Passive Clearing

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Passive clearing is one of two lipid clearing methods for the hydrogel-embedded tissue samples. After completing the initial clearing solution wash for two days to rinse out the excess hydrogel monomers, passive clearing can be employed to slowly remove the lipids and other unattached biomolecules from the embedded tissue sample.

Contents

Methodology

- SDS is ionic detergent that forms micelles (link to wikipedia page?) at given concentration and pH - when embedded tissue sits in clearing solution the micelles passively diffuse in - fatty lipids are not covalently linked to the hydrogel network and will transfer to the micelles and passively diffuse out of the tissue - other biomolecules that were not incorporated into the hydrogel and contain hydrophobic moieties are also likely to be washed out in the micelles - because method relies on passive diffusion in and out, the sample clears from the outside in, which can be observed visually

(thumbnail)
Progression of passive lipid clearing in a whole mouse brain tissue sample


- as sample clears, tissue becomes swollen and increases in size - due to removal of lipid membrane structure constraining acrylamide polymer chains from swelling - without lipids, hydrogel chains prefer to swell as much as possible in aqueous solution and are only constricted by their crosslinks (attached biomolecules and bisacrylamide links) - swelling goes down when sample is placed in mounting solution which is not as attractive to the polyacrylamide chains, so they return to the state that they were polymerized in (anatomical size) - based on evidence from CLARITY-processed brain tissues, swelling and de-swelling process does not appear to affect fine structural details within the tissue, as they can still be observed and look as expected

Advantages and Disadvantages

Advantages

  • Gentle on tissue sample and no risk of damage (unless left for really long periods of time - see clearing solution storage)
  • Cheap - no additional equipment needed, only clearing solution
  • Produces most visually transparent samples - no tissue yellowing as seen in ETC; allows maximum imaging penetration depth and good antibody penetration for immunostaining
  • Easy - can basically "set it and forget it" until complete

Disadvantages

  • Slow process - a few weeks to months depending on sample size and conditions

Procedure

After completing two days of washing with clearing solution and collecting the washes as waste, continue incubation of the hydrogel-embedded tissue sample in 50 mL of clearing solution. Passive clearing will slowly occur regardless of temperature or incubation conditions (slow shaking vs. stagnant).

Strategies to increase clearing speed

While the tissue sample will slowly clear from passive diffusion of micelles as long as it is in the clearing solution, the speed of clearing is highly dependent on several variables including: tissue sample size, tissue type, temperature, and hydrogel composition. While not every variable can be controlled (tissue type, for example), some of the conditions of passive clearing can be set to influence the speed at which the tissue sample clears. Such strategies are as follows:

  • Incubate at higher temperatures -
  • Use smaller samples -
  • Change the clearing solution regularly -
    • Note: waste disposal
  • Apply gentle shaking -
  • Adjust hydrogel composition (for large tissue samples) -

Completion point