Violet laser diode upgrade on the CompuCyte Laser Scanning Cytometer (LSC).
Our CompuCyte LSC has been upgraded with a 405 nm violet diode laser (VLD). This laser is capable of exciting a variety of UV- and violet-excited fluorochromes, including Cascade and Pacific Blue, Cyan Fluorescence Protein and the fluorogenic alkaline phosphatase substrate ELF-97. It can also excite UV-stimulated DNA dyes, including DAPI, Hoechst 33342 and 33258, and the violet-shifted DNA dye Hoechst 34580. Cell cycle analysis can be carried out on the LSC using DAPI, although at a somewhat lower resolution than with true UV excitation.
(Below). Power Technology 30 mW violet laser diode. The laser shown below is identical to violet diode laser mounted on the LSC.
(Below). Lasers on the LSC. The LSC is equipped with a 25 mW argon-ion 488 nm, a 17 mW HeNe 632 nm, and the 15 mW violet diode 305 nm laser. Left photo, the violet diode laser (lower right) and steering optics. Right photo, lasers on. The red HeNe beam is visible and originates from below the steering optics. The violet diode beam is visible perpendicular to the HeNe beam.
Violet-excited bead standards on the LSC: sensitivity and linearity. We use the Molecular Probes InSpeck Blue linearity check beads to determine both detector linearity and relative sensitivity of flow and image cytometers with UV or violet laser sources. The results below show that the PMT response for these standards is linear when excited with the violet laser. The sensitivity based on the lowest fluorescence bead population detectable is similar to the FACSVantage equiped with a similar violet diode laser.
(Below). Molecular Probes InSpeck Blue linearity check beads on the LSC. InSpeck Blue beads were prepared as a cocktail of six beads with descending fluorescent intensities (from an arbitrary 100% down to 0.3%) plus unlabeled beads, then wet-mounted and scanned on the LSC using the violet diode laser. The 10% bead population was the lowest intensity easily distinguishable from the lower-intensity and unlabeled beads. The three brightest bead populations showed good detector linearity.
(Below). Molecular Probes InSpeck Blue linearity check beads on the LSC. Left panel. Although the 10% population was the lowest easily distinguishable bead population with LSC VLD excitation, the 3% and 10% bead populations could be resolved as well (upper left histogram). The unlabeled and 3% bead populations could also be resolved. Right panel. InSpeck bead analysis on the FACSVantage SE, with either a 407 nm krypton-ion laser emitting at 15 mW, or a 408 nm violet diode laser emitting at 15 mW. Sensitivity for this bead standard was comparable between instruments.
Violet-excited DNA content analysis on the LSC. Both suspension and adherent cells can be analyzed for stoichiometric cell cycle on the LSC using the dye binding dyes DAPI or Hoechst 34580. Analysis of DNA standards including chick erythrocytes, trout ejrythrocytes and calf thymus nuclei give both adequate signal C.V.s and DNA content ratios. While cell cycle resolution on the LSC is not comparable to flow cell-based flow cytometers (such as the FACSCalibur or the LSR), it is similar to stream-in-air sorters like the FACSVantage DiVa for UV-excited DNA dyes. DAPI appears to be preferable to Hoechst 34580 for the best DNA content resolution, even with violet excitation.
(Below). DNA content standard analysis on the LSC. Chick erythrocytes (CRBCs), calf thymus nuclei (CTNs) or trout erythrocytes (TRBCs) were labeled with either DAPI or Hoechst 34580 at 5 ug/ml and analyzed as wet mounts on the LSC with violet diode excitation. DNA max pixel versus integral cytograms, and DNA integral histograms are shown with the signlet peak C.V.s indicated.
(Below). DNA content standard linearity on the LSC. Chicken and trout erythrocytes have approximately 30% and 70% DNA content of calf thymus nuclei, respectively. Analysis of DNA content standards with the violet diode at the same detector voltage showed that the PMT was linear across the range of the standards using violet excitation, particularly for DAPI labeling.
(Below). DNA content standard linearity on the LSC. Chicken erythrocyte singlets, doublets, triplets, etc. provide another good linearity check for flow cytometers. This standard also confirmed the linearity of the LSC PMT for violet-excited DAPI DNA labeling.
(Below). L929 cell cycle analysis on the LSC. In the top row below, L929 cells were removed from their tissue culture flask, lysed with NP-40 in PBS to isolate nuclei, then labeled with DAPI at 5 ug/ml in PBS. In the middle and bottom rows, L929 cells were grown up on tissue culture slides, treated with 70% EtOH while still attached to the slides, then labeled with DAPI (middle row) or Hoechst 34580. Cells were then analyzed on the LSC. Although suspension nuclei gave the best cell cycle, attached cells treated with ethanol still gave reasonable resolution..
(Below). Relocation analysis of daughter/apoptotic cells on the LSC. EL4 cells were ethanol treated and labeled with Hoechst 34580 at 5 ug/ml followed by wet mounting and SLC anaysis using the violet laser diode. Cells with G1 DNA content based on Hoechst 34580 integral measurement but increased max pixel signal were then gated and relocated. As previously observed for other DNA probes, these cells are distinguished by their elevated max pixel signal as either daughter cells immediately post-division or apoptotic cells.
These results indicate that the LSC can be used for cell cycle analysis in situations where cell removal from their substrate is detrimental to the prep, or for small numbers of cells.
Violet-excited Cyan Fluorescent Protein (CFP) on the LSC. Cyan Fluorescent Protein (CFP) is an important expressible fluorescent protein used in expression studies and, more recently, as a donor for FRET analysis. CFP is reasonably well excited by violet laser sources (flow cytometric data here). The VLD on the LSC can also excite CFP, and can be used in tandem with the 488 nm laser to do three-color FP analysis of GFP, DsRed and CFP simultaneuously.
(Below). CFP and muliple FP analysis on the LSC. Left panel, a cocktail of NIH 3T3 cells expressing either an empty vector or enhanced CFP (details here). Cells were analyzed with the DAPI filter block containing a 463/50 nm bandpass filter and VLD excitation. Right panel, a cocktail of no FP, GFP, DsRed or CFP-expressing NIH 3T3 cells analyzed on the LSC using dual 488 nm/405 nm excitation. GFP was detected through the FITC filter block (530/30 nm bandpass) and DsRed through the PI filter block (625/28 nm bandpass). Compensation was performed through the software. Done in collaboration with Teresa Hawley and Robert Hawley, American Red Cross Holland Laboratory, Rockville, MD.
ELF-97 on the LSC. Cascade Blue and Pacific Blue are both relatively bright fluorochromes used with violet laser sources on traditional flow cytometers. Unfortunately, both probes are susceptible to photobleaching (like FITC), limiting their usefulness on the LSC. However, the fluorogenic alkaline phosphatase substrate ELF-97 is far more photostable and gives good sensitivity for surface marker detection on the LSC. The FITC filter block was used in place of the DAPI block for ELF-97 detection.
(Below). ELF-97 detection on the LSC. Mouse EL4 cells were labeled with either biotin-anti-CD44 or -CD90 followed by streptavidin conjugated alkaline phosphatase and ELF-97 substrate. Analysis on the LSC was with VLD excitation through the FITC filter block (530/30 nm bandpass).
Click here for more general information about violet laser diodes in flow cytometry.