Green fiber lasers: an alternative to traditional DPSS green lasers for flow cytometry.

Green and yellow diode-pumped solid-state (DPSS) lasers (532 and 561 nm) have become common fixtures on flow cytometers, due to their efficient excitation of phycoerythrin (PE) and its tandems, and their ability to excite an expanding array of expressible red fluorescent proteins. Nevertheless, they have some disadvantages. DPSS 532-nm lasers emit very close to the fluorescein bandwidth, necessitating optical modifications to permit detection of fluorescein and GFP. DPSS 561-nm lasers likewise emit very close to the PE detection bandwidth and also cause unwanted excitation of APC and its tandems, requiring high levels of crossbeam compensation to reduce spectral overlap into the PE tandems. In this article, we report the development of a new generation of green fiber lasers that can be engineered to emit in the range between 532 and 561 nm. A 550-nm green fiber laser was integrated into both a BD LSR II cuvette and FACSVantage DiVa jet-in-air cell sorter. This laser wavelength avoided both the fluorescein and PE bandwidths and provided better excitation of PE and the red fluorescent proteins DsRed and dTomato than a power-matched 532 nm source. Excitation at 550 nm also caused less incidental excitation of APC and its tandems, reducing the need for crossbeam compensation. Excitation in the 550 nm range, therefore, proved to be a good compromise between 532- and 561-nm sources. Fiber laser technology is, therefore, providing the flexibility necessary for precisely matching laser wavelengths to our flow cytometry applications.