The use of UV lasers in fluorescent activated cell sorter analysis

Abstract

Flow cytometry and cell sorting are well-established technologies to identify, analyze and/or sort different populations of cells in basic biological research and clinical diagnosis in pathology. This technology makes use of the principles of light scattering by particles crossing a beam of light, and excitation and fluorescence emission of fluorochromes attached to specific molecules or expressed by cells. All flow cytometers are able to interrogate cells and generate data, but only cell sorters can sort cells in a second stage where electrostatic cell sorting takes place. Flow cytometers and cell sorters use combined systems of fluidics, optics, electronics, and computers. The fluidics system in a typical bench top flow cytometer is used to introduce and focus the particles of interest into the light source (laser) for interrogation. The excitation optics consists of a laser and lenses to shape and focus the beam. The numbers of lasers available in flow cytometry vary. Argon gas is commonly used as an excitation laser. The energized light produced by these lasers emit at 488nm. Many different fluorochromes absorb at 488nm and emit light of longer wavelength in the green (535 nm), orange (585 nm) and red spectrum (600 nm). However, ultraviolet (UV) sensitive or far red fluorochromes require excitation lasers (UV or red diode) that emit in the UV (300-400 nm) or far red (630 nm) range. Typical bench top systems have two lasers (argon and red diode). Addition of a Krypton laser provides additional color capabilities in the violet range. All of these lasers will be broadly addressed in this chapter. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors, intensively focused on UV lasers, which contribute to the performance of these instruments. The remaining sections will focus on clinical-and research-based applications of flow cytometry and cell sorting. © 2014 by Nova Science Publishers, Inc. All rights reserved.