HeNe 633 nm excitation on the LSR II.
The LSR II is equipped with a JDS Uniphase red 633 nm helium-neon laser emitting at 17 mW in the fourth laser position. This laser is more than adequate for exciting a variety of red-excited flurochromes, including APC and its tandem conjugates (including APC-Cy5.5, APC-Alexa Fluor 680, APC-Alexa Fluor 700, APC-Cy7 and APC-Alexa Fluor 750), Cy5, Alexa Fluor 633, Alexa Fluor 647, and the DNA binding dye TO-PRO-3. The red HeNe has a corresponding "trigon" with a default configuration of two PMTs; a third has been added to our instrument. In the default configuration, the LSR II can analyze APC and APC-Cy7, APC and APC-Cy5.5, or APC-Cy5.5 and APC-Cy7 simultaneously. With three PMTs, simultaneous analyis of APC / APC-Cy5.5 / APC-Cy7 is possible.
(Below). HeNe laser trigon configuration. The LSR II HeNe laser trigon in the default configuration, with filters for APC and APC-Cy7 split with a 735 LP dichroic.
(Below). Two-color analysis of APC and APC-Cy7 or APC-Alexa Fluor 750 with the HeNe 633 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 red HeNe laser.
(Below). HeNe laser trigon configuration modified for APC-Cy5.5 (or APC-Alexa Fluor 680 or APC-Alexa Fluor 700) and APC-Cy7 (or APC-Alexa Fluor 750) . The LSR II HeNe laser trigon in modified configuration, with a 710/20 nm filter for APC-Cy5.5 and a signal split with the 735 LP dichroic.
(Below). Two-color analysis of APC-AF700 and APC-Cy7 or APC-Alexa Fluor 750. EL4 cells were labeled with biotin-conjugated CD44 followed by APC-Alexa Fluor 700, 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 red HeNe laser.
(Below). HeNe laser trigon configuration modified for APC, APC-Cy5.5 (or APC-Alexa Fluor 680 or APC-Alexa Fluor 700) and APC-Cy7 (or APC-Alexa Fluor 750) . The LSR II HeNe laser trigon in modified configuration, with a third PMT, a 710/20 nm filter for APC-Cy5.5 and the APC / APC-Cy5.5 split made with a 680 or 695 LP dichroic.
This configuration is being evaluated now.
The red HeNe can be replaced with a yellow 594 nm HeNe laser for up to four-color analysis with Texas Red. Go here to see our initial evaluation.
Go here to download the LSR II User Handbook.
Download Adobe Acrobat Reader here .