http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5042404.html
It depends on the question. I can see for membrane proteins that it doesn't really matter what happens in the bleach zone because you are concerned with the freedom of the unbleached material nearby. But we are thinking about actin filaments and actin binding proteins. If the filaments are saturated with ABP's and you do a FRAP, then it is critically different whether the previously bound proteins have come off or not. Any fixed structural complex requiring binding rather than diffusion of free protein into the bleach zone would have the same issue.
> Good question David. I think we've sort of peripherally gotten onto this one before. I was enquiring about the physical mechanism of bleaching and most of the answers suggested that proteins are not destroyed but rather simply lose the ability to fluoresce. This would be the goal anyway when doing FRAP because burning holes through the tissue really isn't going to teach us much. We are just working on a manuscript that shows differential mobility of many different types of proteins in the plasma membrane. We are saying that these mobility differences are down to different binding interactions in part. If proteins were inactivated and binding partners released, I wouldn't predict such large differences as we are seeing. Mind you, these proteins can reside in very different environments within membranes and in different types of complexes (e.g. lipid rafts) so we are investigating these effects as well.
> The jury is still out and your idea of post-FRAP immunostaining for the fusion protein is one that is not entirely without merit!
Damned by faint praise or maybe this is reviewer speak! I am presuming that "not entirely without merit" is much worse than "has merit" and only slightly better than has no merit. Again, for actin binding, I think it would be important. For membranes, not as likely.
> John.
>
> David Knecht wrote:
>> As long as we are on the topic of bleaching, I have a question about the mechanism. When working with GFP fusion proteins, is there data to indicate which happens when you photobleach:
>> 1. The GFP is inactivated but the fusion protein is not affected
>> 2. The GFP and the fusion protein are both inactivated and the protein leaves it binding site in a complex
>> 3. The GFP and the fustion protein are functionally inactivated, but the fusion protein does not leave its binding site in a complex (binding domain and enzymatic domain behaving differently)
>> What control would you do to confirm this and do people do it? Presumably immunostain after photobleach. It would seem that if you get fast recovery, then 1 and 3 are unlikely. If you don't get fast recovery, then either there is little free protein or 1 or 3 has occurred. Dave
>>
>> On May 12, 2010, at 4:41 AM, John Runions wrote:
>>
>>
>>
>>> Hi Guosheng,
>>>
>>> We have been doing quite a lot of FRAP and photoactivation in tobacco leaves and I just want to add a couple of things to what the others have said. If you are using the older software (pre-ZEN), one small thing that seems to cause the 510 Meta software to get a bug in its brain is the difference between the ROI controller and the Bleach Region controller windows. If you set the ROI for bleaching using the Edit ROI window (accessed from the main LSM window) rather than the Bleach Region window, the bleaching laser won't fire. This is a rather commonplace confusion as the two windows look virtually identical.
>>>
>>> Secondly, what tissue / organelle are you trying to bleach? GFP in the plasma membrane usually bleaches very well indeed leaving a nicely defined image of the ROI but GFP in the ER or cytoplasm FLIPs (Fluorescence Loss in Photobleaching). The cytoplasm and ER are such dynamic systems that during your 50-100 bleaching pulses all of the GFP in the region around the ROI moves through the ROI with the effect of reducing overall GFP intensity in the cell rather than producing a nicely bleached ROI shape.
>>>
>>> My experience with this system is that you won't need anything like 50-100 bleaching scans. Especially since you have indicated that you have a 405 laser (which is wicked). Once you have the system set up properly, you should be able to bleach GFP in tobacco cells with very few pulses indeed. With a 63x objective, we do it with 2-10 iterations of 405 laser set at 25-50% transmission.
>>>
>>> Your results may vary!
>>>
>>> Please write to me if you have any more questions about this.
>>>
>>> All the best, John.
>>>
>>> G.Liu wrote:
>>>
>>>
>>>> Dear all,
>>>>
>>>> We have encountered a problem in our 510Meta for photobleaching. We simply
>>>> could not get the GFP ROI bleached. What we did is as following, anything
>>>> wrong or inappropriate?
>>>>
>>>>
>>>> 1. Collect the Single unsaturated image as usual (normal settings).
>>>>
>>>> 2. Go to Bleach control for bleach settings. Select xx scans before the
>>>> bleach. For GFP, tried 1-50 iteration, defined the Bleach region. All the
>>>> argon laser lines (458, 488, 514) or only 488 to 100% .
>>>>
>>>> 3. Go to Time Lapse--using Manual start, manual stop (input the number of
>>>> scan). Time interval 0 or 1 sec.
>>>>
>>>> 4. Hit StartB.
>>>>
>>>> I can get a series of images but no bleaching was found. The other odd thing
>>>> is that "Bleach" button seems not not working--if hit, it only lasts a few
>>>> seconds then stop. I've tried using diode405 line, it worked once, but I
>>>> can't repeat it anymore.
>>>>
>>>> Any suggestions or advice are greatly appreciated!!
>>>>
>>>> Guosheng
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>> --
>>> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>>>
>>> *********************************
>>> John Runions, Ph.D.
>>> School of Life Sciences
>>> Oxford Brookes University
>>> Oxford, UK
>>> OX3 0BP
>>>
>>> email:
>>>
[hidden email]
>>>
>>> phone: +44 (0) 1865 483 964
>>> Runions’ lab web site
>>>
>>> Visit The Illuminated Plant Cell dot com
>>> Oxford Brookes Master's in Bioimaging with Molecular Technology
>>>
>>>
>>>
>>
>> Dr. David Knecht
>> Department of Molecular and Cell Biology
>> Co-head Flow Cytometry and Confocal Microscopy Facility
>> U-3125
>> 91 N. Eagleville Rd.
>> University of Connecticut
>> Storrs, CT 06269
>> 860-486-2200
>> 860-486-4331 (fax)
>>
>>
>
> --
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
> *********************************
> John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:
[hidden email]
> phone: +44 (0) 1865 483 964
> Runions’ lab web site
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology
>
Dr. David Knecht
91 N. Eagleville Rd.