Hi Griffin,
Granted the DAPI excitation is at the tail of the excitation peak***
when using the 405nm laser line, but there’s a heck of a lot of labeled DNA
in the nucleus to excite. The 405nm laser is essentially designed as a cheap
replacement for an incredibly expensive water cooled uV laser [£50k+ including cooling
system] and this 405nm line is still rarely used for anything other DAPI imaging.
Hoechst is
actually even worse than DAPI with the 405nm laser line, down from 11% to nearer
5% on the excitation peak tail.
In every case I have encountered where the 405nm line laser is
consistently very poor at imaging it requires a long visit by the Zeiss or
Leica engineer [for our German confocal systems]*. Fortunately our confocal systems
are on maintenance contracts as generally an awful lot of DAPI associated
hardware seems to been replaced during ‘repair’ [e.g. lasers, AOTF
& other optics]. This assumes you have done the most a user can do, like
align pinholes [for Zeiss 510s], etc… I always have a perfectly
DAPI/FITC/TRITC labeled Invitrogen BPAE cell and kidney slice** to hand to either
demonstrate to users that ‘the system is working perfectly and it’s
their poor samples’ or, more rarely, that ‘we have a problem Huston’.
The BPAE cells preps seem to store better than the kidney slice, where in the latter
the fluorescence diffuses to ‘incorrect’ locations over time [it
stays bright though]. These Invitrogen slides cost approx £80 and £130
respectively and last up to a year stored in the fridge/freezer.
As other threads have pointed out, DAPI labeling isn’t
always perfectly straight-forward. I’d advise staining separately for
DAPI and avoid the Likes of ‘Vectashield+DAPI’ to keep the
background to the absolute minimum for top notch DAPI images- although use of the
latter shouldn’t produce really naff ‘off’ DAPI images
Keith
*Assuming it was decent at installation – and with our
Zeiss 510 and Leica SP2 confocals DAPI [and even Hoechst] imaging is excellent.
We image at around 10% laser power [30mW 405nm laser].
**http://www.well.ox.ac.uk/cytogenetics/Zeiss.shtml
see our Invitrogen slide images
***http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.uk.html
---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford OX3 7BN,
United Kingdom.
Telephone: +44 (0)1865 287568
Email: [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Perry,
Griffin M
Sent: 23 June 2009 21:31
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
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