Sample Clearing

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After hydrogel tissue embedding, the tissue sample is placed in clearing solution to begin the process of lipid clearing. At this point, the tissue biomolecules, other than lipids, are chemically attached to a hydrogel matrix within the tissue. The lipids are the main source of light scattering within the tissue sample, causing it to be opaque. The clearing solution contains an ionic detergent that collects the lipids and transports them out of the tissue hydrogel, eventually leading to a clear and transparent tissue sample that still contains fine structural details from the biomolecules that are crosslinked in place.

Contents

Clearing solution wash

After hydrogel tissue embedding, all tissue samples undergo a clearing solution wash step for a minimum of 2 days. The purpose of the washing step is to dialyze out the excess hydrogel monomers (formaldehyde, acrylamide, bis-acrylamide, thermal initiator) from inside the tissue. Removal of the monomers is necessary to prevent further polymerization and crosslinking inside the tissue. Also, as a couple of the monomers are toxic, it is best to first rinse them out and dispose of them properly as it will greatly ease handling of the sample and clearing solution during ETC.

Procedure

After removing the newly embedded tissue sample from the bulk hydrogel, place the sample in a container with 50 mL of clearing solution. Incubate at room temperature or 37°C overnight on a shaker/rotator plate.

Replace the 50 mL of clearing solution after 1 day and continue incubation at 37°C with shaking.

Following each 50 mL wash, discard the clearing solution in a liquid waste container in a fume hood. Do not pour the clearing solution from the first 2 washes down the sink as it contains toxic PFA and acrylamide monomers.

Clearing methods and strategies

After the initial 2-day wash in clearing solution to rinse out the hydrogel monomers, the sample is kept in the clearing solution in order to remove the lipids and unattached biomolecules from the hydrogel-embedded tissue. There are two methods for clearing: passive clearing and electrophoretic tissue clearing (ETC). One or both of these methods can be applied to clear the tissue sample. In certain cases, one method may be preferred over another, as discussed in the clearing strategies section below.

Methods

The clearing solution, which contains an ionic detergent that forms micelles in solution, is used to clarify the hydrogel-embedded tissue by removing the unattached fatty lipids. Sample clearing can be performed in two ways:

  • Passive clearing - Basically a continuation of the clearing solution wash step, this method relies on passive diffusion of the lipid-collecting micelles in and out of the tissue by incubating the sample in clearing solution until it appears visually clear.
  • Electrophoretic tissue clearing (ETC) - Taking advantage of the ionic charge on the SDS micelles in the clearing solution, ETC uses an electric field around the sample to actively diffuse the micelles in and out of the tissue for faster clearing.

Clearing strategies

The best clearing method to use for a given tissue sample can depend on an array of variables including sample size, labelling method, processing time constraints, desired imaging depth, and the overall purpose of the tissue sample (i.e. sample used for pilot testing or screening vs. sample for specific data collection).

100% passive clearing

Passive clearing is the simplest and cheapest method of clearing, and it is also the gentlest on the sample tissue. Empirically, samples that undergo only passive clearing have provided the greatest imaging depths and best immunostaining results. In addition, passively cleared samples achieve the best overall transparency. The drawback of using only passive clearing, though, is that it is usually a multi-week long process, the slowest of the clearing strategies.

A 100% passive clearing strategy is recommended for:

  • Small and/or fragile samples (may be risky to place in ETC chamber under high flow)
  • Samples that require immunostaining
  • Samples that require large imaging depths (for example, dorsal to ventral of a whole mouse brain, ~6 mm)
  • High-quality data collection samples

It is also recommended when setting up CLARITY to use passive clearing on a couple initial tissue samples. This will help with troubleshooting the CLARITY process. If encountering difficulties clearing the sample using ETC, samples undergoing passive clearing can be used as a reference to determine if there is a problem earlier in the process during hydrogel embedding (passive clearing samples are not clearing either) or if the problem is with the ETC set-up and conditions.

ETC

ETC is the fastest method of tissue clearing. However, it involves exposing the tissue sample to a high flow rate and an electric field, so there is a risk of sample damage. The start-up equipment cost is high, but if well-maintained, ETC equipment will not need to be replaced very often. It is difficult to obtain complete sample transparency using only ETC as it requires longer exposure times, which can lead to significant yellowing of the sample tissue.

An ETC clearing strategy is recommended for:

  • Larger/whole tissue samples (may take months to clear passively)
  • Transgenic or pre-labelled samples (markers are attached to the hydrogel during embedding)
  • Samples that need to be processed quickly

Passive clearing/ETC combination

A happy medium to receive the benefits of both passive clearing (gentler on sample, better transparency) and ETC (time-saving) is to combine the two methods.

PBST buffer wash after clearing

Purpose of buffer wash

Procedure

Sample storage

Clearing solution

PBST