Integration of a HeNe 543 nm green laser on the LSR II.

Although red 633 nm HeNe lasers have by far seen the most use in flow cytometry, HeNe lasers with other emission wavelengths are also available, including green (543 nm), yellow (594 nm) and orange (612 nm). These HeNe lasers generally do not exceed 5 mW in total power output and have seen only occasional use in flow cytometry.  HeNe 543 nm lasers (sometimes termed "GreNe" lasers) have been occasionally are sometimes installed on both both small benchtop instruments and large sorters with quartz cuvettes; the Beckman Coulter Altra can be equipped with small HeNe 543 nm lasers.   Nevertheless, HeNe lasers other than 633 nm have seen little use in flow cytometry, despite the potential usefulness of their wavelengths; the green HeNe (GreNe) would be particularly useful for flow cytometry.  Green excitation is more optimal for phycoerythrin (PE) excitation than 488 nm blue-green excitation, and 543 nm laser light can excite a wide variety of low molecular weight fluorochromes, including tetramethylrhodamine (TRITC or TAMRA), Cy3, Alexa Fluor 532, Alexa Fluor 546 and Alexa Fluor 555.  Green laser light is also provides more efficient excitation of the expressible protein DsRed.

We have previously evaluated HeNe 594 and 612 nm lasers on a FACStar Plus stream-in-air flow cytometer, and are routinely using a higher-power HeNe 594 nm laser on our LSR II.  Although low-power HeNe lasers are functional on stream-in-air cytometers like the FACStar Plus, 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 low-power lasers a much more feasible prospect,  as illustrated by our yellow HeNe installation.   We have therefore similarly integrated a HeNe 543 nm into the LSR II in place of the traditional red HeNe and used it as an excitation sources for a variety of green-excited fluorochromes.  Excitation of phycoerythrin was found to be much more efficient with this laser than with 488 nm sources, and also gave excellent excitation of other green-excited fluorochromes like Cy3 and DsRed. 

(Below). GreNe 546 nm laser.  We used a stock Research Electro-Optics green 543 nm helium-neon laser with maximum measured power output of 3.0 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..  A 5X beam expansion optic was used to increase the beam cross-section.

(Below).  Green HeNe laser 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.  Beam profiles are shown for the uncorrected beam (left image) or after modification with a 5X beam expansion optic (Newport Instruments).

Excitation of PE, PE tandem conjugates and green-excited low molecular weight fluorochromes with the HeNe 543 nm laser on the LSR II.  The green HeNe was first evaluated for its ability the R- and B-forms of phycoerythrin (PE), as well as several green-excited low molecular weight fluorochromes.  It was then compared to another green excitation source (a Kr 530 nm laser) on the FACSVantage DiVa, and the DPSS 488 nm laser on the same LSR II. 

(Below). HeNe laser trigon configuration.  For this initial evaluation, the LSR II green HeNe was steered into the "red" trigon, outfitted with a 575/26 nm filter in the APC-Cy7 position with no intervening dichroic.

(Below). Linearity bead analysis with the HeNe 543 nm laser.  Molecular Probes Linear Check Carmine linearity calibration beads were used to assess detector sensitivity using the green HeNe laser.  Cocktails of beads ranging from an arbitrary 100% fluorescence down to 0.02% were analyzed by signal transmission directly into "red" trigon APC-Cy7 PMT (no intervening dichroic).

(Below). Comparison of HeNe 543 nm and Kr 530 nm excitation of PE and other green-excited fluorochromes.   The above cells were analyzed either on the LSR II with the green HeNe laser (top row), or on the FACSVantage DiVa with a krypton-ion laser tuned to 530 nm 50 mW output.  Sensitivity with the HeNe 543 nm excitation on the cuvette instrument was found to be superior to higher-power Kr green excitation on the stream-in-air instrument.

(Below). Comparison of HeNe 543 nm and DPSS 488 nm excitation of PE and other green-excited fluorochromes on the LSR II.   The above cells were then analyzed on the LSR II with both the DPSS 488 nm primary laser source emitting at 20 mW output (top row) and the green HeNe laser (bottom row).  PE sensitivity with the HeNe 543 nm excitation was found to be much better than with the DPSS 488 nm source, despite the significant power level differences.

(Below). Comparison of HeNe 543 nm and DPSS 488 nm excitation of PE tandem conjugates on the LSR II.   EL4 cells labeled with biotin-anti-CD44 followed by PE-Cy5, PE-Cy5.5 or PE-Cy7-conjugated streptavidin were then analyzed on the LSR II with both the DPSS 488 nm primary laser source emitting at 20 mW output (top row) and the green HeNe laser (bottom row).  PE sensitivity with the HeNe 543 nm excitation was also found to be much better than with the DPSS 488 nm source.

The green HeNe therefore gives excellent excitation of PE, better than that achieved with a more powerful 488 nm source.  Green excitation sources also tend to excite lower levels of autofluorescence in cells, resulting in higher singal-to-noise ratios.

Multicolor analysis of PE and PE-tandem conjugates with the HeNe 543 nm laser on the LSR II.  Since the green HeNe laser was found to be extremely efficient for PE excitation, multicolor analysis of PE, PE-Cy5.5 and PE-Cy7 was evaluated.   The "red" trigon on the LSR II was modified accordingly with three PMT detectors and the appropriate filters.

(Below). HeNe 543 nm 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). Linearity bead analysis with the HeNe 543 nm laser.  Molecular Probes Linear Check Carmine linearity calibration beads were used to assess detector sensitivity using the green HeNe laser.  Cocktails of beads ranging from an arbitrary 100% fluorescence down to 0.02% were analyzed through both the PE-Cy7, PE-Cy5.5 and PE-Cy5 detectors (left to right).

(Below). Three-color analysis of PE-Cy7, PE-Cy5.5 and PE-Cy5 with the HeNe 543 nm laser.  EL4 cells were labeled with biotin-conjugated CD44 followed by PE-Cy7, PE-Cy5.5 or PE-Cy5-conjugated streptavidin, and a "cocktail" of unlabeled, PE-Cy7, PE-Cy5.5 and PE-Cy5 labeled cells were analyzed using the green HeNe laser.  The green HeNe gave adequate excitation of all tandems.

Analysis of DsRed with the HeNe 543 nm laser on the LSR II.  Although the expressible fluorescent protein DsRed can be excited with a 488 nm laser, best results are obtained with green and yellow laser sources (see data here).  The HeNe 543 nm laser was found to be an excellent excitation source for DsRed on the LSR II, giving similar or better results than other green excitation sources.

(Below). DsRed detection with the HeNe 543 nm laser using different filters.  NIH 3T3 or SP/2 cells expressing DsRed (Clontech) were analyzed on the LSR II wsith the green HeNe laser using 575/26, 585/42 or 610/20 nm filters.  Best results were obtained with the 610/20 nm filter as observed previously.

(Below). DsRed detection with the HeNe 543 nm laser on the LSR II or the Kr 530 nm laser on the FACSVantage DiVa.  NIH 3T3 or SP/2 cells expressing DsRed (Clontech) were analyzed both ont he LSR II with HeNe 543 nm excitation, or on the FACSVantage DiVa with krypton-ion 530 nm excitation.  A 575/26 nm was used in both cases.

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