Confocal Microscopy List

Re: Nuclear Dyes at 405nm?

Posted by David Basiji on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Nuclear-Dyes-at-405nm-tp3144876p3145397.html

Griffin,

The 405 laser diodes can vary in wavelength from unit to unit and since you're exciting DAPI on the tail of it's excitation spectrum, this can give rise a big difference in efficiency from instrument to instrument. Per the Invitrogen spectrum viewer, the nominal excitation efficiency of DAPI at 405 is about 8%, but at 408 it's only 4% and at 402 it's about 11%. That's nearly a three-fold difference in effective excitation efficiency with just a +/-3 nm tolerance on laser wavelength. If feasible, you should check out a different 405 laser in your system.

-David

David Basiji, Ph.D.
President and CEO, Amnis Corporation
2505 Third Ave., Suite 210
Seattle, WA 98121
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Perry, Griffin M
Sent: Tuesday, June 23, 2009 1:31 PM
To: [hidden email]
Subject: Nuclear Dyes at 405nm?

Dear Listserv,

I was wondering if anyone had better results with DAPI @ 405nm than I have. It seems…off. I know it is only getting excited @ 25%, but I know another group that gets good images at the same wavelength. The staining has been uneven with DAPI, but with Topro-3 in the 638nm range, the staining has been beautiful with clearly defined nucleoli. Does anybody use Hoechst 34580? Are there any other alternatives to DAPI @ the 405nm range?

Thanks in advance,

-Griffin