In-vivo and ex-vivo optical clearing methods for biological tissues: review.

Every optical imaging technique is limited in its penetration depth by scattering occurring in biological tissues. Possible solutions to overcome this problem consist of limiting the detrimental effects of scattering by reducing optical inhomogeneities within the sample. This can be achieved either by using physical methods (such as refractive index matching solutions) or by chemical methods (such as the removal of scatterers), based on tissue transformation protocols. This review provides an overview of the current state-of-the-art methods used for both ex-vivo and in-vivo optical clearing of biological tissues. We start with a brief history of the development of the most widespread clearing methods across the new millennium, then we describe the working principles of both physical and chemical methods. Clearing methods are then reviewed, pointing the attention of the reader on both physical and chemical methods, classified based on the tissue size and type for each specific application. A small section is reserved for methods that have already found in-vivo applications at the research level. Finally, a detailed discussion highlighting both the most relevant results achieved and the new ongoing developments in this field is reported in the last part, together with future perspectives for the clearing methodology.