Just a final thought or two on 'Photo-activation' of DAPI.
We regularly capture mFISH karyotypes of metaphase spread chromosomes here
and we always capture with DAPI last - after a complex fluorescence image
acquisition sequence of Spectrum Orange, Far Red [Cy5], Texas Red, Aqua
[DEAC], and FITC fluorescence filter sets. For mFISH capture we always
search for metaphases using the Spectrum Gold filter, and always capture
with DAPI last in the sequence, as the DAPI illumination is known to
seriously bleach the other dyes [according to mFISH manufacturer's Vysis*
and Applied Imaging]. During mFISH capture the FITC signal is particularly
weak**, possibly because it is the last non-DAPI fluorochrome captured [i.e.
bleached]. Typically, the DAPI is always bright, although if we hang around
during capture the other fluorochromes can be seriously 'dimmed' even before
DAPI capture**.
Likewise I've always repeated the dogma to new users that the published
excitation and emission spectra of any fluorochrome can only be taken as a
guide to that actually found in a specimen. The chemistry/biochemistry of
the tissue + added chemicals may affect the spectra measured in any given
sample. Possibly the energy provided by the various illumination lights
might do something to the spectra as well.
Try as I might though I can't easily fit the observed 'photoactivation' of
DAPI into the above two statements [largely because the effect is reversed
by capture sequence]. But I do wonder 'if, by chance, they are related?'
I've tried to reproduce the DAPI 'photo-activation & capture sequence' on a
few of my bright DAPI/FITC samples, and not succeeded so far.
Keith
*Vysis is actually an ex-manufacturer of mFISH paints
**All the chromosomes have a reasonable 'FITC, DEAC etc..' signal, just the
targeted chromosomes aren't quite as 'significantly brighter than the FITC,
DEAC etc.. background' as they should be.
---------------------------------------------------------------------------
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: [log in to unmask]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Cameron Nowell
Sent: 05 May 2009 01:40
To: [log in to unmask]
Subject: Photoactivation/Conversion of DAPI
Hi List,
I think this has been discussed in the past but i have not been able to
find a definitive answer to the problem.
Basically if you have a sample stained with DAPI, after viewing it with
a DAPI filter, the signal can be then detected using a GFP/FITC filter.
I have tried this on samples with nothing but DAPI on them and it still
happens. Suggestions in the past have been to lower the concentration of
DAPI used and to scan/capture the other channels first and DAPI last.
But does anyone out there have any idea why this happens in the first
place? If you look at the spectra of DAPI it is a very good green
emitter (but needs to be excited in the UV-Blue range). It should not be
able to be excited by standard GFP type excitation at 480-490nm. So the
conversion that is happening is shifting the excitation spectra of DAPI
towards the red somehow.
Any ideas?
Cheers
Cam
Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Jerry Sedgewick
Sent: Tuesday, 5 May 2009 1:35 AM
To: [log in to unmask]
Subject: Re: Posting for Basic Confocal Workshop at U of South Carolina
There are still some slots available in this year's Basic Confocal
Workshop hosted by the University of South Carolina. This year's
workshop will be from June 15-19, 2009 and will include a series of
lectures on the theory and applications of confocal microscopy, specimen
preparation, processing confocal images in Photoshop, and 3D
reconstructions using AMIRA. Students will be able to process triple
labeled samples (cell cultures and sections) on site or bring their own
samples to the workshop.
Faculty will include Drs. Jay Jerome (Vanderbilt), Ralph Albrecht (Univ
Wisconsin-Madison), John Mackenzie (North Carolina State Univ), Tom
TrusK (Medical Univ South Carolina) and myself.
Instruments and applications experts from Leica, Nikon, Olympus, Perkin
Elmer, Photometrics, and Zeiss will be available for hands on training
and imaging of samples.
For those contemplating instrumentation proposals as part of the
stimulus or other funding opportunities this is an excellent opportunity
to see several systems side by side and to collect preliminary data on
their instrument of choice.
For further information and registration go to:
http://dba.med.sc.edu/irf/price/irf/irf.htm or contact Anna McNeal
([log in to unmask] <mailto:[log in to unmask]>)
Bob
Bob Price
Research Professor
Dept Cell Biol and Anat
USC School of Medicine
6439 Garner's Ferry Road
Columbia, SC 29208
Tel: 803-733-3392
Admin Tel: 803-253-5822
Fax: 803-733-3212
--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN 55455
(612) 624-6607
[log in to unmask]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and
Output."
Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN 55114
[log in to unmask]
(651) 308-1466
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