High Refractive Index Aqueous Solutions

The disadvantages and limitations of solvent-based clarification have led scientists and researchers to work on methods that do not need to remove water from the tissue. All these water-based techniques, which have been built up, are based on one of these three solutions: [1] simple and inactive submersion of tissue in a solution with a RI near the tissue; [2] removal of tissue fats with tissue water supply (hyperhydration) to reduce the residual tissue RI; and [3] deletion of lipid active or inactive by immersion in an environment with equal RI with tissue.

The simple immersion is such that the specimen is immersed in a solution with a high RI. This method works well in small tissue, but if the texture is large, image quality is not as good as small specimens. In addition, in large tissues, a stronger intensity of clarity is needed. However, in any case, this method is an economical method because the main substance used for clarification is based on glycerol. The RI of glycerol is about 1.4–1.44. In this method, a tissue sample is put in a water-based solution containing a solvent (high RI) to inactive its clarification. Sucrose (28), fructose (56, 176), glycerol (177), 2,2- thiodiethanol (TDE) (178–180), and formamide (59) have been used for this purpose. These biological compounds called the RI match solution (RIMs) have been developed for establishing a proper RI (173). Diatrizoic acid (hypaque) used in FocusClear and Histodenz used in RIMs are complex molecules with an aromatic ring and three iodine atoms.

FocusClear, a simple water-soluble reagent, was invented by Chiang (49) and is used for clearing biological animal/plant/organism tissues. Moreover, its use in 3D imaging of stem cell in mice intestine was reported by FocusClear (181).

TDE, which is a clear-to-yellow, water-soluble and low-viscous liquid, is compatible with immunostaining and can be used for adjusting proper RI in the range of 1.3–1.515 by dissolving certain values. For the first time, TDE was used as a mounting media for tissue clarification for super-resolution microscopy (178). It can also clarify large tissues (176, 179) and is suitability used for confocal due to its feather of suppressing photo-oxidation. Moreover, it has a good tissue clearing ability and is suitable for two photon microscopy (182). One of the problems with TDE is that at high concentrations, the brightness of a number of fluorophores is reduced (178). TDE was used for 3D localization of stem cell in mice gut (183).

SeeDB is a simple immersion water-based optical clearing agent using fructose and thiol that was firstly used on mouse brain for imaging dorsal to the ventral side of it (56). SeeDB contains a saturated solution of fructose in water for reaching its RI to 1.50 (in 37°C) and α-thioglycerol for inhibiting Millard reaction. SeeDB can clear the sample in 3 days and is compatible to many fluorescence proteins and as it is detergent-free, tissues and cellular structures like membrane were preserved and allowed to use lipophilic dye and tracers (56). The light sheet microscopy was not recommended for imaging SeeDB clearing samples due to low resolution (57). 3D human IPs spheres was imaged with SeeDB (184). This protocol was used for 3D imaging of enteroids in vitro (185, 186). 3D imaging of colorectal epithelial stem cells in mice was also done by SeeDB (187). For localization of neural stem cells after transplantation and differentiation in mice brain, SeeDB was used (188). Mammary stem cells was imaged in mice mammary duct by this method (74).