Dear Renato,
even though 405 is clearly suboptimal for DAPI excitation I have never had any problems with any of the lenses in our facility. There is an alternative to DAPI though that even improves the overall efficiency by at least 10fold in our hands, I repost a comment from Jason A. Kilgore on Hoechst 34580 below. Overstaining with Dapi typically generates background caused by binding to other nucleic acid species, extensive illumination with 405 and below can increase autofluorescence, both of which will reduce S/N remarckably.
Concerning your question about the 150xTIRF, that is an objective tuned for single molecule work, I see no reason to use it for conventional or confocal, except maybe for the widefield in combination with a camera with very large pixels, in which case still I would rather try an optovar or other additional magnifying lens before I spend this amount of money.
Best wishes, jens
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Hoechst 34580 would be a better choice than DAPI (or the other Hoechst
dyes), as it excites at 392nm (DAPI excites around 350) and is readily
excited at 405nm.
Hoechst 34580 is available from Molecular Probes (#H21486).
Cheers,
Jason
*Commercial association acknowledged*
Jason A. Kilgore
________________________________
From: Confocal Microscopy List on behalf of Renato Mortara
Sent: Tue 9/22/2009 15:39
To: [log in to unmask]
Subject: DAPI on a 405 laser and 150x 1.45 objective
Hello,
I wonder if I could get some feedback on how two doubts;
1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?
I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.
2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?
Any input will be greatly appreciated,
Best regards,
Renato
Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: 55 11 5579-8306
Fax: 55 11 5571-1095
email: [log in to unmask]
home page: www.ecb.epm.br/~ramortara
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