ABSTRACT
Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.
Subject(s)
Cell Separation/instrumentation , Flow Cytometry/instrumentation , Microscopy, Fluorescence, Multiphoton/instrumentation , Animals , Cell Separation/methods , Equipment Design , Equipment Failure Analysis , Female , Flow Cytometry/methods , Humans , Jurkat Cells , Mice , Mice, Nude , Microscopy, Fluorescence, Multiphoton/methodsABSTRACT
To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.
