Quantitative two -photon flow cytometry in vitro and in vivo.

Abstract

Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. Essentially, all flow cytometry systems in use today use single-photon excitation, employing multiple lasers if multiple dyes are required. This thesis presents the first systematic exploration of two-photon excitation in flow cytometry, especially under nonuniform flow conditions. Two-photon enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. In this thesis, we demonstrate that two-photon excitation in conjunction with a targeted multi-dye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or <italic>in vivo.</italic> 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 measurement under nonuniform excitation. In addition, we present a unique two-beam scanning method to conduct cell size measurements in nonuniform flow. A two-beam, two-channel detection and two-photon excitation flow cytometry (T<super>3</super>FC) system is described. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both <italic>in vitro</italic> in a glass capillary and <italic> in vivo</italic> in the blood stream of live mice. The application of two-photon flow cytometry system to monitor externally injected circulating cancer cell dynamics is shown. The technique also allows us to monitor the fluorescent dye labeling dynamics <italic>in vivo.</italic> This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or <italic>in vivo.</italic>