Violet laser diode excitation on the BD Biosciences LSR II.
The LSR II is equipped with a Coherent VioFlame 408 nm violet diode laser (VLD) in the second position emitting at 25 mW. The VLD is useful for the excitation of the immunophenotyping fluorochromes Cascade Blue and Pacific Blue, Cyan Fluorescence Protein (CFP), and the fluorogenic alkaline phosphatase substrate ELF-97. Incorporation of the VLD into the LSR II allows the addition of violet excited flurochromes for advanced multicolor immunophenotyping, and analysis of multiple fluorescence proteins and fluorescence resonance energy transfer (FRET) using CFP. The VLD and the LSR II collection optics also give surprisingly high-resolution cell cycle analysis using the UV-excited DNA binding dye DAPI. A PMT trigon allows the analysis of up to two violet-excited fluorochromes simultaneously in the default configuration.
(Below). The Coherent VioFlame violet diode laser on the LSC II. The VLD has independent steering optics for easier laser alignment.
(Below). Default configuration for the secondary violet diode detectors. The violet laser diode beam occupies the secondary beam/signal position. Left, the secondary PMT "trigon". Right, the current default configuration for the secondary PMT trigon. This configuration allows simultaneous detection of two blue and green violet-excited fluorochromes, such as Cascade Blue and ELF-97 (illustrated). The "distal" detector slot in the signal path has no PMT or dichroic in the default configuration.
Although the LSR II can detect up to two violet-excited fluorochromes simultaneously in the default position, single fluorochrome detection is best carried out by removing the 505 LP dichroic and sending the signal into the proximal PMT position, since reflection off the dichroic reduces the signal intensity somewhat.
(Below). Analysis of linearity beads in default and modified violet diode trigon configuration. Molecular Probes InSpeck Blue beads were analyzed on the LSR II using VLD excitation and detection through the Cascade Blue 450/50 nm filter. The bead "cocktails" (containing a arbitrary 100% intensity bead population, and populations with decreasing intensity down to 1%, plus unlabeled) were analyzed either with the 505 LP dichroic removed and the 450/50 nm filter placed in front of the the proximal PMT (top row) or with default trigon configuration (bottom row).
Detection of Cascade Blue and Pacific Blue on the LSR II. Cascade Blue and Pacific Blue are violet-excited fluorochromes from Molecular Probes that can be conjugated to antibodies and other proteins. We have previously shown that they are well-excited with low-power violet laser diode sources (see the data here). These fluorochromes also work well on the LSR II. In addition, Cascade Blue or Pacific Blue can be combined with ELF-97 immunolabeling. ELF-97 is a fluorogenic alkaline phosphatase substrate that labels AP-conjugated reagents with a bright green fluorescent precipitate. The emission spectra of ELF-97 is similar to Cascade Yellow, making ELF-97 a potentially useful fluorochrome for polychromatic flow cytometry with UV or violet laser sources.
(Below). Cascade Blue and Pacific Blue detection on the LSR II. EL4 cells were labeled with biotin-conjugated CD44 followed by Cascade Blue or Pacific Blue -conjugated strepavidin. "Cocktails" of unlabeled and labeled cells were then analyzed on the LSR II with the VLD. As demonstrated above, slightly better sensitivity in single color analysis was achieved by removing the intervening dichroic.
(Below). Combined Pacific Blue and ELF-97 detection on the LSR II. EL4 cells were labeled with biotin-conjugated CD44 followed by alkaline phosphatase (AP) or Pacific Blue -conjugated strepavidin. AP-labeled cells were then labeled with ELF-97. "Cocktails" of unlabeled and labeled cells were then analyzed on the LSR II with the VLD (uncompensated cytogram on the left, compensated on the right). Both Cascade Blue and Pacific Blue have good spectral compatibility with ELF-97, requiring minimal compensation.
DNA content analysis with DAPI and the VLD on the LSR II. DAPI is one of the most useful DNA binding dyes for cell cycle analysis, exhibiting relatively low non-specific binding, particularly to RNA. Unfortunely, DAPI excitation requires an ultraviolet laser source, an uncommon item on flow cytometers with quartz flow cells. Nevertheless, several laboratories have observed that DAPI can be reasonably well-excited with violet laser diodes, making the current LSR II configuration potentially useful for measuring DAPI DNA content. The LSR II was therefore tested for DNA content analysis using chicken RBCs, trout RBCs and calf thymus nuclei standards, as well as EL4 cells. DNA content resolution and peak C.V.s were still somewhat poorer than those observed with propidium iodide analysis both on the LSR II itself and on the FACSCalibur, but were within acceptable limits. The future addition of the solid state UV laser to the LSR II bench should ultimately give excellent DNA content resolution for DAPI, based on these preliminary results.
(Below). DNA content analysis with DAPI on the LSR II. Chicken red blood cells (CRBCs), trout red blood cells (TRBCs) and calf thymus nuclei (CTNs) were labeled with DAPI at 5 ug/ml and analyzed on the LSR II with the VLD and the violet diode trigon set up with the dichroic removed and the 450/50 nm in front of the proximal dichroic. Cells were simultaneously analyzed through the LSR II PE channel with 488 nm excitation, and on the BD FACSCalibur with 488 nm excitation and signal collection through the PE channel. Mean fluorescent intensities and C.V.s for the singlet peaks are shown.
(Below). DNA content analysis with DAPI on the LSR II. EL4 cell nuclei were prepared with a solution of NP-40 in PBS and labeled with DAPI at 5 ug/ml, followed by analysis on the LSR II with the VLD and the violet diode trigon set up with the dichroic removed and the 450/50 nm in front of the proximal dichroic. Cells were simultaneously analyzed through the LSR II PE channel with 488 nm excitation, and on the BD FACSCalibur with 488 nm excitation and signal collection through the PE channel. Mean fluorescent intensities and C.V.s for the singlet peaks are shown.
DNA content resolution was lower than with propidium iodide, but surprisingly good given the lower power of the VLD.
Comparison of the Coherent VioFlame VLD on the LSR II to the Power Technology VLD on the FACSVantage DiVa. We have previously mounted a Power Technology 30 mW 408 nm violet diode laser on our FACSVantage DiVa; this configuration gives similar sensitivity to our krypton-ion 407 nm water-cooled laser on the sorter (click here for information and data). Below we've compared the Coherent VioFlame mounted on the LSR II to our Power Technology VLD/FACSVantage DiVa instrument configuration. Sensitivity for Cascade Blue, Pacific Blue and ELF-97 were found to be similar between the two lasers and instruments. Sensitivity for CFP fluorescence was better of the LSRII - this is mainly due to the reduced laser light spillover from the low-power Sapphire laser on the LSR II compared to the more powerful water-cooled argon on the FACSVantage, resulting in lower backgrounds and better signal-to-noise ratios on the LSR II. Detection of both Pacific Blue and CFP on both instruments require a 488 nm notch filter sandwiched with the 463/50 or 485/22 nm emission filters to reduce laser noise and maximize instrument sensitivity.
The same bandpass filters were used for all comparisons. Violet-excited fluoroscence on the LSR II was collected through the most proximal PMT with no intervening dichroic (see above for diagram). Fluorescence on the FACSVantage was also collected with no intervening dichroic.
(Below). Power Technology VLD laser mounted on the FACSVantage DiVa. The VLD is mounted in place of the krypton-ion laser that normally occupies this position on the instrument. Details on mounting the instrument can be found here.
(Below). Cascade Blue and Pacific Blue detection on the FACSVantage DiVa or LSR II with VLD excitation. EL4 cells were labeled with biotin-conjugated CD44 followed by Cascade Blue or Pacific Blue -conjugated strepavidin. Cells were then analyzed on either the FACSVantage DiVa with the Power Technology VLD (top row), or on the LSR II with the Coherent VioFlame (bottom row). 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). Pacific Blue detection. on the FACSVantage DiVa or LSR II with VLD excitation, with a 463/50 nm filter. EL4 cells were labeled with biotin-conjugated CD44 followed by Pacific Blue -conjugated strepavidin. Cells were then analyzed on either the FACSVantage DiVa with the Power Technology VLD (top row), or on the LSR II with the Coherent VioFlame (bottom row). Detection was through a 463/50 nm filter sandwiched with a 488 nm notch filter to block 488 nm laser emission.
(Below). Cyan Fluorescence Protein detection on the FACSVantage DiVa or LSR II with VLD excitation, with a 463/50 nm filter. SP2/0 cells (left and middle histograms) or NIH 3T3 cells (right histograms) expressing enhanced CFP were analyzed on either the FACSVantage DiVa with the Power Technology VLD, or on the LSR II with the Coherent VioFlame, through either a 485/22 nm filter with 488 nm notch filter (top panels), or a 530/30 nm filter (bottom panels). Detection sensitivity was lower on the FACSVantage with the 485/22 nm / 488 nm RB filter combination due to the higher 488 nm laser power and resultant spillover, as indicated by the 530/30 nm analysis, which showed similar sensitivity between instruments.
So, the LSR II was similar in sensitivity to the FACSVantage DiVa for violet laser diode excitation for Cascade Blue and ELF-97. Fluorochromes requiring an emission filter overlapping the 488 nm laser line (including Pacific Blue and CFP) were analyzed with greater sensitivity on instruments with lower 488 nm laser power like the LSR II, although inclusion of a 488 nm notch filter largely obviated this difference.
For more information about violet laser diodes on other flow cytometers in the lab, go here.
Go here to see a comparison of Coherent and Power Technology violet laser diodes on the FACSVantage DiVa.
Click here for information of the violet laser diode on our Compucyte laser scannning cytometer.
Go here to download the LSR II User Handbook.
VLD mounting information and part list.
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