Integration of a HeNe 594 nm yellow laser in the LSR II.
Red helium neon (633 nm) lasers have become important excitation sources for flow cytometry. They efficiently excite a variety of red-excited fluorochromes, including APC and its tandem conjugates APC-Cy5.5 and APC-Cy7, Cy5, Alexa Fluor 633 and 647, the DiI tracer dyes, the dimeric cyanin DNA binding dye TO-PRO-3, and others. HeNe lasers are generally air-cooled, long-lived and simple to operate and maintain. They have largely supplanted the traditional source of orange/red laser excitation, the rhodamine 6G (R6G) dye head laser. Although the RG6 dye head laser provides a valuable range of excitation lines (590 to 620 nm), it has a number of drawbacks; it requires a powerful argon-ion pump laser, employs carcinogenic laser dyes and requires considerable effort to maintain. Red HeNe lasers (generally 20 mW and higher power level) have therefore become common fixtures on cell sorters and laser scanning cytometers.
HeNe lasers with other wavelengths have also been developed; green (543 nm), yellow (594 nm) and orange (612 nm) HeNe lasers are commercially available. HeNe lasers with these longer wavelengths generally cannot exceeded 5 mW in total power output and have not seen extensive use in flow cytometry. HeNe 543 nm lasers (sometimes termed "GreNe" lasers) have been occasionally installed in both small benchtop instruments and large sorters with flow cell capability; the Beckman Coulter Altra can be equipped with small HeNe 543 nm lasers. Nevertheless, longer wavelength HeNe lasers other than 633 nm (specifically 594 and 612 nm) have seen little use in flow cytometry, despite the potential usefulness of their wavelengths; their emission coincides with that of the R6G dye head laser, the optimal excitation source for many red-excited fluorochromes such as Texas Red and APC. HeNe 633 nm lasers cannot excite Texas Red; in addition, the 632 nm line is not spectrally optimal for APC, which has an excitation maxima at 615 nm. Their small size, modest power and cooling requirements and the durability of small HeNe lasers also makes them good candidates as flow cytometry excitation sources.
We have previously evaluated NeNe 594 and 612 nm lasers on a FACStar Plus stream-in-air flow cytometer. Although functional, the poor light-collecting efficiency of stream-in-air collection optics limits the usefulness of most low-power laser sources for these instruments. The BD LSR II uses a FACSCalibur-style quartz flow cell and a novel design focusing objective with a numerical aperture of 1.2; the sensitivity of these collection optics make successful integration of a low-power laser a much more feasible prospect. We have therefore integrated our HeNe 594 nm into the LSR II in place of the traditonal red HeNe and used it as an excitation sources for a variety of orange- and red-excited fluorochromes.
(Below). HeNe 594 nm laser. We used a stock Melles Griot yellow 594 nm helium-neon laser with maximum measured power output of 3.2 mW. The laser was integrated into the LSR II optical bench in place of the supplied HeNe 633 nm laser and aligned in the fourth laser position..
Excitation of APC and APC tandem conjugates with the HeNe 594 nm laser on the LSR II. The yellow HeNe was was first evaluated for its ability to excite APC and its tandem conjugates, a critical requirement for this source.
(Below). HeNe laser trigon configuration. For this initial evaluation, the LSR II HeNe laser trigon was used in the default configuration, with filters for APC and APC-Cy7 split with a 735 LP dichroic.
(Below). Alignment bead analysis. Polyscience Blue Beads (top histograms), Spherotech rainbow beads (middle) and BD Calibrite APC beads (bottom) were used to align the laser and perform daily quality control evaluations. Bead peak C.V.s and Calibrite unlabeled/APC bead separations were comparable to the normal red HeNe laser.
(Below). Linearity bead analysis with the HeNe 594 nm laser. Molecular Probes InSpeck Deep Red linearity calibration beads were used to assess detector sensitity using the yellow HeNe laser. Cocktails of beads ranging from an arbitrary 100% fluorescence down to 0.3%, plus unlabeled, were analyzed through both the APC and APC-Cy7 detectors (top histograms). Increasing the detector gain until the 1% or 0.3% bead population was at the scale maximum (middle and bottom hostogram respectively) showed excellent separation between these dim bead populations and the unlabeled fraction.
(Below). Two-color analysis of APC and APC-Cy7 or APC-Alexa Fluor 750 with the HeNe 594 nm laser. EL4 cells were labeled with biotin-conjugated CD44 followed by APC, APC-Cy7 or APC-Alexa Fluor 750-conjugated streptavidin, and "cocktails" of unlabeled, APC, APC-Cy7 or APC-Alexa Fluor 750 labeled cells were analyzed using the yellow HeNe laser. The yellow HeNe gave excellent excitation of both APC and the tandems (left histograms). Two-color analysis of unlabeled / APC / APC-Cy7 or unlabeled / APC / APC-Alexa Fluor 750 "cocktails" (right cytograms) was possible with minimal compensation.
(Below). Modified HeNe laser trigon configuration. Evaluation of HeNe 594 nm two-color excitation of APC-Cy5.5 or APC-Alexa Fluor 680 and APC-Cy7 were then carried out using the trigon modification shown below, with a 710/20 nm filter for APC-Cy5.5 or APC-Alexa Fluor 680.
(Below). Two-color analysis of APC-Cy5.5 or Alexa Fluor 680 and APC-Cy7 with the HeNe 594 nm laser. EL4 cells were labeled with biotin-conjugated CD44 followed by APC-Cy5.5, APC-Alexa Fluor 680 or APC-Cy7 -conjugated streptavidin, and "cocktails" of unlabeled, APC-Cy5.5, APC-Alexa Fluor 680 or APC-Cy7 labeled cells were analyzed using the yellow HeNe laser. Once again, the yellow HeNe gave excellent excitation of all APC tandems (left histograms). Two-color analysis of unlabeled / APC-Cy5.5 / APC-Cy7 or unlabeled / APC-Alexa Fluor 680 / APC-Cy7 "cocktails" (right cytograms) was possible with compensation.
The HeNe 594 nm laser could therefore excite APC and its tandem conjugates in two-color combinations. To determine if APC and two tandems could be simultaneously analyzed in a three-color combination, a third PMT was added to the HeNe trigon and the filters modified for three color analysis.
(Below). HeNe laser trigon configuration for three-color analysis. A third PMT was added to the distal detector position of the trigon, and the filters and dichroics modified accordingly.
(Below). Alignment bead analysis. Polyscience Blue Beads (top histograms) and Spherotech rainbow beads (bottom) were used to align the laser and perform daily quality control evaluations. Bead peak C.V.s were comparable to the normal red HeNe laser.
(Below). Linearity bead analysis with the HeNe 594 nm laser. Molecular Probes InSpeck Deep Red linearity calibration beads were used to assess detector sensitity using the yellow HeNe laser. Cocktails of beads ranging from an arbitrary 100% fluorescence down to 0.3%, plus unlabeled, were analyzed through both the APC-Cy7, APC-Cy5.5 and APC detectors (top to bottom).
(Below). Three-color analysis of APC, APC-Cy5.5 and APC-Cy7 with the HeNe 594 nm laser. EL4 cells were labeled with biotin-conjugated CD44 followed by APC, APC-Cy5.5 or APC-Cy7-conjugated streptavidin, and "cocktails" of unlabeled, APC, APC-Cy5.5 and APC-Cy7 labeled cells were analyzed using the yellow HeNe laser. The yellow HeNe still gave excellent excitation of both APC and the tandems (top histrograms). Three-color analysis of unlabeled / APC / APC-Cy5.5 / APC-Cy7 "cocktails" (lower three cytograms) was possible with compensation.
An important problem with using red-excited flurochromes is spectral interference by the fluorochrome PE-Cy5, the phycoerythrin Cy5 tandem conjugate. Since Cy5 is excited by red laser sources such as the red HeNe, its coincidental emission with APC usually requires a large level of color compensation to control spectral bleedover and allow simultaneous use of PE-Cy5 and APC. Since the yellow HeNe emits a shorter wavelength than is optimal for Cy5 excitation, it was expected that APC / PE-Cy5 crossbeam compensation problem should be reduced using this laser. PE-Cy5 bleedover and the resulting crossbeam compensation requirement was therefore evaluated for the APC and APC tandem detectors. Spectral overlap of the fluorochrome PerCP into the APC and APC tandem detectors was similarly evaluated.
(Below). Crossbeam compensation between PE-Cy5 and APC and its tandems. EL4 cells were labeled with biotin-conjugated CD44 followed by PE-Cy5-conjugated streptavidin, and analyzed using the yellow HeNe laser, observing the spectral overlap into the APC and APC tandem detectors. The crossbeam compensation was then adjusted to correct for spectral overlap, and the value noted below. Overlap into the APC (top cytograms), APC-Cy5.5 (middle) and APC-Cy7 (below) are shown, both without (left column) and with compensation (right column).
(Below). Crossbeam compensation between PE-Cy5 and APC and its tandems. As above, EL4 cells were labeled with biotin-conjugated CD44 followed by PerCP-conjugated streptavidin, and analyzed using the yellow HeNe laser, observing the spectral overlap into the APC and APC tandem detectors. The crossbeam compensation was then adjusted to correct for spectral overlap, and the value noted below. Overlap into the APC (top cytograms), APC-Cy5.5 (middle) and APC-Cy7 (below) are shown.
Crossbeam compensation requirements for PE-Cy5 and APC and its tandems using the yellow HeNe were at acceptable levels. As expected, the crossbeam compensation requirements for PerCP were much lower. Comparisons to a red HeNe source are in progress.
Excitation of Texas Red and Alexa Fluor 594 with the HeNe 594 nm laser on the LSR II. The main advantage of the yellow HeNe laser for flow cytometer is the ability to excite Texas Red and its more recent analog Alexa Fluor 594, useful low molecular weight fluorochromes that cannot be excited with red HeNe sources. Texas Red or Alexa Fluor 594 can theoretically be combined with APC and it tandems to do four-color analysis off one yellow HeNe laser, a significant advantage when doing polychromatic flow cytometer (seven colors and beyond).
(Below). HeNe laser trigon configuration for Texas Red or Alexa Fluor 594 detection. To initially evaluate the ability of the yellow HeNe to excite Texas Red and Alexa Fluor 594, all dichroics were removed from the HeNe trigon, and a 630/22 nm filter inserted in front of the proximal detector. Although a 610 nm would be more optimal from a detection perspective, the spectral proximity to the 594 nm line resulted in high backgrounds and necessitated a longer wavelength filter.
(Below). Detection of Texas Red and Alexa Fluor 594 on the LSR II with the HeNe 594 nm laser. The yellow HeNe gave excellent excitation of both Texas Red and Alexa Fluor 594.
Combining Texas Red or Alexa Fluor 594 with APC and its tandems for three- and four-color analysis with the HeNe 594 nm laser on the LSR II. The ability of the yellow HeNe to excite Texas Red and Alexa Fluor 594 opens the possibility of three- and four-color analysis with APC and its tandems. The HeNe trigon is limited to three-color analysis using the configurations shown below. The yellow laser was found to excite Texas Red or Alexa Fluor 594, APC and APC-Cy7 simultaneously with good signal separation and reasonable compensation.
(Below). HeNe laser trigon configuration for Texas Red or Alexa Fluor 594 detection with APC and APC-Cy7. This configuration requires a custom 635 LP dichroic.
(Below). Linearity bead analysis with the HeNe 594 nm laser. Molecular Probes Carmine LinearFlow linearity calibration beads were used to assess detector sensitity using the yellow HeNe laser. Cocktails of beads ranging from an arbitrary 100% fluorescence down to 0.02% were analyzed through both the Texas Red, APC and APC-Cy7 detectors.
(Below). Three-color analysis of Texas Red or Alexa Fluor 594, APC and APC-Cy7 using the HeNe 594 nm laser. EL4 cells were labeled with biotin-conjugated CD44 followed by Texas Red, Alexa Fluor 594, APC or APC-Cy7-conjugated streptavidin, and "cocktails" of unlabeled, Texas Red, APC and APC-Cy7 (left panels) or Alexa Fluor 594, APC and APC-Cy7 (right panels) labeled cells were analyzed using the yellow HeNe laser. A customized 630 LP dichroic was used to split the Texas Red or Alexa Fluor 594 and APC signals.
The yellow laser was also able to excite Texas Red, APC and APC-Cy5.5 simultaneously, also with reaonable compensation levels. This suggests that excitation of four fluorochromes (Texas Red, APC, APC-Cy5.5 and APC-Cy7) should be possible with the replacement of the current trigon with an octagon.
(Below). HeNe laser trigon configuration for Texas Red or Alexa Fluor 594 detection with APC and APC-Cy5.5. Analysis was done with the default 680 LP dichroic shown, or with a custom 695 LO dichroic.
(Below). Linearity bead analysis with the HeNe 594 nm laser. Molecular Probes Carmine LinearFlow linearity calibration beads were used to assess detector sensitity using the yellow HeNe laser. Cocktails of beads ranging from an arbitrary 100% fluorescence down to 0.02% were analyzed through both the Texas Red, APC and APC-Cy5.5 detectors. 680 LP (upper row) or 695 LP (lower row) dichroics were used to separate the APC and APC-Cy5.5 signals.
(Below). Three-color analysis of Texas Red or Alexa Fluor 594, APC and APC-Cy5.5 using the HeNe 594 nm laser. EL4 cells were labeled with biotin-conjugated CD44 followed by Texas Red, APC or APC-Cy5.5-conjugated streptavidin, and "cocktails" of unlabeled, Texas Red, APC and APC-Cy.5 labeled cells were analyzed using the yellow HeNe laser. 680 LP (left panels) or 695 LP (right panels) dichroics were used to separate the APC and APC-Cy5.5 signals.
(Below). Proposed HeNe laser octagon configuration for Texas Red or Alexa Fluor 594 detection with APC, APC-Cy5.5 and APC-Cy7. We are planning to substitute a PMT octagon for the existing trigon, for four-color analysis with the yellow HeNe.
Configuration of this design is in progress.
Excitation of low molecular weight red-excited fluorochromes with the HeNe 594 nm laser on the LSR II. One possible drawback of the yellow HeNe laser is its reduced ability to excite several low molecular weight red-excited fluorochromes, including Cy5 and Alexa Fluor 633 and Alexa Fluor 647. The results show some reduction in sensitivity for these probes compared to the red HeNe, which should be taken into account in both flurochrome and laser choice.
(Below). Cy5, Alexa Fluor 633 and Alexa Fluor 647 excitation with the NeNe 594 nm laser. EL4 cells were labeled with biotin-conjugated CD44 followed by APC, Cy5, Alexa Fluor 633 or Alexa Fluior 647 conjugated streptavidin, and analyzed as "cocktails" with unlabeled cells with either the HeNe 594 laser (top histograms) or the HeNe 632 nm (bottom).
Click here to view information on the standard JDS Uniphase red 633 nm HeNe laser that comes with the LSR II.
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