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

Abstract

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.

Fig. 1.

Graph showing the trend of published papers on tissue clearing during the last century. Data obtained from Scopus (www.scopus.com). The recent developments of aqueous solutions by Tuchin and associates, as well as the development of tissue transformation techniques, provoked a high widespread of the field, identified by the two abrupt variations in slope occurring in the mid 90’s and 2013, respectively.

Fig. 2.

(top) Usage of different clearing methods in papers focused on specific biological applications. The center and right pie charts show usage of clearing methods in combination with light-sheet fluorescence microscopy and confocal laser-scanning microscopy, respectively. (bottom) Timeline of the use of different clearing techniques for biological applications. The details of the papers used for this literature analysis can be found in Data File 1.

Fig. 3.

(a) Whole mouse brain before and after FDISCO clearing. (b) Thy1-GFP-M mouse brain images with insets at high resolution (c) 3D visualization of the vasculature in the mouse brain labeled by injection of CD31-A647 antibody. Images modified with permission from Qi et al [44].

Fig. 4.

(a) Whole mouse heart before and after CLARITY clearing. (b) Representative fluorescent microscope images of Rainbow heart at a different stage of development E14.5, P1, and P21 expressing Cre under the control of early cardiovascular progenitor transcription factors Mesp1 and Nkx2.5. Images modified with permission from Sereti et al. [67].

Fig. 5.

In-vivo two-photon cortical imaging of mouse brain through an optically cleared intact skull. Before imaging, the skull was topically treated with 10% collagenase (10% EDTA for P21-P30 aged mice) for 5-10 minutes and then 80% glycerol was dropped onto the skull. (a) Orthogonal (x–z) projections of dendrites through the intact skull, before and after skull optical clearing, demonstrating that the depth is obviously enhanced after clearing (the imaging parameter and data processing were the same). Scale bar = 10 μm. (b) The depth when imaging the dendrites of Thy1-YFP neurons, before and after skull optical clearing (P30, n = 10 mice; statistical method: one-way analysis of variance (ANOVA); Po0.001). Images modified with permission from Zhao et al. [75].

Fig. 6.

Transdermal imaging of blood circulation in mouse through optically cleared skin using laser speckle contrast imaging (LSCI). LSCI provides low or high contrast depending if the imaged scatterers are moving or stationary, respectively. Optical clearing was obtained using topical application of polyethylene glycol (PEG-400) alone or in combination with 10% Thiazone as chemical enhancer. Photographs (top row) and laser speckle temporal contrast maps (bottom row) of in vivo rat skin at the initial state, 4, 12, 24, 40 min after treatment of different OCAs, and 2 min after treatment of saline. (a) PEG-400, (b) mixed solution of PEG-400 and Thiazone. (c) Dynamic temporal contrast in five specific areas: 1, 2 in a (treatment with PEG-400), 3, 4, and 5 in b (treatment with a mixture of PEG-400 and Thiazone), before and after the application of optical clearing agents and saline. Here, 1, 3, and 4 are vessel areas, while 2 and 5 are no-vessel areas. Images modified with permission from Zhu et al. [70].