DPSS 561 nm yellow-green laser on the BD LSR II
We recently tested a Melles Griot DPSS 561 nm laser on our BD LSR II. This recently developed laser emits in the yellow-green range, at a longer wavelength than traditional DPSS green lasers. Yellow-green 561 nm laser emission more efficiently excited both the R- and B-forms of phycoerythrin, as well as the fluorescent protein DsRed and it more recent variants. Yellow-green excitation also causes produces less autofluorescence in cells, further increasing the signal-to-noise ratio for PE and DsRed detection. Since 561 nm excitation is well-separated from fluorescein and GFP emission, this laser causes virtually no crossover contamination in the green detection channel, allowing simultaneous fluorescein or GFP detection without blocking optics (as are usually required for DPSS 532 nm sources.
(Below). DPSS 561 nm yellow-green solid state laser. This engineering prototype from Melles Griot emits at 10 mW.
(Below). DPSS 561 nm laser installed on the BD LSR II. This laser installed on our BD LSR II in the position normally reserved for our DPSS 532 nm green laser. The laser beam was directed into the path normally used for our HeNe yellow or red laser, which is removed when a DPSS is used. Four-axis rotation and translation allows alignment in addition to the downstream laser optics.
(Below). DPSS 561 nm laser installed on the BD LSR II. Installation schematic for the DPSS 561 nm installation. Beam was directed down the path normally reserved for the HeNe red or yellow lasers (the fourth position).
Phycoerythrin. Although traditionally excited with the 488 nm laser sources found on most flow cytometers, phycoerythrins (PE) excitation maxima actually falls in the green to yellow range of the visible spectra. Green lasers generally provide better PE excitation than 488 nm blue-green sources, and are used in an increasing number of flow cytometers. Changing to a yellow green source like the DPSS 561 nm further increases sensitivity for PE, since this source falls at the maximum cusp of the PE excitation spectra. Green and green-yellow lasers also result in less cellular autufluorescence from endogenous molecules such as riboflavins; this also increases the signla-to-noise ratio for PE detection. In actual practice, DPSS 561 nm excitation increase PE sensitivity several-fold over 488 nm excitation (below). Detection sensitivity of the B-form of phycoerythrin (which has a green-shifted excitation spectrum) was particularly pronounced.
(Below). Excitation-emission spectra for riboflavin and R- phycoerythrin. Green and particularly yellow-green laser sources would be expected to give better PE excitation, as well as reduced autofluorescence from endogenous such as riboflavins.
(Below). R- and B-phyocoerythrin detection with DPSS 561 nm excitation. EL4 cells were labeled with biotin-anti-CD25 or CD90, followed by streptavidin conjugated to either the R- or B-form of phycoerythrin. Cells were then analyzed on the BD LSR II with either 488 nm (top row) or 561 nm (bottom row) excitation, using the same emission filter (610/20 nm). Mean fluorescent intensities for background (black/open peaks) and specific labeling (grey filled peaks) are indicated.
DsRed. Similarly, the fluorescent protein DsRed is usually excited with 488 nm lasers but "prefers" a longer wavelength excitation source (see our section on DsRed). DPSS 561 nm excitation increased sensitivity for DsRed many-fold (below).
(Below). Excitation-emission spectra for DsRed. DPSS 561 nm excitation falls closer to the excitation maxima for DsRed than shorter wavelength sources.
(Below). DsRed detection with DPSS 561 nm excitation. SP2/0 (left histograms) and NIH 3T3 cells (middle and right histograms) constituitively expressing DsRed (BD Clontech) were analyzed on the BD LSR II with either 488 nm (top row) or 561 nm (bottom row) excitation using the same emission filter (610/20 nm). Mean fluorescent intensities for background (black/open peaks) and specific labeling (grey filled peaks) are indicated. For NIH 3T3 cells, detector gain was decreased in the right histograms to bring the 561 nm excited signals on scale.
Simultaneous analysis of fluorescein or GFP. Green lasers on flow cytometers often preclude or complicate the detection of fluorescein or GFP, due to detector contamination by scattered laser light. DPSS 561 nm laser light does not appear to contaminate the fluorescein/GFP detector (530/30 nm bandpass filter).
(Below). Fluorescein/GFP detector status with DPSS 561 nm laser. Fluorescein low-intensity MESF microspheres (Bang Laboratories) were analyzed on the BD LSR II with 488 nm excitation and detection through the fluorescein detector (530/30 nm), with the installed DPSS 561 nm laser both off and on (left and right histogram). DPSS 561 nm laser light had no apparent effect on sensitivity in the green channel based on microsphere resolution.
