Investigation of the Feasibility of an Optical Imaging System for the Application of In Vivo Flow Cytometry

Abstract

This thesis investigates the feasibility of employing an optical imaging system for the application of in vivo flow cytometry for detecting rare circulating tumor cells (CTCs) in vasculature. This investigation presented used three optical imaging configurations: a Nikon Eclipse E600 fluorescence microscope with a PIXIS 2048B CCD camera; a Nikon Eclipse E600 fluorescence microscope with a ThorLabs DCC 3240N CMOS camera; and a custom built confocal microendoscope with a ThorLabs DCC 3240N CMOS camera. These systems were employed to gain insight as to what signal to noise ratios and sensitivities are required to sufficiently detect fluorescently labeled cancer cells. This work presents general concepts of fluorescence and confocal microscopy, the experimental setups employed, and experimental measurements and results obtained. The experimental measurements involved the following: the simulation of flow cytometry by imaging green fluorescent microspheres, with a fluorescence excitation range of 505-515 nm and a diameter of 15µm, in a square crit tube moving on a translational stage, and imaging a selection of cells that included MCF10A breast cells (non-cancerous), OVCAR3 ovarian cancer cells, and patient derived xenogram (PDX) breast cancer cells, which express folate-receptor proteins on their surface. We fluorescently labeled these cells with the introduction of a new folate-receptor targeted fluorescent contrast agent OTL38, made by On Target Laboratories. The results established that we were able to image and detect fluorescence microspheres with a minimum signal to noise ratio (SNR) of 2.3 using the ThorLabs DCC 3240N camera on the Nikon Fluorescence microscope. We were able to image and detect the cells used on all three system configurations. Analyzing the different cell uptake efficacies of the contrast agent OTL38, we established that the SNR levels were variable when imaging PDX breast cancer cells. We propose future work to investigate possible effects on the variability of SNR results, as well as, and future steps in designing a real-time optical fluorescence imaging system for in vivo flow cytometry.