mesoSPIM_preparing_CUBIC_samples
Samples cleared using CUBIC can be mounted a) inside a small cuvette, b) held by a clamp; c) glued to a sample holder; d) mounted in an agarose block, or a combination of these. If a small cuvette is used, the material of cuvette depends on CUBIC version, see below.
This is the latest version of CUBIC with higher RI (1.52) and thus better index matching for most biological tissues. The inner cuvette (if used) should be made of optical glass (RI 1.52) and filled with CUBIC-R+, M3294 mounting oil from TCI, or ~3:2 mix of HIVAC F-4 (RI 1.55) : mineral oil (Sigma-Aldrich, M8410, RI 1.47) Matsumoto et al, 2019. The outer cuvette can be filled with the same oil as the inner cuvette (RI 1.52).
This is a version of CUBIC with lower RI (1.47, similar to CLARITY) used for expansion microscopy. If inner cuvette is used, its material should be quartz (RI=1.46). The cuvette can be filled with M3292 mounting oil from TCI, or suitable mineral oil (e.g. M8410 from Sigma-Aldrich, RI 1.47). The outer cuvette can be filled with M3292 or mineral oil as well, but not with CUBIC-X reagent, because the latter will evaporate, changing its RI and making residue. See further Murakami et al, 2018 and Matsumoto et al, 2019.
If the sample is sufficiently stiff, or mounted inside an agarose block (see below), it can be also imaged directly, without the inner cuvette, by using a clamp similar to iDISCO samples and dipping it directly into the outer cuvette filled with matching oil. Alternatively, the clamp with sample can be placed inside the inner cuvette.
CUBIC samples can be mounted in agarose block to mechanical stability and long-term preservation, check video at CUBIC website and in Matsumoto et al, 2019. The agarose block is then dipped into the immersion cuvette with matching oil for imaging.
The clamp can be 3D printed from our design files (IPT, STL, STP). We use industrial SLS printer and PA12 (polyamide, nylon) for high-resolution printing of teeth and other small features.
Sample can be also glued to a weight (similar to CLARITY mounting) with cyanoacrylate glue. This will make it stable inside the cuvette and prevent floating, but the glue can damage parts of the sample.
The cuvettes and holders can be cleaned with hot soapy water and rinsed with distilled water.
The combination of CUBIC-L / CUBIC-R+ (for delipidation and RI matching, repectively) is the recommended protocol suitable for most users. The CUBIC-X should be used with expansion protocol.
CUBIC reagents supplier is TCI Chemicals that provides CUBIC Tissue Clearing Kit and CUBIC-HV Staining Kit.
We thank Prof. Etsuo Susaki for reviewing this information.