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

Passive clearing works by simply incubating the tissue sample in the clearing solution for a long duration of time until the lipids are washed out and the sample looks transparent. The main component of the tissue sample clearing solution is sodium dodecyl sulfate (SDS), an ionic detergent that forms micelles at a given concentration and pH. When the hydrogel-embedded tissue sits in the clearing solution, the SDS micelles passively diffuse into the tissue. Inside the tissue, fatty lipids are not covalently linked to the hydrogel network, and so they 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 of the tissue in the micelles. Because this clearing method relies on passive diffusion of the micelles in and out of the tissue, the sample tends to clear on the outside first and continue inwards, which can be observed visually.

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

As the sample clears, the tissue becomes swollen and increases in size. This is due to the removal of the lipid membrane structure which constrains the acrylamide polymer chains making up the hydrogel matrix from swelling. Without the lipids, the hydrogel chains prefer to swell as much as possible in aqueous solution and are only constricted by the hydrogel crosslinks, in this case the attached biomolecules and bis-acrylamide links. Swelling goes down when the sample is placed in the mounting solution which is not as attractive to the polyacrylamide chains, so they return to the state that they were polymerized in (sample returns to anatomical size). Based on evidence from CLARITY-processed brain tissues, the swelling and de-swelling process does not appear to affect fine structural details within the tissue as they can still be observed and do not exhibit any deformations.

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 the 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 - Tissue clearing is significantly faster at 37°C (~6 weeks for a mouse brain) as opposed to room temperature (~6 months for a mouse brain). The temperature can also be raised up to about 55°C for even faster clearing. Incubation at 60°C or higher may lead to negative effects regarding the hydrogel integrity and fluorescence retention of pre-labelled biomolecules.
  • Use smaller samples - Smaller tissue samples clear much faster than larger samples. This is one reason why sample sectioning is recommended immediately after hydrogel embedding prior to sample clearing. At 37°C, 1 mm thick brain slices passively clear in 1-2 weeks as opposed to around 6 weeks of clearing time for a whole mouse brain. While not always possible for every application, if only a smaller volume of the tissue sample is needed for data collection, it is recommended to section this out before passive clearing to help cut down on the incubation time needed.
  • Change the clearing solution regularly - Replacing the clearing solution in the sample container regularly (either daily or once every few days) can help to increase the passive clearing speed.
    • Note: Washes from the first two days are disposed of as waste because they contain hydrogel monomers. Afterwards, the clearing solution washes are safe for sink disposal.
  • Apply gentle shaking - While the sample will clear if left stagnant in the solution, placing the container on a gentle shaker or rotator plate is recommended to aid passive micelle diffusion for faster clearing.
  • Adjust hydrogel composition (for large tissue samples) - Decreasing the acrylamide or bis-acrylamide concentration in the hydrogel solution before sample preparation and hydrogel embedding will result in a hydrogel matrix that is less crosslinked and more porous. The increased porosity of such embedded tissue samples will aid in passive diffusion of the SDS micelles in and out of the tissue, and therefore the passive clearing speed will also increase. Do note, however, that decreased crosslinking in the hydrogel will also result in a greater loss of unattached proteins and biomolecules during sample clearing.

Completion point

As the sample clears during incubation, visually check for clearing by holding the container up to the light. In the early to middle stages of clearing, certain areas of the tissue will look transparent (most likely the outer edges of the sample) while other areas will still appear opaque. Once the entire span of tissue appears see-through, the sample can be removed from the clearing solution and placed in PBST buffer for the next step in CLARITY processing.