title: Violet laser diodes in flow cytometry: an update.
abstract: BACKGROUND
  In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton-ion lasers on stream-in-air cytometers. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific Blue, but may have been insufficient for applications requiring higher levels of photon saturation, such as low-level expression of Cyan Fluorescent Protein (ECFP) in CFP-YFP FRET applications. In this follow-up study, we have tested more powerful VLDs emitting at 55 mW, and a beam-merged dual module VLD with 100 mW combined output, for their ability to excite a variety of violet-excited fluorochromes, including CFP.
  
  
  METHODS
  A dual module VLD (two linear polarized VLDs with their beams merged by a polarized beam combiner) emitting at 404 nm was mounted on a BD FACSVantage DiVa stream-in-air cytometer. The individual polarized 55 mW beams or the 100 mW combined beams were used to analyze PBMCs labeled with the violet-excited probes Cascade Blue, Alexa Fluor 405, Cascade Yellow and Pacific Orange dyes. Violet-excited fluorescent microsphere mixtures with decreasing fluorescence levels were also used to detect the minimum sensitivity threshold and precision of these lasers. VLD excitation on a gel-coupled cuvette flow cytometer was used as a sensitivity baseline.
  
  
  RESULTS
  The dual module 100 mW VLD gave both sensitivity and precision levels approaching that observed for lower-power sources on a cuvette cytometer. Single polarized VLD modules at 55 mW gave slightly decreased sensitivity for the microspheres standards and all the tested fluorochromes compared to the 100 mW source.
  
  
  CONCLUSIONS
  While 55 mW laser sources performed adequately in the stream-in-air format, increasing the power to 100 mW did give a small but detectable increase in instrument sensitivity. This sensitivity level approached that of cuvette systems.
authors.affiliation: Experimental Transplantation and Immunology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, USA.
authors.forename: William
authors.initials: W
authors.lastname: Telford
authors.forename: Veena
authors.initials: V
authors.lastname: Kapoor
authors.forename: James
authors.initials: J
authors.lastname: Jackson
authors.forename: Walter
authors.initials: W
authors.lastname: Burgess
authors.forename: Gayle
authors.initials: G
authors.lastname: Buller
authors.forename: Teresa
authors.initials: T
authors.lastname: Hawley
authors.forename: Robert
authors.initials: R
authors.lastname: Hawley
mesh_terms: D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods / Q000639:trends; D005456:Fluorescent Dyes; D006801:Humans; D007834:Lasers; D007963:Leukocytes, Mononuclear / Q000166:cytology; D051379:Mice; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes