Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Abstract

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a lightscattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI;(b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted. K E Y W O R D Simaging, light scattering, optical clearing, optical microscopy, spheroids.

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

“… N ( Jianru et al., 2015 ) RTF Triethanolamine, formamide A Embryonic brain, embryo. N Short (h-1 d) Y Y ( Yu et al., 2018 ) Scale Urea, Triton X-, and glycerol A Mouse embryo, heart N Very long N.D. N ( Hama et al., 2011 ) ScaleA2 Urea, glycerol, Triton X- A Mouse brain Expansion Long (w) Incompatible with tropomyosin IHC N ( Decroix et al., 2015 ) ( Richardson and Lichtman, 2015 ) ScaleS Sorbitol-based Scale A Mouse brain Slight expansion Medium (6 d) Y N ( Hama et al., 2015 ) ( Wan et al., 2018 ) ScaleSQ 9.1 M Urea, 22.5% (w/w) sorbitol A Mouse brain Strong expansion Short (h) Y Y (slightly) ( Wan et al., 2018 ) ( Costa et al., 2019 ) SCM CLARITY with a larger concentration of VA-044 O Mouse heart Expansion Long (2–3 w) Y N ( Matryba et al., 2019 ; Richardson and Lichtman, 2017 ) sDISCO Hydroxytoluene, THF, BABB, DBE, propyl gallate O Mouse brain Shrinkage (30% approx.) Medium (1 w) N.D. N ( Hahn et al., 2019 ) SeeDB 80.2...…”

Toward Better Medical Diagnosis: Tissue Optical Clearing

“…The fact that glycerol has a refractive index about 1.4 makes the use of a solution of glycerol and water (80% of glycerol, n = 1.44) suitable for tissue optical clearing. This clarifies the basic idea of using aqueous solutions for tissue clearing [14]. Glycerol based solution is not the only effective approach but also sugar solutions including glucose, fructose and sucrose have been widely used as aqueous clearing agents [15].…”

“…Benzyl Alcohol, Benzyl Benzoate (BAAB) [11], 3D Imaging of Solvent-Cleared Organs (3DISCO) [12] and Polyethylene (PEG)-Associated Solvent System (PEGASOS) [13] are the most common organic solvent-based clearing techniques. Although the organic agents can make the tissue sample transparent in a relatively short time "may be hours", this category suffers from some drawbacks such as high toxicity, tissue shrinkage and endogenous fluorescence quenching [4] [14].…”

Improvement of light penetration in biological tissue using an ultrasound-induced heating tunnel

“…The diameters smaller than (diameter = 1 mm), equal to (diameter = 2 mm), and larger than (diameter = 4, 8, 16 mm) the beam width of the incident light were simulated. The relationship between the fluence improvement and temperature rise (5,10, and 20 ℃) was investigated by setting the initial temperature of the cube from 37, 32, and 22 ℃ (the scatter coefficient (μs) of the cube = 15.7, 16.6, 18.4 mm-1 followed the experiment results of Cletus 15 ). On the other hand, during the HIFU heating process, the actual heating area will not be a homogeneous region and instead will be a multilayer shape with different temperature gradient 30 and the max temperature increasing can only reach 10 ℃ considering not to induce the inertial cavitation effect in the scattering medium.…”

“…The existing methods for decreasing scattering can typically be divided into two types: wavefront shaping 6-8 and optical clearing [9][10][11] . In a wavefront shaping process, ultrasound could be used as a guide to modulate and refocus light.…”