Confocal Microscopy List

Re: Nuclear Dyes at 405nm?

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Nuclear-Dyes-at-405nm-tp3144876p3148265.html

Some manufacturers - including Nikon - supply VC (violet corrected) planapochromats designed to optimise the image from short wavelengths. The improvement relative to a regular planapochromat is substantial. If you are going to do a lot of 405nm work it's worth paying the (non trivial) cost of such an objective.

No commercial affiliation,

Guy Cox

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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Phone +61 2 9351 3176     Fax +61 2 9351 7682
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     http://www.guycox.net

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, 24 June 2009 7:18 PM
To: [hidden email]
Subject: Re: Nuclear Dyes at 405nm?

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

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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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