Development of oblique plane microscopy and its application to time-lapse high-content imaging of live oncogene-expressing spheroids
File(s)
Author(s)
Rowe-Brown, Leo Nathan
Type
Thesis
Abstract
Oblique plane microscopy (OPM) is a technique characterised by its ability to image 3D specimens with low photodamage while being compatible with standard sample-mounting techniques. Here, instrumentation and automation for OPM is presented to achieve automated four-colour imaging, compared to two-colour in the previous implementation. Additionally, an existing sample pre-find method was developed to locate samples in 3D using custom autofocus and spheroid detection scripts.
This OPM setup was used in a collaborative project to acquire four-channel, 3D time-lapse image data of 170–180 spheroids comprising up to three populations of breast cell lines, each expressing different oncogenes: HER2WT, BRAFV600E and HER2D16. An analysis pipeline was developed that segmented nuclei in 3D using Cellpose and performed cell-level, time-resolved analysis of the progression of each cell population. This marks the first instance of studying the competition between cells expressing different oncogenes in ~170 spheroids in parallel.
Finally, the light collection efficiency of OPM and dual-view OPM (dOPM) systems was investigated using a vectorial ray-tracing simulation. Unlike previously published methods, this simulation models the tilt of the mirror and a thin-film mirror coating for the first time. For the dOPM system considered (1st objective 60×/1.2 NA water, 2nd/3rd objective 50×/0.95 NA air) with an OPM angle of 35°, the calculated collection efficiencies relative to the primary microscope alone were (to 3 decimal places) 0.471, 0.331 and 0.329 for excitation polarised in the illumination sheet plane, perpendicular to the plane, and unpolarised respectively. Despite additional losses at the polarising beamsplitter in dOPM, for OPM these were lower at 0.298, 0.248 and 0.277.
Preliminary experimental measurements of relative collection efficiency showed trends similar to the simulations but were 22% lower than simulation after including manufactures’ transmission data for the additional dOPM optics. Further experiment is needed to determine the source of the residual losses.
This OPM setup was used in a collaborative project to acquire four-channel, 3D time-lapse image data of 170–180 spheroids comprising up to three populations of breast cell lines, each expressing different oncogenes: HER2WT, BRAFV600E and HER2D16. An analysis pipeline was developed that segmented nuclei in 3D using Cellpose and performed cell-level, time-resolved analysis of the progression of each cell population. This marks the first instance of studying the competition between cells expressing different oncogenes in ~170 spheroids in parallel.
Finally, the light collection efficiency of OPM and dual-view OPM (dOPM) systems was investigated using a vectorial ray-tracing simulation. Unlike previously published methods, this simulation models the tilt of the mirror and a thin-film mirror coating for the first time. For the dOPM system considered (1st objective 60×/1.2 NA water, 2nd/3rd objective 50×/0.95 NA air) with an OPM angle of 35°, the calculated collection efficiencies relative to the primary microscope alone were (to 3 decimal places) 0.471, 0.331 and 0.329 for excitation polarised in the illumination sheet plane, perpendicular to the plane, and unpolarised respectively. Despite additional losses at the polarising beamsplitter in dOPM, for OPM these were lower at 0.298, 0.248 and 0.277.
Preliminary experimental measurements of relative collection efficiency showed trends similar to the simulations but were 22% lower than simulation after including manufactures’ transmission data for the additional dOPM optics. Further experiment is needed to determine the source of the residual losses.
Version
Open Access
Date Issued
2024-02-28
Date Awarded
01/06/2024
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Dunsby, Chris
Publisher Department
Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)