ReViMS: Software Tool for Estimating the Volumes of 3-D Multicellular Spheroids Imaged Using a Light Sheet Fluorescence Microscope

Abstract

Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. However, there remains a lack of validated methods for quantifying the volumes of 3-D multicellular aggregates. Here, we present Reconstruction and Visualization from Multiple Sections (ReViMS), an open-source, user-friendly software for automatically segmenting z-stacks of fluorescence images and estimating the volumes of 3-D multicellular spheroids. To assess the precision and accuracy of the volume estimates obtained with ReViMS, we used several cancer spheroids imaged with LSFM. Both the precision and accuracy were >95%, demonstrating the effectiveness of ReViMS.

Machine learning reveals heterogeneous responses to FAK, Rac, Rho, and Cdc42 inhibition on vascular smooth muscle cell spheroid formation and morphology

“…The automated analysis could be used to cluster or group the spheroids based on their morphologies and would serve as a way to study differences in drug responses that may arise amongst spheroids. Several spheroid image analysis platforms have been developed to automatically analyze high-contrast spheroids imaged with brightfield or fluorescence microscopes to quantify spheroid volumes and morphological parameters [40][41][42]. These softwares are, however, limited in analyzing the morphology of drug-disturbed VSMC spheroids as follows: i) It is very challenging to detect low contrast edges of the disturbed VSMC spheroids from bright-field or phase-contrast images.…”

3D-Cell-Annotator: an open-source active surface tool for single cell segmentation in 3D microscopy images

“…In order to obtain the ground truth for this dataset, we asked three expert annotators to segment the 77 z-stacks. For each stack we averaged their 3D segmentations by utilizing ReViMS (Piccinini et al 2017, De Santis et al 2019), a 3D extension of STAPLE (Warfield et al 2004). We segmented the cells in a semi-automatic way by manually initializing with three contours on the three orthogonal axes, and then visually adjusting the parameters during surface evolution.…”

Open-Source Tools for Volume Estimation of 3D Multicellular Aggregates

“…ReViMS is an open-source, user-friendly software for estimating the volume of cell aggregates with irregular shape and organoids (i.e., multicellular aggregates that self-organise into ex vivo mini-organs [2]) by automatically segmenting single slices of z-stacks of fluorescence images [12]. ReViMS is written in MATLAB and has been designed for biologists: it allows reconstruction of the external surface of a cell aggregate and estimation of its volume without any programming skills.…”

“…In order to assess the accuracy of volume instances estimated with ReViSP, ReViMS, MITK, and OpenSegSPIM, we used the three cancer multicellular aggregates already considered in [12]. These multicellular aggregates were generated using A549 cells (lung adenocarcinoma human cell line, American Type Culture Collection, Rockville, MD, USA).…”

“…In this work, we compared several open-source tools today available for estimating the volume of 3D multicellular aggregates. In addition, we presented a new version of the Reconstruction and Visualization from Multiple Sections (ReViMS version 2.0) tool [12]. Besides providing functions for supporting manual annotation, we implemented: (a) a new active contouring method for automatic object segmentation; and (b) a new module for "averaging" different 2D/3D segmentations, caused for instance by different human annotators or computational algorithms.…”