Formaldehyde-fixed samples as FRAP controls for reversible GFP, YFP photobleaching?

[Stanislav Vitha]

Hello,

Some FRAP protocols recommend using formaldehyde-fixed samples as controls
to check for reversible photobleaching of the fluorescent protein, reasoning
that there is no diffusion in fixed samples, thus the recovery of
fluorescence in the bleached area is attributed to reversible
photobleaching. From that follows that if no FRAP is observed in the fixed
samples, reversible photobleaching is negligible, and FRAP in the live
sample represents protein turnover (diffusion).

Is this reasoning correct? I wonder if it possible that fixation
(crosslinking) alters the photochemical properties of the fluorescent
protein, eliminating thus reversible photobleaching even if the protein is
normally capable of it.

Thanks for your input.

Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University

[Mark Cannell]

A very valid concern. If seems to me that if photobleaching were due to
oxidation, some reversal might be due to the reducing agents in the cell
that will be be destroyed by fixation.

Cheers


Stanislav Vitha wrote:

> Hello,
>
> Some FRAP protocols recommend using formaldehyde-fixed samples as controls
> to check for reversible photobleaching of the fluorescent protein, reasoning
> that there is no diffusion in fixed samples, thus the recovery of
> fluorescence in the bleached area is attributed to reversible
> photobleaching. From that follows that if no FRAP is observed in the fixed
> samples, reversible photobleaching is negligible, and FRAP in the live
> sample represents protein turnover (diffusion).
>
> Is this reasoning correct? I wonder if it possible that fixation
> (crosslinking) alters the photochemical properties of the fluorescent
> protein, eliminating thus reversible photobleaching even if the protein is
> normally capable of it.
>
> Thanks for your input.
>
> Stanislav Vitha
> Microscopy and Imaging Center
> Texas A&M University
>  
>  

[Arne Seitz]

Hi,

what you call reversible photobleaching is most likely accumulation of non-fluorescent triplet states. They have a much longer lifetime than the excited singlet states.
Fixation is definitively changing the properties of GFP. Organic solvent even seems to destroy GFP completely.

http://www.bio.davidson.edu/courses/Molbio/restricted/02GFPwow/GFPwowpg1.html

How the lifetime of triplet states is influenced by fixation is to my knowledge not investigates in detail but I would assume that they are definitely influenced by their chemical environment and thereby also by fixation. Thus doing FRAP experiments on fixed samples is maybe not the best control.

IMHO it is much better to prolong the number of postbleach frames until you have a equilibrium between electronic ground state, excited singlet state and triplet state. Depending on your imaging conditions this might take 20-30 frames. Please keep in mind that the proportion of triplet states will depend on the imaging conditions (pixel dwell time, laser intensity etc.) so that it might also be worth playing around with these parameters in order to have less triplet states.

Cheers Arne


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stanislav Vitha
Sent: jeudi 2 septembre 2010 00:09
To: [hidden email]
Subject: Formaldehyde-fixed samples as FRAP controls for reversible GFP, YFP photobleaching?

Hello,

Some FRAP protocols recommend using formaldehyde-fixed samples as controls
to check for reversible photobleaching of the fluorescent protein, reasoning
that there is no diffusion in fixed samples, thus the recovery of
fluorescence in the bleached area is attributed to reversible
photobleaching. From that follows that if no FRAP is observed in the fixed
samples, reversible photobleaching is negligible, and FRAP in the live
sample represents protein turnover (diffusion).

Is this reasoning correct? I wonder if it possible that fixation
(crosslinking) alters the photochemical properties of the fluorescent
protein, eliminating thus reversible photobleaching even if the protein is
normally capable of it.

Thanks for your input.

Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University
 

[ChrisWilms]

Reversible "photobleaching" of XFPs has been described previously and  
it appears to be a speciality of fluorescent proteins. I'm not sure  
it's simply a matter of triplet state accumulation. No matter what it  
is, I do agree that fixation will interfere with this process. My  
advice would be to move to an XFP that is less prone to darkstates.

Best, Christian

On/off blinking and switching behaviour of single molecules of green  
fluorescent protein
Dickson, R. M. and Cubitt, A. B. and Tsien, R. Y. and Moerner, W. E.
Nature 388 (1997), p355--358

Imaging individual green fluorescent proteins
Pierce, D. W. and Hom-Booher, N. and Vale, R. D.
Nature 388 (1997), p338--338

Fluorescence correlation spectroscopy reveals fast optical excitation-
driven intramolecular dynamics of yellow fluorescent proteins
Schwille, P. and Kummer, S. and Heikal, A. A. and Moerner, W. E. and  
Webb, W. W.
PNAS 97 (2000), p151-156

> what you call reversible photobleaching is most likely accumulation  
> of non-fluorescent triplet states. They have a much longer lifetime  
> than the excited singlet states.

[Gert van Cappellen-2]

When you start to excite GFP's (with a high laser power) you will drive
a part of the molecules in the off-state. The molecules will remain in
this state for a couple of seconds. This effect can be measured both by
molecules that are mainly bound, for instance H2b-GFP as in fixed
samples. Off course fixation might influence the properties of the
molecules in the off state. Good FRAP analysis software takes this off
state into account.

Hope this helps a bit,
Gert van Cappellen

Stanislav Vitha schreef:

> Hello,
>
> Some FRAP protocols recommend using formaldehyde-fixed samples as controls
> to check for reversible photobleaching of the fluorescent protein, reasoning
> that there is no diffusion in fixed samples, thus the recovery of
> fluorescence in the bleached area is attributed to reversible
> photobleaching. From that follows that if no FRAP is observed in the fixed
> samples, reversible photobleaching is negligible, and FRAP in the live
> sample represents protein turnover (diffusion).
>
> Is this reasoning correct? I wonder if it possible that fixation
> (crosslinking) alters the photochemical properties of the fluorescent
> protein, eliminating thus reversible photobleaching even if the protein is
> normally capable of it.
>
> Thanks for your input.
>
> Stanislav Vitha
> Microscopy and Imaging Center
> Texas A&M University
>  
>
>