3D Spheroid Imaging and Analysis

Images were acquired on the Zeiss Axio Observer Z1 microscope, using the 63 × Plan-Apochromat (NA 1.4) oil, the 40 × LD Plan-Neofluar (NA 0.6) and the 20 × Plan-Apochromat (NA 0.8) objectives, the AxioCam MRm Rev.3 camera, and the software package AxioVision version 4.8.2 (all from Zeiss, Göttingen, Germany). Filter sets and related staining were as follows: (1) Alexa Fluor 488—filter set 38 HE, (2) Alexa Fluor 555—filter set 43 HE, (3) Phalloidin-Alexa Fluor 660—filter set 50, and (4) DAPI—filter set 49 (all filter sets from Zeiss). Summary images of spheroids were acquired by fluorescence microscopy in four colors with 35 z-slices per spheroid. Classification of spheroids: Spheroid 3D structure, polarity and lumen formation was determined manually on blinded/masked images by three raters using 4-color z-stacks, as described previously [23 (link)], and discrepant rating discussed, before unmasking and calculation of distribution.