Cy3 photobleaching DAPI

[Gareth Howell]

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Dear list members,
I was wondering if anyone could explain something that I have seen for many years now and dismissed, but now feel the need to try and find an answer for (must be getting old!)

Why is it if one has a very intense Cy3 staining on cells can this be detected in the DAPI channel, and as some of my facility's users have noted, actually bleaches DAPI signal. It seem counter-intuitive for bleed-through to occur 'backwards' through the spectrum. Have other people seen or reported this? Please accept my apologies if this is a dumb question but I just can't work it out!

Gareth
University of Leeds
UK.

[Guenter Giese]

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Gareth,

you may have observed anti-Stokes fluorescence. I also run across such a
phenomenon about a year ago with GFP (543 nm excitation, 495-537 nm spectral
detection with LEICA SP2).

By literature search I found a reference dealing with anti-Stokes
fluorescence (I do not know if this is the first description of this
phenomenon in microscopy):

Saito, K., Tokunaga, M., Iwane, A. H., and Yanagida, T. (1997). Dual-colour
microscopy of single fluorophores bound to myosin interacting with
fluorescently labelled actin using anti-Stokes fluorescence. J Microsc 188,
255-263.

Best,

Guenter



------------------------------------------
Dr. Guenter Giese
Light Microscopy Facility Manager
Dept. of Biomedical Optics
MPI fuer Medizinische Forschung Jahnstr. 29
D-69120 Heidelberg, Germany
Phone [phone] (Fax: -325)
e-mail: [hidden email]  

> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List
> [mailto:[hidden email]] Im Auftrag von Gareth Howell
> Gesendet: Freitag, 11. Januar 2008 12:38
> An: [hidden email]
> Betreff: Cy3 photobleaching DAPI
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
>
> Dear list members,
> I was wondering if anyone could explain something that I have
> seen for many years now and dismissed, but now feel the need
> to try and find an answer for (must be getting old!)
>
> Why is it if one has a very intense Cy3 staining on cells can
> this be detected in the DAPI channel, and as some of my
> facility's users have noted, actually bleaches DAPI signal.
> It seem counter-intuitive for bleed-through to occur
> 'backwards' through the spectrum. Have other people seen or
> reported this? Please accept my apologies if this is a dumb
> question but I just can't work it out!
>
> Gareth
> University of Leeds
> UK.
>
>

[Karl Garsha]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Gareth,

In order to shed light on this mysterious observation I think it will be
important to know the bandpass cutoffs for the excitation filters,
dichroics and emission filters being used to image your fluorochromes.

My educated guess would be that there is likely some overlap between the
  DAPI emission bandpass filter and the Cy3 emission, as might be the
case with a long-pass emission filter for DAPI, there could also be some
direct excitation of the Cy3 depending on the bandpass profile for the
DAPI exciter. Another possibility is backscatter from the Cy3 excitation
getting into the DAPI detection band. I'm not sure if you are using some
sort of dual bandpass and selecting single exciters or exactly what the
configuration is, but  the key is probably something to do with the
convolution of the bandpass filters with the actual absorption and
emission profiles of the two dyes. Also, one has to keep in mind that
there is some slope to filter cut-off specifications, what this means is
that full blocking (or full bandpass) is not achieved right at the
cutoff boundary, rather about 10-nm bandwidth on either side of the
boundary number is usually required to ensure full blocking capability
(the cut-off/cut-on can be steeper for newer filter production
technologies).

As far as the bleaching phenomenon, it could be true bleaching due to
direct absorption of excitation light, which is orders of magnitude
brighter than any fluorescence emission, or, if it is not witnessed
through the eyepieces, it could be due to a digital camera auto-scaling
the image brightness values to accommodate the brighter Cy3 signal
within the dynamic range. If the camera is set to auto-scale, assuming
you are picking up the Cy3 signal in the DAPI channel or using a
dual-emission bandpass, the DAPI signal could appear to vanish in real
time on the digital image.

Although there may be other possibilities, years of experience
troubleshooting multiplexed filter configurations leads me to suspect
the filter combinations as the most likely explanation, with camera
auto-scaling possibly being a factor.

It doesn't seem nearly as likely to me that Cy3 fluorescence is somehow
blue-shifting and then being absorbed by DAPI to such a degree that it
is causing photobleaching. If this were the case, you might be able to
quantify the Cy3 signal getting brighter as the DAPI bleaches with a
good CCD camera, although you would have to confirm that there is no
autoscaling going on with the imaging hardware or software in order to
measure this.

Cheers,
Karl

Gareth Howell wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list members,
> I was wondering if anyone could explain something that I have seen for
> many years now and dismissed, but now feel the need to try and find an
> answer for (must be getting old!)
>
> Why is it if one has a very intense Cy3 staining on cells can this be
> detected in the DAPI channel, and as some of my facility's users have
> noted, actually bleaches DAPI signal. It seem counter-intuitive for
> bleed-through to occur 'backwards' through the spectrum. Have other
> people seen or reported this? Please accept my apologies if this is a
> dumb question but I just can't work it out!
>
> Gareth
> University of Leeds
> UK.
>

--
Karl Garsha
Research Microscopy Specialist
US-Southwest Region
Leica Microsystems-Life Sciences Research
www.leica-microsystems.com