Internal research and development projects.
Low power lasers in flow cytometry. We have a laboratory-wide project integrating new low-power lasers into our flow cytometers, and carrying out rigorous perfomance evaluations on these potentially useful excitation sources. Results from our near-UV, violet and blue diode and green and yellow HeNe evaluations are available below...
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Near UV laser diodes (NUVLD). These 370 - 380 nm sources are under development by several manufacturers, and may ultimately prove to be useful replacements for bulkier, more expensive UV gas lasers. We tested a Power Technology 372 nm NUVLD on our LSR II for DAPI DNA content measurement and other UV-excited applications. The preliminary results are presented here. |
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Violet laser diodes (VLD). Violet laser diodes emit in the near-UV to violet range (395 - 410 nm). They may make useful, low-cost substitutes for more expensive water-cooled gas lasers in flow and image cytometry applications. See our evaluations of these laser sources on the FACSVantage DiVa, the LSC and the LSR II, and a comparison between Coherent and Power Technology VLDs. |
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Blue laser diodes (BLD). Blue laser diodes have been around as long as violets, but have not been widely incorporated into flow cytometers. Go here to see our evaluation of this laser on the BD LSR II for CFP excitation. |
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NEW! DPSS 532 nm laser on the BD LSR II. We have permanently installed a diode-pumped solid state (DPSS) 532 nm green laser on our BD LSR II. This laser provides better excitation for phycoerythrin and DsRed, with lower autofluorescence backgrounds. It also gives us access to low molecular weight fluors such as Cy3, TAMRA, and Alexa Fluor 532, 546 and 555. Go here for sample data. |
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Green 543 nm helium neon lasers (GreNe). This green laser source can be mounted on our LSR II for exceptionally sensitive phycoerythrin detection. The GreNe can also be used to excite rhodamine-coupled reagents, Cy3 and the green-excited Alexa Fluor dyes (including 532, 546 and 555). Go here for more information on this laser. |
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NEW! DPSS 561 nm laser on the BD LSR II. We have tested a Melles Griot diode-pumped solid state (DPSS) 561 nm yellow-green laser on our BD LSR II. This laser provides better excitation of phycoerythrin and DsRed with lower autofluorescence backgrounds, and allows the simultaneous analysis of fluorescein or GFP without the laser light contamination encountered with green laser sources. More information is here. |
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Yellow 594 nm helium neon lasers (YeNe) on the LSR II. We now use a yellow 594 nm HeNe laser on our LSR II in place of the traditional red laser; this source provides excellent APC and APC tandem conjugation, and can also excite Texas Red and Alexa Fluor 594 nm. Yellow excitation also reduces the crossbeam compensation required between PE-Cy5 and APC. Click here for more information about these lasers on the LSR II. |
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NEW! Yellow 594 nm helium neon lasers (YeNe) on the LSC. We have also installed a yellow 594 nm HeNe laser on our Compucyte laser scanning cytometer (LSC) for excitation of Texas Red and Alexa Fluor 594. This allows easier transfer of epifluorescence and confocal applications (which frequently use these yellow-excited fluorochromes) to the LSC format. For more information, go here. |
Other projects...
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Fluorochromes on the LSC. The long laser dwell time required for laser scanning cytometry places special demands on fluorochromes for photostability and resistance to photobleaching. Click here to see our LSC photobleaching evaluations for a variety of fluorochromes. |
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Evaluating novel fluorochromes and labeling methods. The Zenon labeling system from Molecular Probes allows easy fluorochrome conjugation to mouse antibodies; click here to see our initial evaluation of the system. We have also tested the PBXL series of stabilized cyanobacteria photosystem II antennae complexes developed by Martek Biosciences, the CryptoFluor series of low molecular weight phycobiliproteins of the same source, and the tandem conjugate PerCP-Cy5.5 developed by BD Biosciences. We also describe methodology for using the fluorogenic alkaline phosphatase substrate ELF-97 for both detection of endogenous and antibody-conjugated AP activity. |
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Caspase assays for flow and image cytometry. We have evaluated several flow assays for the detection of functional caspase activity in apoptotic cells, including intregration of these assays into other cell death detection systems. These include OncoImmunin's PhiPhiLux caspase 3 assay and Pharmingen's anti-active caspase 3 antibody. Go here to see sample data. |
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CTL- and NK-mediated cytolysis for flow and image cytometry. Flow or image cytometric detection of caspase 6 activation represents an earlier and more sensitive assay for cytolytic activity than chromium release. Click here for information on both flow and image cytometry assays. |
