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 light-scattering 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.
Highlights
The results demonstrated that the best protocol for the spheroids immunofluorescence labeling included: (a) fixation of the spheroids during 15 min with 4% paraformaldehyde at room temperature (RT); (b) detergent‐based permeabilization by Triton X‐100, during 15 min, at RT; (c) blockage of unspecific binding sites with a solution of 0.1% bovine serum albumin, 0.2% Triton X‐100, 0.05% Tween‐20% and 10% goat serum for 1 hr at RT; (d) incubation of the first antibody at 37°C during 18–24 hr; (e) incubation of the second antibody at 37°C during 4 hr; (f) dehydration and clearing by benzyl alcohol/benzyl benzoate (BABB) during 15 min; and (g) imaging by mDSLM (Smyrek & Stelzer, 2017)
For more than 100 years, optical clearing methods have been used for the clearing of large biological samples obtained from animals
Optical clearing methods started to be explored for allowing the whole imaging of large 3D spheroids, as reviewed in this article
Summary
To perform the imaging of intact spheroids researchers can employ optical clearing methods before samples are analyzed by microscopy. Grist et al (2016) applied SeeDB to clear MCF‐7 breast cancer spheroids with 370 ± 90 μm of the diameter that were transfected with fluorescent ubiquitination‐based cell cycle indicator (FUCCI) for imaging by using 2PM.
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