Fwd: Re: Photoactivation/Conversion of DAPI

[George McNamara]

Hi Keith,

Comments:

• You should acquire the FITC channel first - the Aqua channel is the most likely source of inactivation of FITC. I typically recommend acquiring longest to shortest wavelength fluorophores when possible when using multi-filter sequential acquisition. On the LSM510/UV, I most often set up 4-color acquisitions as Cy5+Alexa Fluor 488 in the first track and Alexa Fluor 568 + DAPI in the second track, to acquire in less time than four track acquisition.
•  If you are using FITC in 2009 - why? Switch to Alexa Fluor 488 or Rhodamine 110 (the latter is Spectrum Green), or DyLight 488 or HiLyte Fluor 488.
•  You are not using FITC - this is a reactive dye and the ITC should be long gone by the time you coverslip and look.
•  You might want to invest the time, money and effort to find a mounting medium that is not fluorescent. One obvious candidate: 2,2'-thiodiethanol (Staudt et al 2007, search the listserv for protocols). I also strongly recommend applying the DAPI during a wash step, and not having it in the mounting medium.
•  Published spectra are usually acquired on spectrofluorimeters, which are a heck of a lot more accurate than the things on most researchers spectral imaging microscopes (with the exception of Jeremy Lerner's PARISS).
•  I hope you have minimal "tissue" on your metaphase spreads.
•  if "added chemicals" are affecting your experiments, I suggest you leave them out and find (and publish) alternatives.
•  The energy from wavelengths longer than the absorption wavelength of FITC will not affect FITC directly (unless you are using a multiphoton excitation microscope). Other fluorophores, and possibly endogenous fluorophores and chromophores (which should be minimal on metaphase chromosome preparations) could produce oxygen (or other) radicals when absorbing at their preferred wavelengths.
• I would refer to the DAPI phenomenon as photoconversion, not photo-activation. The latter implies the chemical was not fluorescent at any wavelengths until activated.
•  You might want to trade in your mFISH system and reagents for a SKY system and SKY kits. SKY has been simultaneous five fluorophore acquisition since 1997 (followed by a high resolution DAPI image for inverse G-banding view of the chromosomes). Or, send your specimens to Paul Edwards at Cambridge University ( http://www.path.cam.ac.uk/pages/edwards/) for SKYing.

best wishes,


George

Date:         Mon, 18 May 2009 10:12:52 +0100
From: Keith Morris <[hidden email]>
Subject: Re: Photoactivation/Conversion of DAPI
To: [hidden email]


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:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 
-----Original Message-----
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Cameron Nowell
Sent: 05 May 2009 01:40
To: [hidden email]
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
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