A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids.

Akos Diosdi,Maria Harmati,Krisztian Koos,Timea Toth,Filippo Piccinini,Dominik Hirling,Maria Kovacs,Peter Horvath,Krisztina Buzas

doi:10.1016/j.csbj.2021.01.040

Abstract

3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment.

Highlights

Two dimensional (2D) monolayer cell cultures have been used extensively as model systems to evaluate the efficacy of compounds in drug discovery studies for decades
We asked ten microscopy experts to visually evaluate the sharpness of the images and we correlated their evaluations with the results of the seven metrics
Histogram threshold metric distinguished between the optical clearing protocols, but the results did not match the ground truth: the ScaleA2 and the CUBIC protocols did not improve transparency compared to the uncleared group, which is in contrast to the results for the experts’ evaluation

Summary

Introduction

Two dimensional (2D) monolayer cell cultures have been used extensively as model systems to evaluate the efficacy of compounds in drug discovery studies for decades. ⇑ Corresponding author at: Synthetic and Systems Biology Unit, Biological and preserve the structure of the cells, making this model system remarkable for many biological research fields, such as drug discovery, tumor biology or immunotherapy [5,6,7]. Despite their advantages exploited in screening studies, large-scale image acquisition is still challenging in case of 3D samples. Since LSFM provides high imaging speed with remarkably low photobleaching and high penetration depth [11], the technique is well suited for the imaging of large spheroids, typically up to 500 mm [12]

Objectives

Results

Conclusion