Automated Microscopy for 3D Organoid Analysis

Detailed image acquisition and analysis procedures for acquiring images in 96-well plates were previously described [73 (link)]. Bright field microscopy was conducted using a retrofitted Nikon Eclipse Ti-U microscope using a ×4 objective lens and a motorized stage (Prior Scientific, H117). Bright field image stacks for each individual well were captured using a Nikon DS-Ri2 camera (Nikon Instruments Inc., MQA17000) driven by the NIS-Elements software (Nikon). The depth images of each FOV (well) were then stacked using the EDF algorithm in FIJI. Images were taken on most days for 9 days. The processed images were curated manually with the objects identified labelled either as S (spheroids) or O (organoids). The parameters were measured by drawing a line around the object. The average counts per well and the proportion of organoids were calculated.

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Nguyen P.M., Dagley L.F., Preaudet A., Lam N., Giam M., Fung K.Y., Aizel K., van Duijneveldt G., Tan C.W., Hirokawa Y., Yip H.Y., Love C.G., Poh A.R., Cruz A.D., Burstroem C., Feltham R., Abdirahman S.M., Meiselbach K., Low R.R., Palmieri M., Ernst M., Webb A.I., Burgess T., Sieber O.M., Bouillet P, & Putoczki T.L. (2019). Loss of Bcl-G, a Bcl-2 family member, augments the development of inflammation-associated colorectal cancer. Cell Death and Differentiation, 27(2), 742-757.

Publication 2019

Eclipse Ti-U microscope DS-Ri2 camera NIS-Elements software

Lens Microscope Organoids

Corresponding Organization :

Other organizations : Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Olivia Newton-John Cancer Wellness & Research Centre, La Trobe University

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Variable analysis

independent variables

Microscopy technique (bright field microscopy)

dependent variables

Counts of spheroids and organoids per well
Proportion of organoids

control variables

Objective lens (×4)
Motorized stage (Prior Scientific, H117)
Camera (Nikon DS-Ri2)
Imaging software (NIS-Elements)
Image processing software (FIJI)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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