Comparison of Power Technology and Coherent violet diode lasers on the FACSVantage DiVa
We have previously mounted a Power Technology 408 nm violet laser diode (VLD) on our FACSVantage DiVa, replacing the existing water-cooled krypton-ion laser for applications requiring violet excitation (data here). The VLD was found to work as well as the larger krypton-ion for several violet-excited fluorochromes, includign Cascade and Pacific Blue, ELF-97 and Cyan Fluorescence Protein. Coherent now manufacturers a VLD, the VioFlame, which possesses a smaller, more elliptical beam spot and a higher post-circularization power level (25 mW for our laser, compared to an 18 mW maximum for our Power Technology. We mounted both lasers on the FACSVantage and compared their ability to excite several fluorochromes. Despite differing beam shapes and power levels, the lasers performed comparably.
The Power Technology laser was set to 408 nm, 15 mW post-circularization optic power level. The Coherent VioFlame was factory-set to 408 nm, 25 mW post-circularization optic power level. The same bandpass filters were used for all comparisons. Fluorescence on the FACSVantage was collected with a single reflection off a 100% mirror, with no intervening dichroic. Alignments were performed with Polyscience 2 um yellow-green beads. PMT voltage levels were held constant for both laser installations.
(Below). Power Technology (above) or Coherent (below) VLD lasers beam profiles. Profiles were collected with a WinCamD CCD beam profiling system (DataRay, Inc.) with a ND4.0 filter between the laser and the CCD element. Lasers were mounted on the FACSVantage in the beam orientation shown.
(Below). Power Technology (above) or Coherent (below) VLD lasers mounted on the FACSVantage DiVa. The VLDs are mounted in place of the krypton-ion laser that normally occupies this position on the instrument. Power Technology Details on mounting the instrument can be found here (a downloadable document with illustrations and part list is here).
(Below). Cascade Blue and Pacific Blue detection on the FACSVantage DiVa with Power Technology or Coherent VLD excitation. EL4 cells were labeled with biotin-conjugated CD44 or CD90 followed by Cascade Blue or Pacific Blue -conjugated strepavidin. Cells were then analyzed on either the FACSVantage DiVa with the Power Technology (top row) or Coherent VioFlame (bottom row) VLD. Detection was through the same 440/10 nm filter. Median fluorescence intensity (MFI) values are shown at the top of the histograms, and the ratio is in boldface.
(Below). Cascade Blue and Pacific Blue detection on the FACSVantage DiVa with Power Technology or Coherent VLD excitation through a 463/50 nm filter. EL4 cells were labeled with biotin-conjugated CD44 or CD90 followed by Pacific Blue -conjugated strepavidin. Cells were then analyzed on either the FACSVantage DiVa with the Power Technology (top row) or Coherent VioFlame (bottom row) VLD. Detection was through the same 463/50 nm filter sandwiched with 1 488 nm notch filter. Median fluorescence intensity (MFI) values are shown at the top of the histograms, and the ratio is in boldface. This filter was more optimal than the 440/10 above but required the 488 nm notch filter to block 488 nm laser light.
For more information about violet laser diodes on other flow cytometers in the lab, go here.
Go here for the violet laser diode evaluation on the BD LSR II.
We have also evaluated a violet laser diode installed in our Compucyte laser scanning cytometer; the results can be seen here.
VLD mounting information and part list.
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