction of the incident beam.

http://www.fasebj.org/content/29/1_Supplement/632.1

Transparent water with bubbles of transparent air

Opaque, milky appearance

Transparent air with drops of transparent water

Opaque, milky appearance

RIAIR = 1.0 RIWATER = 1.33

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Cell RI = 1.38

Cleared tissue is treated to homogenize its refractive index. This leads to a greater amount of light propagated in the direction of the incident beam.

http://logosbio.com/x_clarity/x_clarity/image_gallery.php#pro_taps

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Refractive index of materials used in microscopy

Cells 1.38

Glycerol mounting media 1.43Vectashield 1.457Fluoromount G 1.40Mowiol 1.49Prolong Gold fresh 1.39

cured 1 day 1.40cured 4 days 1.44

Solvent based clearing solution 1.52Aqueous based clearing solution 1.45

Cover glass 1.52

Water 1.33Silicon oil 1.40Glycerol 1.45Oil 1.52

There are many techniques developed to clear tissue. Most clearing protocols aim to:

replace ICF/ECF with RIMS

remove lipids with solvents or detergents

generally retain proteins

How do you choose the right clearing protocol for your tissue?

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Summary of Tissue Clearing Methods

1. Solvent Based Clearing (BABB, 3Disco, iDisco)

● relatively fast

Richardson and Lichtman, Cell 162, 2015

● passive diffusion method

● tissue immersed in dehydrating solvent, ICF/ECF removed

● immunostaining possible but requires Ab validation to test compatibility

● endogenous fluorescence quenched, proteins generally retained

● high RI ~1.56

● immersion in clearing solvent removes lipid

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● RI ~ 1.47

Aqueous Immersion (ClearT, SeeDB, FRUIT, TDE)

Richardson and Lichtman, Cell 162, 2015

● specimen immersed in clearing solution

● large tissues take a long time – weeks, months?

● suitable for small tissues ~0.5 mm

●ICF/ECF replaced, lipid usually retained

● difficult to work with high [C] / viscosity solutions

● endogenous fluorescence, immunostaining in some techniques

● tissue hydrated, some techniques deform the sample

● time to clear measured in days, weeks, months

Hyperhydration (Scale, CUBIC)

Richardson and Lichtman, Cell 162, 2015

● passive diffusion of immersion fluid containing detergent, urea orformamide, glycerol or sucrose

● replaces ICF/ECF, removes lipids

● endogenous fluorescence or fluorescent protein preserved

● immunostaining possible with CUBIC technique

● hyperhydration lowers RI 1.37- 1.38

● some tissue expansion as proteins are hydrated

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● technically demanding but fast, clears the tissue well, results in a robustsample with RI 1.45 – 1.47

Hydrogel Embedding (sClarity, xClarity, PACT/PARS)

Richardson and Lichtman, Cell 162, 2015

● ICF/ECF replaced via cardiac perfusion of cold acrylamide and crosslinker

● stabilizes tissue by forming a pore-containing tissue-gel hybrid

● lipids removed with SDS (passive, ETC or stochastic electrotransport)

● endogenous fluorescence, fluorescent protein and or immunostaining

● deformation with some techniques: ETC vs. stochastic electrotransport

● protein and antibody retention depends on hydrogel pore size, which can bemodified by adjusting the concentration of acrylamide monomer

Some Immunostaining Considerations

● cost of the large amounts of Ab and other reagents can be significant https://www.braindecoder.com/post/a-

visual-history-of-neurons-1089282606#

● clearing technique used, compatibility

● in hydrogel embedded tissue, depends on gel pore size

● use of primary Ab, primary + secondary, Ab fragments, aptamers

● solvent based techniques require Ab validation

● antibody concentration needs to be optimized

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Troubleshooting

-If there’s a brighter ring around the edges and not much stainingdeeper in the sample, [Ab] is too high non-specific staining can lead to poor diffusion

-If there’s no ring background AND no staining deeper in the sampleantibody is depleted by the antigen [Ab] is too low or antigen is too concentrated for whole-mount staining

- weak but visible staining increase incubation time

-don’t use antibodies raised in mice. Secondary IgG binds to endogenous mouse IgG in vasculature

- [secondary IgG] ≤ [primary IgG]

NATURE METHODS | VOL.11 NO.12 | DECEMBER 2014 | 1211

Imaging Whole Mount Tissues

● Need a microscope with optical sectioning capability

● Need to match RI of lens with RI of cleared tissue, immersion fluid

● High NA, LWD lenses

● Clearing solvents can damagelenses. Samples should be in a sealed imaging chamber

RI adjustable lens

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Low Resolution Imaging

Use a macroscope with optical sectioning ability● apotome● light sheet illumination

Could use two photon or confocal with low resolution, LWD lens but won’t be closely RI-matched

http://focuspi.com/products-solutions/advanced-imaging-systems/115-2

High Resolution Imaging

For specimens < ~500 µm could use two photon equipped with an appropriate lens. RI-matching critical for high resolution imaging

How long to image a whole cleared mouse brain at high resolution on a two photon or confocalmicroscope?

AOMF Zeiss LSM710 confocal/2-photon microscope

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53 days???

Analysis of Large Files (>50GB+) Clarity video from the Deisseroth study, Nature, 2013 (3400µm stack)

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http://hcbi.fas.harvard.edu/

A light sheet microscope is much faster

Can produce amazing high resolution images of whole mount tissues

How long to image?

Aaaahhhh...only a couple of hours

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High resolution images of large, whole mount tissues can be HUGE4.2 x 106 frames = 30 TB per colour channelLow resolution from 10 Gb – 1 TBComputer needs to be fast, lots of RAM, lots of storageNeed good rendering software – Arivis (hours), optimized Fiji (overnight), Imaris (?)