Application of novel laser technology for flow cytometric analysis

Telford, William

Abstract

Flow cytometers rely almost exclusively on lasers as a source of excitation for fluorescent probes. While the coherence and power level of lasers makes them ideal sources for illuminating individual cells, their discrete wavelengths limit the range of visible light that is available for fluorescent probe excitation. Even the most modern flow cytometers typically provide no more than four discrete laser wavelengths, with the traditional blue-green 488 nm and red 633 to 640 nm lines being the most common. Even with these multi-laser instruments, coverage of the ultraviolet-to-infrared spectrum is far from complete, with large excitation gaps that exclude many useful fluorescent probes. This is in large part due to the limited number of wavelengths available from existing laser technology that is compatible with fluorescence instrumentation. Three recent laser technologies now provide the means to minimize or eliminate the gaps in excitation for flow cytometry. First, improvements in diode-pumped solid state (DPSS) laser technology now provide an enormous variety of discrete laser lines, many applicable for flow cytometric analysis. Green 532 nm lasers and yellow-green 561 nm lasers are becoming common fixtures on cytometers; improvements in cavity design and doping are increasing this wavelength range even further into the yellow to orange range, and below 488 nm into the blue. The development of fiber lasers, where an easily doped fiber optic constitutes the lasing cavity, has also increased the range of wavelengths available from solid state sources. Second, quasi-continuous wave supercontinuum white light lasers are now available that emit continuously from the violet to the infrared, allowing selective filtering of the wavelength of interest for excitation purposes. Third, hollow fiber laser technology is allowing the design of specific fiber lasers to emit specific series of wavelengths, allowing directed design of laser sources for flow cytometry. In this project, we evaluate a wide varity of novel laser technology, including solid-state diode and diode-pumped (DPSS) laser technology, fiber lasers, and non-linear supercontinuum laser technology. If applicable for flow cytometry and relevant to the needs of CCR investigators, this laser technology is integrated into flow cytometric instrumentation and validated in real-life biomedical applications. This validated technology is then made available to CCR investigators.