Improved diffuse fluorescence flow cytometer prototype for high sensitivity detection of rare circulating cells in vivo

Abstract. Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×103 cells/mL. In combination, these improvements resulted in an overall cell counting sensitivity of about 1 cell/mL or better in vivo

A Dedicated Low-Cost Fluorescence Microfluidic Device Reader for Point- of-Care Ocular Diagnostics

Microfluidic fluorescence assay devices show great promise as preclinical and clinical diagnostic instruments. Normally, fluorescence signals from microfluidic chips are quantified by analysis of images obtained with a commercial fluorescence microscope. This method is unnecessarily expensive, time consuming, and requires significant operator training, particularly when considering future clinical translation of the technology. In this work, we developed a dedicated low cost fluorescence microfluidic device reader (FMDR) to read sandwich immunofluorescence assay (sIFA) devices configured to detect vascular endothelial growth factor ligand concentrations in ocular fluid samples. Using a series of sIFA calibration standards and a limited set of human ocular fluid samples, we demonstrated that our FMDR reader has similar sensitivity and accuracy to a fluorescence microscope for this task, with significantly lower total cost and reduced reading time. We anticipate that the reader could be used with minor modifications for virtually any fluorescence microfluidic device