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Abstract

Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light absorbing substances. Although tissue clearing was first reported in 1911, it was not widely used in light microscopy until 21th century, because instrumentation of that time did not permit to acquire and handle images of thick (mm to cm) samples as whole. Rapid progress in optical instrumentation, computers and software over the last decades made micrograph acquisition of centimeter-thick samples feasible. This boosted tissue clearing use and development. Numerous diverse protocols have been developed. They use organic solvents or water-miscible substances, such as detergents and chaotropic agents; some protocols require application of electric field or perfusion with special devices. There is no "best-for-all" tissue clearing method. Depending on the case, one or another protocol is more suitable. Most of protocols require days or even weeks to complete, thus choosing an unsuitable protocol may cause an important waste of time. Several inter-dependent parameters should be taken into account to choose a tissue clearing protocol, such as: 1) required image quality (resolution, contrast, signal to noise ratio etc), 2) nature and size of the sample, 3) type of labels, 4) characteristics of the available instrumentation, 5) budget, 6) time budget, and 7) feasibility. Present review focusses on the practical aspects of various tissue clearing techniques. It is aimed to help non-experts to choose tissue clearing techniques which are optimal for their particular cases.