From CLARITY Wiki
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.
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. FocusClear solution is the best choice, and is especially recommended for thick tissue samples like a whole mouse brain, as it has shown to provide the optimal tissue transparency and maximum imaging depths. Other solutions with a refractive index of ~1.45, such as 85% glycerol, also optically match the embedded tissue, but the sample can still exhibit a bit of cloudiness in these solutions, especially when it is several millimeters thick. Often, the sample will appear transparent on the outer edges and cloudy towards the center. So, FocusClear alternatives seem to be applicable when only needing to image near the surface of a thick sample or when imaging thin tissue sections (<1-2 mm thick).
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.
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 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.
Imaging of CLARITY-processed tissue samples can be performed using standard confocal microscopy. Objectives with long working distances and high numerical aperture (NA) will be desired for maximum imaging depth and high resolution. There are no limitations to the type of objective (air, water, or oil-immersion) used.
- Commonly used microscopy technique
- Supports image tiling to cover large tissue areas
- No CLARITY-specific modifications needed, just optimal objectives
- Slow processing time, especially for high resolution, high sampling, and large tissue sections
- Illuminates entire sample during imaging - leads to photobleaching after a couple days (also important when attempting large imaging depths)
Light sheet microscopy
Light sheet microscopy is another three-dimensional imaging technique that can be used for clarified samples. However, the current commercially available light sheet systems are designed for very small tissue samples and are not optimized for CLARITY-processed tissues.
- Fast processing times
- No sample photobleaching
- Resolution comparable to confocal microscopy
- Not currently commercially available
Sample storage after imaging
After imaging, sample storage in the mounting solution is not recommended. If left for long periods of time, the imaging chamber will slowly dry out from evaporation through the putty and silicone elastomer, which will also dry out the sample. Also, if the sample was not washed thoroughly in PBST to remove all the residual SDS micelles from the clearing solution, a white precipitate can slowly develop when the sample is in FocusClear. The precipitation is irreversible and will act as an opaque barrier if the sample needs to be imaged again later.
To store the sample for future use after imaging, it is recommended to disassemble the imaging chamber and place the tissue sample back in PBS or PBST for long-term storage (or for a second round of immunostaining and imaging). Either PBS or PBST can be used when transferring the sample from mounting solution since there are no micelles that need to be removed (but PBST should always be used when transferring from clearing solution). A small amount of sodium azide can also be added to the PBS buffer to prevent bacterial growth and contamination. The sample can be stored at room temperature or 4°C.
- Note: The tissue sample will swell to a larger size and turn cloudier again when placed back in PBS.
- Note: If additional clearing is desired, the tissue sample can be returned to the clearing solution for further sample clearing (passive clearing is recommended at this point) after being washed for a day or two in PBS.
Two-dimensional and three-dimensional image analysis of clarified tissue samples can be performed using any open source (free) or commercially available imaging software. Most programs are compatible with files from the majority of confocal microscope companies. They mainly differ in their user interface (and ease of use) and price.