Development of the in vivo flow cytometer

Abstract

An in vivo flow cytometer has been developed that allows the real-time detection and quantification of circulating cells containing fluorescent proteins or labeled with fluorochrome molecules in live animals, without the need to extract blood samples. A stationary laser beam is focused by a cylindrical lens to a slit of light that is then demagnified and focused across a blood vessel by an achromat and microscope objective. Fluorescent cells are excited one by one as they flow through the excitation laser light slit, creating a burst of fluorescence whose width is inversely proportional to their velocity. The fluorescence signal is detected through a confocal slit aperture using a photomultiplier tube. The analog signal from the photomultiplier tube is then digitized, filtered, and recorded as a function of time onto a computer. Computer programs post- process the data for the presence of cell signal, as well as various aspects of the cell signal such as height, width, and temporal location of the signal peak. Two in vivo flow cytometers have been built: a single-slit, single-color system and a two- slit, two-color system. The single-slit, single-color system provides excitation at 632 nm, and the two-slit, two-color system provides excitation at 632 nm and 473 nm. The two- slit, two-color system can operate in several different modes: single-slit at 632 nm or 473 nm, double-slit at 632 nm or 473 nm, and double-slit with one excitation slit at 632 nm and the other at 473 nm.

(cont.) Thus far, the single-slit, single-color system has been used to study the circulation kinetics of different prostate cancer and leukemia cell lines with different metastatic potential, as well as the effect of different host environments (i.e., mouse versus rat). In addition, the device has been used to develop a new in vivo labeling method of white blood cells that does not result in significant depletion of the labeled cells, allowing for the possibility of autoimmune and transplant rejection studies. The two-slit, two-color system is being used to track two different cell populations, or one cell population labeled with two different markers, one of which can be the green fluorescent protein.