Original Article
Near-Ultraviolet Laser Diodes for Brilliant Ultraviolet Fluorophore Excitation William G. Telford*
Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received 16 February 2015; Revised 5 April 2015; Accepted 15 April 2015 Additional Supporting Information may be found in the online version of this article. *Correspondence to: William Telford, Ph.D., Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10-CRC Room 3-3297 East, 10 Center Drive, Bethesda, MD 20892, USA. E-mail: [email protected] Preliminary data from this project were presented at the CYTO 2015 International Society for the Advancement of Cytometry conference in Glasgow Scotland, June 23–27, 2015. The author has no financial interests in any company or technology cited in this study. Published online 30 April 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22686 Published 2015 Wiley Periodicals Inc. on behalf of ISAC.
Cytometry Part A 87A: 11271137, 2015
Abstract Although multiple lasers are now standard equipment on most modern flow cytometers, ultraviolet (UV) lasers (325–365 nm) remain an uncommon excitation source for cytometry. Nd:YVO4 frequency-tripled diode pumped solid-state lasers emitting at 355 nm are now the primary means of providing UV excitation on multilaser flow cytometers. Although a number of UV excited fluorochromes are available for flow cytometry, the cost of solid-state UV lasers remains prohibitively high, limiting their use to all but the most sophisticated multilaser instruments. The recent introduction of the brilliant ultraviolet (BUV) series of fluorochromes for cell surface marker detection and their importance in increasing the number of simultaneous parameters for high-dimensional analysis has increased the urgency of including UV sources in cytometer designs; however, these lasers remain expensive. Near-UV laser diodes (NUVLDs), a direct diode laser source emitting in the 370–380 nm range, have been previously validated for flow cytometric analysis of most UV-excited probes, including quantum nanocrystals, the Hoechst dyes, and 40 ,6-diamidino-2-phenylindole. However, they remain a little-used laser source for cytometry, despite their significantly lower cost. In this study, the ability of NUVLDs to excite the BUV dyes was assessed, along with their compatibility with simultaneous brilliant violet (BV) labeling. A NUVLD emitting at 375 nm was found to excite most of the available BUV dyes at least as well as a UV 355 nm source. This slightly longer wavelength did produce some unwanted excitation of BV dyes, but at sufficiently low levels to require minimal additional compensation. NUVLDs are compact, relatively inexpensive lasers that have higher power levels than the newest generation of small 355 nm lasers. They can, therefore, make a useful, cost-effective substitute for traditional UV lasers in multicolor analysis involving the BUV and BV dyes. Published 2015 Wiley Periodicals, Inc. This article is a US Government work and is in the public domain in the United States of America.
Key terms flow cytometer; ultraviolet laser; near-ultraviolet laser diode; NUVLD; brilliant ultraviolet dye
MODERN flow cytometers rely on multiple laser wavelengths to excite the wide variety of fluorescent probes available for cellular analysis (1). In addition to the traditional blue–green 488 nm laser wavelength red lasers, usually red laser diodes in the 635–645 nm range and violet laser diodes, which emit between 400 and 410 nm, are the most frequent additions (2–4). Violet laser diodes have been particularly useful for increasing the number of fluorescent probes that can be used simultaneously (5). The brilliant violet (BV) series of fluorochromes, recently developed by BD Sirigen (San Diego, CA), are a series of synthetic fluorescent polymers that can be excited by violet laser diodes and emit from the long violet range to the long red. The seven BV dyes (BV421, BV510, BV570, BV605, BV650, BV711, and BV785) are spectrally compatible with each other and can be used for simultaneous labeling. With these probes, simultaneous high-dimensional 17-color analysis can be practically achieved using flow cytometers equipped with only blue–green, red, and violet lasers. The increasing number of characterized cell surface markers on immune cells has led to a need for the analysis of even larger numbers of simultaneous parameters.
Original Article The brilliant ultraviolet (BUV) series of fluorescent probes, also from Sirigen, has recently been developed as an additional set of fluorescent to be added to the existing array of fluorophores. These dyes can be excited using an ultraviolet laser in the 325– 365 nm range. At the time of writing this article, four BUV dyes have been commercially released, including BUV395, BUV496, BUV737, and BUV805. This set of probes can allow the addition of four additional fluorescent parameters to existing immunophenotyping panels. Using these and other fluorochromes, high-dimensional panels with up to 27 fluorescent parameters have been achieved, with higher numbers being developed. The practical use of the BUV dyes is nevertheless complicated by the absence of ultraviolet lasers on most commercial flow cytometers. Earlier flow cytometers were equipped with argon-ion, krypton-ion, or helium–cadmium lasers that could be tuned to an ultraviolet line in the 325–365 nm range. These large ion lasers allowed the analysis of ultraviolet excited fluorescent probes like the calcium indicator indo-1 and the DNA binding dyes Hoechst 33258, Hoechst 33342, and 4,6-diamidino-2-phenylindole, permitting their use early in the history of flow cytometry (6–10). These water-cooled ion lasers were expensive and required extensive maintenance; as a result, they were and remain uncommon on commercial instruments. The recent revolution in solid-state laser technology has resulted in many improvements for flow cytometer; lasers are now smaller, cheaper, easier to use, and far more reliable (8). Ultraviolet lasers on flow cytometers are now almost always Nd:YVO4 frequency-tripled diode pumped solid state (DPSS) lasers emitting at 355 nm, and are available on high-end research systems. This wavelength is ideal for all ultravioletexcited fluorescent probes, including the BUV dyes. However, even solid-state ultraviolet lasers remain a very expensive option for flow cytometer. The comparatively low cost of violet laser diodes (about 1/10th the cost of a UV 355 nm laser) greatly aided their widespread introduction into “standard” cytometer systems, where they are now almost universal. This has not yet happened with the ultraviolet laser. Like the violet laser diode, there is a simpler direct diode laser alternative to traditional UV lasers available in the nearultraviolet range. Near-ultraviolet laser diodes (NUVLDs) were developed shortly after the violet laser diode and have similar semiconductor chemistry (11). NUVLDs emit in the 370–385 nm range, somewhat longer in wavelength than that of Nd:YVO4 DPSS units. Like violet laser diodes, they are easier to manufacture than DPSS lasers, can be built in very small packages, and are far less expensive than DPSS lasers. NUVLDs are currently available in single-mode laser modules emitting up to 50 mW, and multimode modules that exceed 100 mW, power levels appropriate for both cuvette and jet-inair cytometer systems. Despite their slightly longer wavelength, NUVLDs have been validated for use in flow cytometry for most UV-excited fluorophores (12). They excite quantum nanocrystals, Hoechst 33258 and 33342 and 40 ,6-diamidino-2-phenylindole-labeled cells as well as more conventional ultraviolet lasers (13–15). They have been used for Hoechst side population detection of stem cells in mouse bone marrow. NUVLDs 1128
are currently available as an option on several high-end cytometer systems. However, they have not seen widespread usage as an ultraviolet laser substitute. Although they can be used to excite almost all ultraviolet-excited fluorescent probes, there is one exception: the intracellular calcium chelator indo1. The calcium-bound and calcium-free forms of indo-1 emit at 390 nm and 500 nm, respectively. The emission range for the calcium-bound chelator is, therefore, too close to the laser wavelength, resulting in poor excitation (12,13). Historically, cytometer manufacturers have used indo-1 detection as a legacy requirement for ultraviolet laser excitation, because it was once a widely used fluorescent probe. Although it is used far less today, the indo-1 benchmark remains, and NUVLDs have not been widely adopted as an ultraviolet laser substitute. The development of the BUV range of fluorescent probes has caused renewed urgency for an inexpensive ultraviolet laser source. In this study, we compared traditional DPSS ultraviolet lasers with NUVLD sources as excitation sources for the BUV dyes. We also determined whether the slightly longer NUVLD wavelength produced any unwanted BV excitation, potentially complicating multicolor panel development with both dye series. This study showed that NUVLDs were excellent alternatives to traditional solid-state UV sources for this application.
MATERIALS AND METHODS Cells Mouse splenocytes and human peripheral blood mononuclear cells (PBMCs) were used for both single fluorochrome labeling and multicolor panels in this study. Mouse splenocytes were obtained from young (6–18 weeks old) C57B/6 randomly selected male and female mice maintained in the NIH Animal Facility and euthanized according to NIH guidelines. Human PBMCs were obtained from discarded clinical apheresis flow-through products from the NIH Division of Transfusion Medicine regulated by NIH guidelines, frozen, stored in vapor phase liquid nitrogen, and thawed for use in each experiment. Antibodies and Labeling The following fluorochrome-conjugated anti-mouse antibodies were used in this study: BUV395 anti-mouse CD4 (clone GK1.5, BD Pharmingen, Torrey Pines, CA); BUV496 anti-mouse/human B220/CD45R (BD Pharmingen); biotinconjugated anti-mouse CD44 (clone IM7, BD Pharmingen), BUV737 anti-human CD4 (clone OKT4, BioLegend, San Diego, CA), biotin anti-human CD3 (clone OKT3, BD Pharmingen), BUV805 anti-human CD20 (clone 2H7, BD Pharmingen), BV421 anti-mouse CD3 (clone 145-2C11, BioLegend), BV510 anti-mouse CD8a (clone 53-6.7, BioLegend), BV711 anti-mouse/human CD11b (clone M1/70, BioLegend), BV785 anti-human CD45RO (clone UCHL1, BioLegend), BUV737 streptavidin (BD Pharmingen), and BUV805 streptavidin (BD Pharmingen). Cells were labeled in phosphate-buffered saline (PBS) containing 2% fetal bovine serum at 48C, and washed with this buffer by centrifugation at 400g for 5 min. Human cells were labeled with all antibodies NUVLD Lasers for BUV Dye Excitation
Original Article simultaneously. Mouse splenocytes were labeled individually with PBS/2% fetal bovine serum centrifugation washes between labels. Cells were fixed with 2% paraformaldehyde in PBS and analyzed within 24 h. Some labeling experiments were carried out using antibody-coupled capture microspheres (VersaChrome beads, Beckman-Coulter, Miami, FL).
Figure 1. Optical configuration for BD Biosciences LSR II used in this study. Two PMT trigon clusters were aligned with either the UV 355 nm or NUVLD 375 nm laser, and a violet laser diode emitting at 405 nm. Filters and dichroics are shown and were configured as described in the Materials and Methods section.
Flow Cytometry All analyses were carried out on a LSR II flow cytometer (BD Biosciences, San Jose, CA) equipped with a mode-locked Nd:YVO4 frequency-tripled 355 nm laser emitting at 20 mW (ILX Lightwave/Newport, Bozeman, MT), a NUVLD 375 nm emitting at 11 mW (Power Technology, Alexander, AR) and a violet laser diode 405 nm 50 mW (Melles Griot, Carlsbad, CA). The UV laser and NUVLD were aligned on the same optical path to the same fiber-coupled pinhole and detector trigon cluster on the LSR II. The violet laser diode was aligned to a separate detector cluster. The NUVLD was equipped with a 375/8 nm “clean-up” bandpass filter directly in front of the laser output (Semrock, Rochester, NY). Laser power was confirmed with a UV-enhanced silicon photodiode detector and
Figure 2. Comparison of BUV395, BUV496, and BUV737 excitations using labeled mouse splenocytes and either the UV 355 nm laser (top three rows) or NUVLD 375 nm laser (bottom row). Gray traces depict unlabeled cells, and black traces depict labeled cells. Filters are as indicated in Figure 1 and the Materials and Methods section. The staining index for each positive peak is indicated in each histogram. Staining indices for BUV 395 anti-mouse CD4 and BUV496 anti-mouse CD45R are for the positive peak only, and BUV737 anti-mouse CD44 is for the entire population.
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Figure 3. (a) Optical configuration for determining incidental excitation of BV421 and BV510 by UV laser source. (b) Excitation of BV421 and BV510 labeled VersaChrome compensation microspheres by the UV 355 nm laser (top dotplot), NUVLD 375 nm (middle dotplot), or violet laser diode 405 nm (bottom dotplot). Staining indices for each axis are shown to the left of each dotplot. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
power meter (Newport, Irvine, CA), and laser alignment was confirmed with Spherotech Rainbow 8 microspheres (Spherotech, Lake Forest, IL). Both UV 355 nm and NUVLD lasers were aligned to the same detector trigon equipped with indicated filters. BUV395 was detected using either a 390/18 nm bandpass filter for the UV 355 nm laser, or a 405/30 nm bandpass filter for both laser sources, and a 405 nm notch filter to block incidental violet laser light. BUV495 was detected with a 515/30 nm bandpass filter, and BUV737 with a 740/13 nm filter (Semrock). BUV395 and BUV496 signals were separated with a 450-nm longpass dichroic, and BUV496 and BUV737 with a 640 longpass dichroic (Semrock). For these experiments, BV421 was detected through a 450/50 nm filter and BV510 through a 530/30 nm filter, separated with a 505 nm longpass dichroic. For simultaneous BUV737 and BUV805 detection, BUV805 was detected through a 819/44 nm bandpass filter, and separated from BUV737 using a 750 longpass dichroic (Semrock). For BUV737/BUV805/BV711/BV785 experiments, BV711 was detected through a 710/50 nm filter and BV785 through a 780/60 nm filter, with a 735 nm longpass dichroic. For some 1130
experiments, the BV421 and BV510 filters above were installed in the detector cluster normally used for BUV detection, to assess incidental excitation of the BV dyes by the UV 355 nm laser and NUVLD sources. For these same experiments, the violet laser was also aligned to the UV/ NUVLD detector cluster for comparison purposes. For all experiments, PMT voltage levels were kept fixed between comparisons. All data were acquired in DiVa 5.0.3 (BD Biosciences) and analyzed using FlowJo ver. 7.6.5 for Windows (FlowJo LLC, Ashland, OR). All fluorescence compensation was done automatically using the spillover algorithms in FlowJo. Labeling levels were expressed using staining indices (SIs) calculated as previously described (16).
RESULTS The flow cytometer in this study was equipped with both a solid-state Nd:YVO4 frequency-tripled 355 nm laser emitting at 20 mW and a NUVLD 375 nm emitting at 11 mW. Either laser could be switched on as needed, and were both aligned to the same fiber coupled pinhole behind the flow cell, and NUVLD Lasers for BUV Dye Excitation
Original Article
Figure 4. Five-color labeling of mouse splenocytes using directly conjugated antibodies with BUV395, BUV496, BUV737, BV421, and BV510. (a) Analysis with UV 355 nm laser and a 390/18 nm filter for BUV395. (b) Analysis with UV 355 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. (c) Analysis with NUVLD 375 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. Spillover matrices for each analysis are shown below the dotplots. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the same set of three PMT detectors (arranged in a trigon cluster customarily used on BD Biosciences cytometers). In addition, this instrument had a violet laser diode 405 nm installed and emitting at 50 mW, aligned to a separate pinhole Cytometry Part A 87A: 11271137, 2015
and separate set of PMTs. The optical configuration for this instrument is shown in Figure 1. Using this optical layout, it was possible to compare not only the UV 355 nm laser and NUVLD for BUV dye excitation, but also the incidental 1131
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Figure 4. Continued
excitation and spillover of spectrally adjacent BV 421 and 510 dyes into the BUV detectors. Alignment for all these lasers was verified using Spherotech 8 Rainbow microspheres prior to each experiment (data not shown). The ability of both the traditional UV 355 nm laser and NUVLD to excite BUV395, BUV496, and BUV737 labeled mouse splenocytes is shown in Figure 2. Labeling efficiency 1132
was expressed as SI (see Materials and Methods section), shown with each histogram. In the top row, cells were excited with the UV 355 nm laser source and detected through a 390/ 18 nm filter usual for BUV395, and no other optics. Because a violet 405 nm laser was present in the system, a 405 nm restriction bandpass (RB) or notch filter was then included in the laser path to exclude any violet laser light spillover into NUVLD Lasers for BUV Dye Excitation
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Figure 4. Continued
the BUV395 detector; these data are shown in the second row. No violet laser light spillover was, therefore, present using the 390/18 nm filter. Although a 390/18 nm filter would work with the 355 nm laser, its bandwidth would be too close to a NUVLD 375 nm laser. Therefore, a 405/30 nm filter was substituted for Cytometry Part A 87A: 11271137, 2015
it, along with a 405 nm RB blocker described earlier (third row). With the 355 nm laser, this new filter and blocker worked as well as the 390/18 nm filter (third row). When the NUVLD was substituted for the UV 355 nm laser in the fourth row, sensitivity was at least equal to or slightly better that with the 355 nm source (bottom row). It should be noted that the 1133
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Figure 5. (a) Optical configuration for LSR II for simultaneous analysis of BUV737, BUV805, BV711, and BV785. (b) Comparison of BUV737 and BUV805 excitation using labeled mouse splenocytes and either the UV 355 nm laser (top row) or NUVLD 375 nm laser (bottom row) using the above filters. Left column BUV737 anti-CD4; middle and right columns, BUV anti-mouse CD3 and CD20 labeling. Gray traces depict unlabeled cells and black traces depict labeled cells. The staining index for each positive peak is indicated in each histogram. Staining indices for each labeling are shown. (c) Four-color labeling of human PBMCs using directly conjugated antibodies with BUV737, BUV805, BV711, and BV785 using UV 355 nm laser (top dotplots) or NUVLD 375 nm laser (bottom dotplots). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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NUVLD Lasers for BUV Dye Excitation
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Figure 5. Continued
NUVLD emitted at a lower power level than the 355 nm laser (11 mW versus 20 mW) still demonstrated equal or better excitation of all three BUV dyes. This was also shown using a series of anti-human BUV conjugates used to label human PBMCs (Supporting Information Figure S1). This high degree Cytometry Part A 87A: 11271137, 2015
of excitation even with lower laser power levels may be due to the intrinsically high efficiency of the BUV dyes. The replacement of the conventional 390/18 nm filter with a relatively wide 405/30 nm filter was necessary for the NUVLD, and a 405 nm RB filter was essential to keep violet laser light out of 1135
Original Article the BUV395 detector. The NUVLD was also equipped with a 375/8 nm clean-up filter. These modifications were critical for NUVLD use in a multilaser system. Although the NUVLD gave excellent BUV dye excitation, its slightly longer wavelength ran the risk of exciting the BV dyes better than the 355 nm line. This would be an undesirable situation, because increased incidental excitation of the BV dyes would likely increase their spillover into adjacent BUV detectors, increasing the requirement for fluorescence compensation or spillover between these probes. The problem is illustrated in Figure 3. BV421- and BV510-labeled antibody capture beads were analyzed using 355 nm (top dotplot), NUVLD 375 nm (middle dotplot), or violet 405 nm (bottom dotplot) laser light. For these experiments, all lasers were aligned to the same detector cluster equipped with BV421 (450/50 nm) and BV510 (530/30 nm) filters. As expected, the violet laser gave excellent excitation of both BV421 and BV510 (as indicated by the SIs on each plot), whereas the 355 nm laser excited both dyes poorly. The NUVLD 375 nm laser, however, gave suboptimal excitation, but greater than the 355 nm source. The NUVLD did, therefore, cause unwanted excitation of the BV dyes, which would increase signal overlap into the adjacent BUV detectors. The substitution of a longer and wider 405/30 nm filter for BUV395 also increased the possibility of increased spillover of BV421 and BV510 signals into the BUV395 and BUV496 detectors, as well as less separation between BUV395 and BUV496. To determine whether this overlap and the filter changes for BUV395 would be problematic for multicolor analysis, mouse splenocytes were labeled with a five-color antibody panel consisting of BUV395, BUV496, BUV737, BV421, and BV510 conjugated antibodies and analyzed on the LSRII using the detector configuration shown in Figure 1. The results are shown in Figure 4. Figure 4a shows the five-color panel with UV 355 nm excitation using a 390/18 nm filter for BUV395; Figure 4b with UV 355 nm excitation and a 405/30 nm BUV395 filter with a 405 nm RB blocker; and Figure 4c for NUVLD 375 nm excitation with the same 405/30 nm filter and blocker. The spillover values are shown for each panel in the accompanying tables. First, the staining pattern is almost identical for all three excitation and filter conditions. The NUVLD laser data could not be distinguished from the UV 355 nm results. The NUVLD with filter modification did produce a higher level of BV421 spillover into the BUV 395 detector, from
Near-Ultraviolet Laser Diodes for Brilliant Ultraviolet Fluorophore Excitation William G. Telford*
Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received 16 February 2015; Revised 5 April 2015; Accepted 15 April 2015 Additional Supporting Information may be found in the online version of this article. *Correspondence to: William Telford, Ph.D., Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10-CRC Room 3-3297 East, 10 Center Drive, Bethesda, MD 20892, USA. E-mail: [email protected] Preliminary data from this project were presented at the CYTO 2015 International Society for the Advancement of Cytometry conference in Glasgow Scotland, June 23–27, 2015. The author has no financial interests in any company or technology cited in this study. Published online 30 April 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22686 Published 2015 Wiley Periodicals Inc. on behalf of ISAC.
Cytometry Part A 87A: 11271137, 2015
Abstract Although multiple lasers are now standard equipment on most modern flow cytometers, ultraviolet (UV) lasers (325–365 nm) remain an uncommon excitation source for cytometry. Nd:YVO4 frequency-tripled diode pumped solid-state lasers emitting at 355 nm are now the primary means of providing UV excitation on multilaser flow cytometers. Although a number of UV excited fluorochromes are available for flow cytometry, the cost of solid-state UV lasers remains prohibitively high, limiting their use to all but the most sophisticated multilaser instruments. The recent introduction of the brilliant ultraviolet (BUV) series of fluorochromes for cell surface marker detection and their importance in increasing the number of simultaneous parameters for high-dimensional analysis has increased the urgency of including UV sources in cytometer designs; however, these lasers remain expensive. Near-UV laser diodes (NUVLDs), a direct diode laser source emitting in the 370–380 nm range, have been previously validated for flow cytometric analysis of most UV-excited probes, including quantum nanocrystals, the Hoechst dyes, and 40 ,6-diamidino-2-phenylindole. However, they remain a little-used laser source for cytometry, despite their significantly lower cost. In this study, the ability of NUVLDs to excite the BUV dyes was assessed, along with their compatibility with simultaneous brilliant violet (BV) labeling. A NUVLD emitting at 375 nm was found to excite most of the available BUV dyes at least as well as a UV 355 nm source. This slightly longer wavelength did produce some unwanted excitation of BV dyes, but at sufficiently low levels to require minimal additional compensation. NUVLDs are compact, relatively inexpensive lasers that have higher power levels than the newest generation of small 355 nm lasers. They can, therefore, make a useful, cost-effective substitute for traditional UV lasers in multicolor analysis involving the BUV and BV dyes. Published 2015 Wiley Periodicals, Inc. This article is a US Government work and is in the public domain in the United States of America.
Key terms flow cytometer; ultraviolet laser; near-ultraviolet laser diode; NUVLD; brilliant ultraviolet dye
MODERN flow cytometers rely on multiple laser wavelengths to excite the wide variety of fluorescent probes available for cellular analysis (1). In addition to the traditional blue–green 488 nm laser wavelength red lasers, usually red laser diodes in the 635–645 nm range and violet laser diodes, which emit between 400 and 410 nm, are the most frequent additions (2–4). Violet laser diodes have been particularly useful for increasing the number of fluorescent probes that can be used simultaneously (5). The brilliant violet (BV) series of fluorochromes, recently developed by BD Sirigen (San Diego, CA), are a series of synthetic fluorescent polymers that can be excited by violet laser diodes and emit from the long violet range to the long red. The seven BV dyes (BV421, BV510, BV570, BV605, BV650, BV711, and BV785) are spectrally compatible with each other and can be used for simultaneous labeling. With these probes, simultaneous high-dimensional 17-color analysis can be practically achieved using flow cytometers equipped with only blue–green, red, and violet lasers. The increasing number of characterized cell surface markers on immune cells has led to a need for the analysis of even larger numbers of simultaneous parameters.
Original Article The brilliant ultraviolet (BUV) series of fluorescent probes, also from Sirigen, has recently been developed as an additional set of fluorescent to be added to the existing array of fluorophores. These dyes can be excited using an ultraviolet laser in the 325– 365 nm range. At the time of writing this article, four BUV dyes have been commercially released, including BUV395, BUV496, BUV737, and BUV805. This set of probes can allow the addition of four additional fluorescent parameters to existing immunophenotyping panels. Using these and other fluorochromes, high-dimensional panels with up to 27 fluorescent parameters have been achieved, with higher numbers being developed. The practical use of the BUV dyes is nevertheless complicated by the absence of ultraviolet lasers on most commercial flow cytometers. Earlier flow cytometers were equipped with argon-ion, krypton-ion, or helium–cadmium lasers that could be tuned to an ultraviolet line in the 325–365 nm range. These large ion lasers allowed the analysis of ultraviolet excited fluorescent probes like the calcium indicator indo-1 and the DNA binding dyes Hoechst 33258, Hoechst 33342, and 4,6-diamidino-2-phenylindole, permitting their use early in the history of flow cytometry (6–10). These water-cooled ion lasers were expensive and required extensive maintenance; as a result, they were and remain uncommon on commercial instruments. The recent revolution in solid-state laser technology has resulted in many improvements for flow cytometer; lasers are now smaller, cheaper, easier to use, and far more reliable (8). Ultraviolet lasers on flow cytometers are now almost always Nd:YVO4 frequency-tripled diode pumped solid state (DPSS) lasers emitting at 355 nm, and are available on high-end research systems. This wavelength is ideal for all ultravioletexcited fluorescent probes, including the BUV dyes. However, even solid-state ultraviolet lasers remain a very expensive option for flow cytometer. The comparatively low cost of violet laser diodes (about 1/10th the cost of a UV 355 nm laser) greatly aided their widespread introduction into “standard” cytometer systems, where they are now almost universal. This has not yet happened with the ultraviolet laser. Like the violet laser diode, there is a simpler direct diode laser alternative to traditional UV lasers available in the nearultraviolet range. Near-ultraviolet laser diodes (NUVLDs) were developed shortly after the violet laser diode and have similar semiconductor chemistry (11). NUVLDs emit in the 370–385 nm range, somewhat longer in wavelength than that of Nd:YVO4 DPSS units. Like violet laser diodes, they are easier to manufacture than DPSS lasers, can be built in very small packages, and are far less expensive than DPSS lasers. NUVLDs are currently available in single-mode laser modules emitting up to 50 mW, and multimode modules that exceed 100 mW, power levels appropriate for both cuvette and jet-inair cytometer systems. Despite their slightly longer wavelength, NUVLDs have been validated for use in flow cytometry for most UV-excited fluorophores (12). They excite quantum nanocrystals, Hoechst 33258 and 33342 and 40 ,6-diamidino-2-phenylindole-labeled cells as well as more conventional ultraviolet lasers (13–15). They have been used for Hoechst side population detection of stem cells in mouse bone marrow. NUVLDs 1128
are currently available as an option on several high-end cytometer systems. However, they have not seen widespread usage as an ultraviolet laser substitute. Although they can be used to excite almost all ultraviolet-excited fluorescent probes, there is one exception: the intracellular calcium chelator indo1. The calcium-bound and calcium-free forms of indo-1 emit at 390 nm and 500 nm, respectively. The emission range for the calcium-bound chelator is, therefore, too close to the laser wavelength, resulting in poor excitation (12,13). Historically, cytometer manufacturers have used indo-1 detection as a legacy requirement for ultraviolet laser excitation, because it was once a widely used fluorescent probe. Although it is used far less today, the indo-1 benchmark remains, and NUVLDs have not been widely adopted as an ultraviolet laser substitute. The development of the BUV range of fluorescent probes has caused renewed urgency for an inexpensive ultraviolet laser source. In this study, we compared traditional DPSS ultraviolet lasers with NUVLD sources as excitation sources for the BUV dyes. We also determined whether the slightly longer NUVLD wavelength produced any unwanted BV excitation, potentially complicating multicolor panel development with both dye series. This study showed that NUVLDs were excellent alternatives to traditional solid-state UV sources for this application.
MATERIALS AND METHODS Cells Mouse splenocytes and human peripheral blood mononuclear cells (PBMCs) were used for both single fluorochrome labeling and multicolor panels in this study. Mouse splenocytes were obtained from young (6–18 weeks old) C57B/6 randomly selected male and female mice maintained in the NIH Animal Facility and euthanized according to NIH guidelines. Human PBMCs were obtained from discarded clinical apheresis flow-through products from the NIH Division of Transfusion Medicine regulated by NIH guidelines, frozen, stored in vapor phase liquid nitrogen, and thawed for use in each experiment. Antibodies and Labeling The following fluorochrome-conjugated anti-mouse antibodies were used in this study: BUV395 anti-mouse CD4 (clone GK1.5, BD Pharmingen, Torrey Pines, CA); BUV496 anti-mouse/human B220/CD45R (BD Pharmingen); biotinconjugated anti-mouse CD44 (clone IM7, BD Pharmingen), BUV737 anti-human CD4 (clone OKT4, BioLegend, San Diego, CA), biotin anti-human CD3 (clone OKT3, BD Pharmingen), BUV805 anti-human CD20 (clone 2H7, BD Pharmingen), BV421 anti-mouse CD3 (clone 145-2C11, BioLegend), BV510 anti-mouse CD8a (clone 53-6.7, BioLegend), BV711 anti-mouse/human CD11b (clone M1/70, BioLegend), BV785 anti-human CD45RO (clone UCHL1, BioLegend), BUV737 streptavidin (BD Pharmingen), and BUV805 streptavidin (BD Pharmingen). Cells were labeled in phosphate-buffered saline (PBS) containing 2% fetal bovine serum at 48C, and washed with this buffer by centrifugation at 400g for 5 min. Human cells were labeled with all antibodies NUVLD Lasers for BUV Dye Excitation
Original Article simultaneously. Mouse splenocytes were labeled individually with PBS/2% fetal bovine serum centrifugation washes between labels. Cells were fixed with 2% paraformaldehyde in PBS and analyzed within 24 h. Some labeling experiments were carried out using antibody-coupled capture microspheres (VersaChrome beads, Beckman-Coulter, Miami, FL).
Figure 1. Optical configuration for BD Biosciences LSR II used in this study. Two PMT trigon clusters were aligned with either the UV 355 nm or NUVLD 375 nm laser, and a violet laser diode emitting at 405 nm. Filters and dichroics are shown and were configured as described in the Materials and Methods section.
Flow Cytometry All analyses were carried out on a LSR II flow cytometer (BD Biosciences, San Jose, CA) equipped with a mode-locked Nd:YVO4 frequency-tripled 355 nm laser emitting at 20 mW (ILX Lightwave/Newport, Bozeman, MT), a NUVLD 375 nm emitting at 11 mW (Power Technology, Alexander, AR) and a violet laser diode 405 nm 50 mW (Melles Griot, Carlsbad, CA). The UV laser and NUVLD were aligned on the same optical path to the same fiber-coupled pinhole and detector trigon cluster on the LSR II. The violet laser diode was aligned to a separate detector cluster. The NUVLD was equipped with a 375/8 nm “clean-up” bandpass filter directly in front of the laser output (Semrock, Rochester, NY). Laser power was confirmed with a UV-enhanced silicon photodiode detector and
Figure 2. Comparison of BUV395, BUV496, and BUV737 excitations using labeled mouse splenocytes and either the UV 355 nm laser (top three rows) or NUVLD 375 nm laser (bottom row). Gray traces depict unlabeled cells, and black traces depict labeled cells. Filters are as indicated in Figure 1 and the Materials and Methods section. The staining index for each positive peak is indicated in each histogram. Staining indices for BUV 395 anti-mouse CD4 and BUV496 anti-mouse CD45R are for the positive peak only, and BUV737 anti-mouse CD44 is for the entire population.
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Figure 3. (a) Optical configuration for determining incidental excitation of BV421 and BV510 by UV laser source. (b) Excitation of BV421 and BV510 labeled VersaChrome compensation microspheres by the UV 355 nm laser (top dotplot), NUVLD 375 nm (middle dotplot), or violet laser diode 405 nm (bottom dotplot). Staining indices for each axis are shown to the left of each dotplot. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
power meter (Newport, Irvine, CA), and laser alignment was confirmed with Spherotech Rainbow 8 microspheres (Spherotech, Lake Forest, IL). Both UV 355 nm and NUVLD lasers were aligned to the same detector trigon equipped with indicated filters. BUV395 was detected using either a 390/18 nm bandpass filter for the UV 355 nm laser, or a 405/30 nm bandpass filter for both laser sources, and a 405 nm notch filter to block incidental violet laser light. BUV495 was detected with a 515/30 nm bandpass filter, and BUV737 with a 740/13 nm filter (Semrock). BUV395 and BUV496 signals were separated with a 450-nm longpass dichroic, and BUV496 and BUV737 with a 640 longpass dichroic (Semrock). For these experiments, BV421 was detected through a 450/50 nm filter and BV510 through a 530/30 nm filter, separated with a 505 nm longpass dichroic. For simultaneous BUV737 and BUV805 detection, BUV805 was detected through a 819/44 nm bandpass filter, and separated from BUV737 using a 750 longpass dichroic (Semrock). For BUV737/BUV805/BV711/BV785 experiments, BV711 was detected through a 710/50 nm filter and BV785 through a 780/60 nm filter, with a 735 nm longpass dichroic. For some 1130
experiments, the BV421 and BV510 filters above were installed in the detector cluster normally used for BUV detection, to assess incidental excitation of the BV dyes by the UV 355 nm laser and NUVLD sources. For these same experiments, the violet laser was also aligned to the UV/ NUVLD detector cluster for comparison purposes. For all experiments, PMT voltage levels were kept fixed between comparisons. All data were acquired in DiVa 5.0.3 (BD Biosciences) and analyzed using FlowJo ver. 7.6.5 for Windows (FlowJo LLC, Ashland, OR). All fluorescence compensation was done automatically using the spillover algorithms in FlowJo. Labeling levels were expressed using staining indices (SIs) calculated as previously described (16).
RESULTS The flow cytometer in this study was equipped with both a solid-state Nd:YVO4 frequency-tripled 355 nm laser emitting at 20 mW and a NUVLD 375 nm emitting at 11 mW. Either laser could be switched on as needed, and were both aligned to the same fiber coupled pinhole behind the flow cell, and NUVLD Lasers for BUV Dye Excitation
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Figure 4. Five-color labeling of mouse splenocytes using directly conjugated antibodies with BUV395, BUV496, BUV737, BV421, and BV510. (a) Analysis with UV 355 nm laser and a 390/18 nm filter for BUV395. (b) Analysis with UV 355 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. (c) Analysis with NUVLD 375 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. Spillover matrices for each analysis are shown below the dotplots. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the same set of three PMT detectors (arranged in a trigon cluster customarily used on BD Biosciences cytometers). In addition, this instrument had a violet laser diode 405 nm installed and emitting at 50 mW, aligned to a separate pinhole Cytometry Part A 87A: 11271137, 2015
and separate set of PMTs. The optical configuration for this instrument is shown in Figure 1. Using this optical layout, it was possible to compare not only the UV 355 nm laser and NUVLD for BUV dye excitation, but also the incidental 1131
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Figure 4. Continued
excitation and spillover of spectrally adjacent BV 421 and 510 dyes into the BUV detectors. Alignment for all these lasers was verified using Spherotech 8 Rainbow microspheres prior to each experiment (data not shown). The ability of both the traditional UV 355 nm laser and NUVLD to excite BUV395, BUV496, and BUV737 labeled mouse splenocytes is shown in Figure 2. Labeling efficiency 1132
was expressed as SI (see Materials and Methods section), shown with each histogram. In the top row, cells were excited with the UV 355 nm laser source and detected through a 390/ 18 nm filter usual for BUV395, and no other optics. Because a violet 405 nm laser was present in the system, a 405 nm restriction bandpass (RB) or notch filter was then included in the laser path to exclude any violet laser light spillover into NUVLD Lasers for BUV Dye Excitation
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Figure 4. Continued
the BUV395 detector; these data are shown in the second row. No violet laser light spillover was, therefore, present using the 390/18 nm filter. Although a 390/18 nm filter would work with the 355 nm laser, its bandwidth would be too close to a NUVLD 375 nm laser. Therefore, a 405/30 nm filter was substituted for Cytometry Part A 87A: 11271137, 2015
it, along with a 405 nm RB blocker described earlier (third row). With the 355 nm laser, this new filter and blocker worked as well as the 390/18 nm filter (third row). When the NUVLD was substituted for the UV 355 nm laser in the fourth row, sensitivity was at least equal to or slightly better that with the 355 nm source (bottom row). It should be noted that the 1133
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Figure 5. (a) Optical configuration for LSR II for simultaneous analysis of BUV737, BUV805, BV711, and BV785. (b) Comparison of BUV737 and BUV805 excitation using labeled mouse splenocytes and either the UV 355 nm laser (top row) or NUVLD 375 nm laser (bottom row) using the above filters. Left column BUV737 anti-CD4; middle and right columns, BUV anti-mouse CD3 and CD20 labeling. Gray traces depict unlabeled cells and black traces depict labeled cells. The staining index for each positive peak is indicated in each histogram. Staining indices for each labeling are shown. (c) Four-color labeling of human PBMCs using directly conjugated antibodies with BUV737, BUV805, BV711, and BV785 using UV 355 nm laser (top dotplots) or NUVLD 375 nm laser (bottom dotplots). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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NUVLD Lasers for BUV Dye Excitation
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Figure 5. Continued
NUVLD emitted at a lower power level than the 355 nm laser (11 mW versus 20 mW) still demonstrated equal or better excitation of all three BUV dyes. This was also shown using a series of anti-human BUV conjugates used to label human PBMCs (Supporting Information Figure S1). This high degree Cytometry Part A 87A: 11271137, 2015
of excitation even with lower laser power levels may be due to the intrinsically high efficiency of the BUV dyes. The replacement of the conventional 390/18 nm filter with a relatively wide 405/30 nm filter was necessary for the NUVLD, and a 405 nm RB filter was essential to keep violet laser light out of 1135
Original Article the BUV395 detector. The NUVLD was also equipped with a 375/8 nm clean-up filter. These modifications were critical for NUVLD use in a multilaser system. Although the NUVLD gave excellent BUV dye excitation, its slightly longer wavelength ran the risk of exciting the BV dyes better than the 355 nm line. This would be an undesirable situation, because increased incidental excitation of the BV dyes would likely increase their spillover into adjacent BUV detectors, increasing the requirement for fluorescence compensation or spillover between these probes. The problem is illustrated in Figure 3. BV421- and BV510-labeled antibody capture beads were analyzed using 355 nm (top dotplot), NUVLD 375 nm (middle dotplot), or violet 405 nm (bottom dotplot) laser light. For these experiments, all lasers were aligned to the same detector cluster equipped with BV421 (450/50 nm) and BV510 (530/30 nm) filters. As expected, the violet laser gave excellent excitation of both BV421 and BV510 (as indicated by the SIs on each plot), whereas the 355 nm laser excited both dyes poorly. The NUVLD 375 nm laser, however, gave suboptimal excitation, but greater than the 355 nm source. The NUVLD did, therefore, cause unwanted excitation of the BV dyes, which would increase signal overlap into the adjacent BUV detectors. The substitution of a longer and wider 405/30 nm filter for BUV395 also increased the possibility of increased spillover of BV421 and BV510 signals into the BUV395 and BUV496 detectors, as well as less separation between BUV395 and BUV496. To determine whether this overlap and the filter changes for BUV395 would be problematic for multicolor analysis, mouse splenocytes were labeled with a five-color antibody panel consisting of BUV395, BUV496, BUV737, BV421, and BV510 conjugated antibodies and analyzed on the LSRII using the detector configuration shown in Figure 1. The results are shown in Figure 4. Figure 4a shows the five-color panel with UV 355 nm excitation using a 390/18 nm filter for BUV395; Figure 4b with UV 355 nm excitation and a 405/30 nm BUV395 filter with a 405 nm RB blocker; and Figure 4c for NUVLD 375 nm excitation with the same 405/30 nm filter and blocker. The spillover values are shown for each panel in the accompanying tables. First, the staining pattern is almost identical for all three excitation and filter conditions. The NUVLD laser data could not be distinguished from the UV 355 nm results. The NUVLD with filter modification did produce a higher level of BV421 spillover into the BUV 395 detector, from
