Application of novel laser technology for flow cytometric analysis

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

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. A major focus of our Core is to harness novel laser technology for application to enhanced detection technologies for flow cytometry. Four recent laser technologies now provide the means to minimize or eliminate the gaps in excitation for flow cytometry. First, improvements in diode and 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. Our Core was the first group to employ yellow 561 nm lasers for flow cytometry and continues to improve on this technology. Many visible single wavelength lasers have been similarly evaluated and integrated into accessible cytometers for investigator use. Second, quasi-continuous wave supercontinuum (SC) 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. Our group has integrated SC lasers into flow cytometers, allowing unlimited access to any visible wavelength for fluorochrome excitation. This technology is available to CCR and NCI investigators with specialized analysis needs. Related fiber laser technology, including high gas pressure hollow fiber lasers that produce powerful discrete laser wavelength, have also been investigated and reported as possible multi-wavelength laser sources for flow cytometry. Fourth, deep ultraviolet (DUV) and near infrared (NIR) laser sources are being evaluated to expand the wavelength range of lasers available for flow cytometry, and to expand the fluorochrome range for high-dimensional flow cytometric analysis. Our Core is driving DUV laser development and has devised the mans to integrate these non-standard laser sources into flow cytometers. We are also pursuing fluorescent probe options to provide targets for these laser sources. Once again, this work potentially expands the utility of flow cytometry for both novel fluorescent probes and increasing the reach of polychromatic analysis.