Revision as of 22:33, 20 April 2014

Following sample clearing in the clearing solution, the hydrogel-embedded tissue is washed in PBST buffer to remove SDS micelles. If the sample was labelled with fluorescent proteins prior to CLARITY processing (from a transgenic mouse line, for example), the proteins will be attached to the hydrogel matrix, and the sample can be imaged immediately after the PBST washing step. Otherwise, the clarified samples can be labelled via immunostaining and then prepared for imaging.

Sample preparation

To prepare the hydrogel-embedded tissue sample for imaging, it is first removed from PBST solution and placed in a mounting solution. After reaching an equilibrium swelling state in the mounting solution, the sample is mounted in a chamber containing the same solution for imaging.

Place in mounting solution

Right before imaging, the tissue sample should be placed in a mounting solution. The purpose of the mounting solution is to optically match the refractive index of the cleared tissue (~1.45). In other words, the mounting solution removes the cloudiness that is observed when the cleared tissue is swollen in either clearing solution or PBS. It is in the mounting solution that the sample should look the most optically transparent, and therefore can be probed to the most extended depths with the microscope.

In addition to refractive index matching, the mounting solution also reverses the tissue swelling that occurs in clearing solution and PBS (after clearing). After several hours in the mounting solution, the sample should return to its anatomical size.

Choosing a mounting solution will depend mostly on the thickness of the tissue sample and the desired penetration depth for imaging.

Mounted sample in imaging chamber (an unclear brain is shown for contrast)

Procedure

Remove the tissue sample from PBST where it was swollen for washing/staining/storage. Place the sample in a few milliliters of the mounting solution. Only enough solution to fully immerse the sample is needed (~4 mL for a whole mouse brain, for example). Incubate the sample with gentle shaking at either room temperature or 37°C for several hours or overnight. It is not recommended to leave the tissue sample in mounting solution for extended periods of time before imaging, but a day or two should be fine as long as the PBST washes were completed to rinse out residual SDS micelles from the sample.

Mount in imaging chamber

Once the tissue appears visually transparent and has de-swelled back to its anatomical size, the sample is ready for mounting. A sealed imaging chamber is constructed to hold the sample and mounting solution. It is important to keep the sample swollen in the mounting solution throughout imaging or it will dry out.

Imaging techniques

Imaging should be performed using a microscopy technique that focuses on one plane within the sample at a time. Standard light microscopy will not be suitable for the optically transparent, intact tissue samples as light will penetrate the entire sample and fluorescence will be emitted from everywhere. Thus, the signal from the focused plane will be diluted and probably lost by the signal coming from everywhere else in the sample, causing the sample to look like a blob of autofluorescence under the light microscope.

Confocal microscopy

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Advantages

Disadvantages

Light sheet microscopy

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Advantages

Disadvantages

Sample storage after imaging

Image analysis