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

Abstract

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 TM cuvette and FACSVantage DiVa TM 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. Published 2009 Wiley-Liss, Inc. (1-4). A good example of this is the recent trend toward the incorporation of green and yellow laser sources into flow cytometers. Diode-pumped solid-state (DPSS) 532-nm green lasers are now common fixtures on modern flow cytometers (5). These lasers are mainly used to replace the traditional 488 nm excitation of phycoerythrin (PE) and its tandems. While 488-nm laser sources have traditionally been used to excite both fluorescein and PE for multicolor flow cytometry, this wavelength does not provide optimal excitation of PE, acting instead on a minor shoulder in the PE excitation spectrum. Green 532-nm laser light emits closer to the excitation maximum for PE, resulting in substantially improved signal-to-background ratios and detection sensitivity for PE, and in particular the PE tandem conjugates. Green lasers also excite less cellular autofluorescence, further improving fluorochrome sensitivity. Finally, green lasers provide substantially better excitation of the shorter wavelength red fluorescent proteins than traditional 488-nm sources, including DsRed and dTomato, and can excite other useful probes like the rhodamine derivatives (6,7).

Novel performance of fiber lasers: tunable operation in visible range

“…For applications the most interesting are the possibilities to use these lasers as light sources in confocal microscopy and flow cytometry. The first tests have shown very good results [15]. Fig.…”

Near‐ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation

“…All fluorescence compensation was done automatically using the spillover algorithms in FlowJo. Labeling levels were expressed using staining indices (SIs) calculated as previously described (16).…”

Half-Watt single frequency yellow 561 nm and yellow-green 553 nm DPSS lasers with record 19% optical conversion efficiency

“…The 561 nm wavelength was proposed several years ago, but it appears that a shorter wavelength, such as 553 nm could be in some cases a better compromise [1]. It is clear that both wavelengths will be used in flow cytometry.…”

“…Flow cytometry requires wavelengths in the range of 550 nm to 565 nm in order to excite phycoerythrin (PE) and its tandems and red fluorescent proteins such as DsRed and dTomato and to avoid fluorescence bandwidth of fluorescein and GFP [1]. The 561 nm wavelength was proposed several years ago, but it appears that a shorter wavelength, such as 553 nm could be in some cases a better compromise [1].…”