Photobleach (FRAP) problem in Zeiss 510Meta

[G.Liu]

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

[leoncio vergara]

Are these live or fixed samples?  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of G.Liu
Sent: Tuesday, May 11, 2010 3:21 PM
To: [hidden email]
Subject: Photobleach (FRAP) problem in Zeiss 510Meta

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



--
View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038048.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Cselenyak Attila]

Were 50 iterations enough at the previous experiments? Sometimes I
need 100 iterations with 488 laser line at 100% with living cells.
Have you seen any bleaching in the whole image?

Best,

Attila

On Tue, May 11, 2010 at 10:21 PM, G.Liu <[hidden email]> 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
>
>
>
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038048.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>

[Julio Vazquez]

Guosheng,

This is how we do it.

Julio.

VII. Photobleaching a region (FRAP).

A photobleaching experiment is a standard time series, with an additional bleaching step either at the beginning of the series (Method 1) , or at some arbitrary position between the pre-bleach and post-bleach frames (Method 2). 

Method 1.

1. Select ACQUIRE>TIME SERIES. Define all parameters as indicated in Section VI.

2. Select "EDIT BLEACH" in main menu

3. Click DEFINE REGION. This will open a BLEACH REGION control panel.

4. Under INTERACTIVE ROI DEFINITION, choose desired shape, and draw shape in your sample image.

5. Under BLEACH PARAMETERS, choose # ITERATIONS (number of bleach scans), for instance 50.

6. Under EXCITATION OF BLEACH  (Bleach Control window),  adjust laser transmission for the bleach step. (experiment to find parameters that provide good bleaching within the ROI, with minimal effect outside ROI). Typically, you will use 100% for the 543 and/or 633. The Argon laser is stronger, so you may want to try somewhere around 30-50%, as a start.

7. To bleach your region, click BLEACH in the BLEACH CONTROL window. You can see the laser scanning (no image will be collected while bleaching).

8. Run your time series.

Method 2. 

Add Step 5b to the procedure above:

5b. Under BLEACH PARAMETERS, click BLEACH AFTER NUMBER OF SCANS. Fill in the number of control (pre-bleach scans). 

This number will also be counted as part of the total number of time points in the time series settings. 

in Step 7, click STARTB in the TIME SERIES CONTROL window. This will start your time series, and will include your bleach routine after the specified number of scans.

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org

On May 11, 2010, at 1:21 PM, 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

-- 

View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038048.html

Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[G.Liu]

The sample is tobacco leaf (live).
And I have contacted Zeiss specialist. Due to no internet hookup for the confocal computer, I did not expect a remote LIVE online trouble shooting.

[leoncio vergara]

As it was pointed out in another response, you may need more than 50 iterations to bleach. Also in live cells you may have problems bleaching if the GFP labeled molecule diffuses back into the ROI. You need to bleach faster than the recovery rate (or bleach completely the cell or all comunicating compartments, which may defeat the purpose of a kinetic experiment). This a lot like trying to make a hole in water :)

On a scanning microscope the bleach rate depends not only on the laser power applied but also on the shape and size of the ROI. Since the scan in the x direction is faster compared to the y direction, an ROI oriented with the longer dimension horizontaly will scan faster than another ROI of identical size and shape but oriented with the longest dimension vertically. Remember that when drawing the ROIs to minimize the bleaching time.

Also rmemeber that the scan region is always rectangular no matter what the shape of the ROI. If you draw an elliptical or free shape ROI, the scanned area will always be the circumscribed rectangle, for pixels outside the ROI but inside that rectangle, the AOTF will will simply block the light but the pixel will still be scanned.

Once you setup the parameters and the ROI, use the "test bleach" button in the Bleach settings window to test if the bleach works before running your time lapse experiment (in another cell of course).

Hope this helps  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of G.Liu
Sent: Tuesday, May 11, 2010 4:04 PM
To: [hidden email]
Subject: Re: Photobleach (FRAP) problem in Zeiss 510Meta

The sample is tobacco leaf (live).
And I have contacted Zeiss specialist. Due to no internet hookup for the confocal computer, I did not expect a remote LIVE online trouble shooting.
--
View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038275.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Julio Vazquez]

Guosheng, 

I think we are a bit confused as to whether you are unable to run the Photobleaching procedure with the LSM software, or whether you are actually not seeing bleaching. The methods I sent you explain how we run the method. you can see whether the procedure is working because you should see the laser scanning the sample during the bleach procedure (with no image collected at the same time). 

To find out the best conditions for bleaching, it is best to bleach a small region, so you can photobleach quickly and start imaging very soon afterwards (especially if you incorporate the bleach routine in your time lapse). Yu can also just use high zoom and high laser power and run a time lapse over a very small region (that is acquire maybe 50 frames without delay in between). You should see intensity drop as you keep imaging (and you will how many iterations are needed for e.g. 50% bleaching). You can then reduce laser power to normal levels and repeat time lapse to see if you have recovery. Once you have determined that, you can set your bleaching/imaging parameters accordingly.

--

Julio 

On May 11, 2010, at 2:04 PM, G.Liu wrote:

The sample is tobacco leaf (live).

And I have contacted Zeiss specialist. Due to no internet hookup for the

confocal computer, I did not expect a remote LIVE online trouble shooting.

-- 

View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038275.html

Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Axel Kurt Preuss]

It could be it s not aligned and you are bleaching in a different spot
Best to take a fluorescent slide and test xy

Thanks

Axel

Axel K Preuss PhD, A*Star IMCB-Central Imaging Facility 6-19B   Sent
from. [phone]

On May 12, 2010, at 4:19 AM, "G.Liu" <[hidden email]> 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
>
>
>
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038048.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com
> .

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

[John Runions]

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



  

[Knecht, David]

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: [phone]
> 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)

[John Runions]

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!  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: [phone]
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)
  

[Knecht, David]

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.  

On May 12, 2010, at 1:11 PM, John Runions wrote:

> 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: [phone]
>>> 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: [phone]
> 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)

[Gert van Cappellen]

Looking  to your protocol I have the impression that your lasers do have
a rather low power. For normal excitation you use 30-50% where we
normally use 0.5-1% (0.8 µW) with our Zeiss LSM510's. If we go higher in
power we start bleaching with normal scanning. For bleaching we normally
need 1-5 iterations to bleach more than 50% of the signal (gfp). For
this the argon laser should have a power higher than 1.5 mW measured on
a 10x lens. Below I added a link to a strip FRAP protocol that one of
our users made
http://www.erasmusmc.nl/oic-cs/627229/LSM510Strip-FRAPprotocol.pdf

Good luck with your experiments,
Gert van Cappellen

--
W.A. (Gert) van Cappellen
Optical Imaging Centre http://www.erasmusmc.nl/oic


Julio Vazquez schreef:

> Guosheng,
>
> This is how we do it.
>
> Julio.
>
>
> *VII. Photobleaching a region (FRAP).*
>
> A photobleaching experiment is a standard time series, with an
> additional bleaching step either at the beginning of the series
> (Method 1) , or at some arbitrary position between the pre-bleach and
> post-bleach frames (Method 2).
>
> *Method 1.*
>
> 1. Select ACQUIRE>TIME SERIES. Define all parameters as indicated in
> Section VI.
>
> 2. Select "EDIT BLEACH" in main menu
>
> 3. Click DEFINE REGION. This will open a BLEACH REGION control panel.
>
> 4. Under INTERACTIVE ROI DEFINITION, choose desired shape, and draw
> shape in your sample image.
>
> 5. Under BLEACH PARAMETERS, choose # ITERATIONS (number of bleach
> scans), for instance 50.
>
> 6. Under EXCITATION OF BLEACH  (Bleach Control window),  adjust laser
> transmission for the bleach step. (experiment to find parameters that
> provide good bleaching within the ROI, with minimal effect outside
> ROI). Typically, you will use 100% for the 543 and/or 633. The Argon
> laser is stronger, so you may want to try somewhere around 30-50%, as
> a start.
>
> 7. To bleach your region, click BLEACH in the BLEACH CONTROL window.
> You can see the laser scanning (no image will be collected while
> bleaching).
>
> 8. Run your time series.
>
>
> *Method 2. *
>
>
> Add Step 5b to the procedure above:
>
> 5b. Under BLEACH PARAMETERS, click BLEACH AFTER NUMBER OF SCANS. Fill
> in the number of control (pre-bleach scans).
>
> This number will also be counted as part of the total number of time
> points in the time series settings.
>
> in Step 7, click STARTB in the TIME SERIES CONTROL window. This will
> start your time series, and will include your bleach routine after the
> specified number of scans.
>
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> 1100 Fairview Ave. N.,  mailstop DE-512
> Seattle, WA 98109-1024
>
> http://www.fhcrc.org
>
>
>
> On May 11, 2010, at 1:21 PM, 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
>>
>>
>>
>> --
>> View this message in context:
>> http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038048.html
>> Sent from the Confocal Microscopy List mailing list archive at
>> Nabble.com.
>

[Claire Brown]

I would suggest you fix some cells and then test out your bleaching protocol. In my experience with live cells much of the recovery is faster than you would think. When I first started bleaching focal adhesions I didn't think I was bleaching but it was just recovering too fast.

I usually use a long box only 20 or so pixels high. As others have mentioned the confocal scans the entire line anyway and just turns on the observation or bleach intensity in the ROIs so you may as well make the region long in y and get lots of data. With the box small you should have a fast enough scan time to see the bleach.

Also take care with live cells not to bleach too much. When you use successive bleaches (like 50) you deplete a whole region of the cell and your kinetics may be dominated by diffusion back into the ROI rather than the dynamics in the ROI itself. This much laser light can also cause phototoxicity to the cells. I usually recommend one bleach scan if you can manage and never more than 10. Remember the shape of the recovery curve should be the same whether you bleach 20% or 100% of the fluorescence (assuming you are not depleting the entire region of fluorescence when you bleach more). So less is better.

Claire

[Straatman, Kees (Dr.)]

Just an idea. If you label with two FPS, let say GFP and RFP. Bleach the GFP. If your proteins come off your ratio GFP:RFP will be restored to pre-bleach levels. If the protein does not come off, your RFP signal increases when new protein binds and the GFP:RFP will decrease.

Kees

Dr K.R. Straatman
Senior Experimental Officer
College of Medicine, Biological Sciences and Psychology
University of Leicester
http://www.le.ac.uk/biochem/microscopy/home.html


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht
Sent: 12 May 2010 18:43
To: [hidden email]
Subject: Re: Photobleach (FRAP) problem in Zeiss 510Meta

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.  

On May 12, 2010, at 1:11 PM, John Runions wrote:

> 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: [phone]
>>> 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: [phone]
> 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)

[Chen, Mei Ling]

Dear Guosheng,
Did you make the right registration of the ROI on Bleach edit and Time
series control windows?
We have had very good and stable results of FRAP from LSM510 and LSM510
META microscopes.
Mei Ling

On Tue, May 11, 2010 3:21 pm, 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
>
>
>
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038048.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>

[srprasad]

The protocol link you posted does not work.

[G.Liu]

Dear All,

Thanks you very much for all of your input (comments, suggestions, protocols etc ) in regard to the photobleaching in our Zeiss510. The good news is that we got some progress on this but bad news is that a new question needs to be addressed: the movement of the bleaching subject/organelle.

We are using transiently GFP-expressed tobacco leaves and want to bleach whole or partial nucleus. However the nucleus seems to move at lease about 1-2um in a 5min period. we tried to use larger pinhole and larger ROI but the plane of focus is still an issue (so only saw partial bleach). Zeiss specialist suggest to stick the leaves on the slide with krazy glue and surrounded by something to avoid the leave floating/vibrating (we are using inverted microscope). We tried but the movement was still noticeable. Have some of you meet a similar problem and how did it solved?

Meantime I am want to install a macro that could track the cell movement (developed by Ellenberg). Any experience on it?

Thank you in advance.

[John Runions]

!!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
some distance from the cells, this will kill them!!!

I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
if nuclear movement is an issue.  This, of course depends on what your
experiment is.  If you are trying to observe FRAP of a protein or
mechanism that transports on microfilaments, then depolymerising the actin
won't work.

John.



Dear All,

Thanks you very much for all of your input (comments, suggestions,
protocols
etc ) in regard to the photobleaching in our Zeiss510. The good news is
that
we got some progress on this but bad news is that a new question needs to
be
addressed: the movement of the bleaching subject/organelle.

We are using transiently GFP-expressed tobacco leaves and want to bleach
whole or partial nucleus. However the nucleus seems to move at lease about
1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
the plane of focus is still an issue (so only saw partial bleach). Zeiss
specialist suggest to stick the leaves on the slide with krazy glue and
surrounded by something to avoid the leave floating/vibrating (we are
using
inverted microscope). We tried but the movement was still noticeable. Have
some of you meet a similar problem and how did it solved?

Meantime I am want to install a macro that could track the cell movement
(developed by Ellenberg). Any experience on it?

Thank you in advance.
--
View this message in context:
http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5051254.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.



*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site
(http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)

Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)

Oxford Brookes Master's in Bioimaging with Molecular Technology
(http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)

[John Runions]

Also, I meant to say -

There are much better ways to immobilize whole tissues and leaf pieces
than with glue.  Try mounting the leaf piece in low-melting temperature
agarose and then sealing the slide with VALAP (which is vaseline / lanolin
/ paraffin wax mixed 1:1:1 and heated to homogenize.  You use a hot metal
spatula to 'draw' the warm liquid VALAP around the edges of the coverslip
and then it sets and seals the coverslip without being toxic to living
cells).   Tissue will stay happy and immobilized for hours in this setup.


!!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
some distance from the cells, this will kill them!!!

I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
if nuclear movement is an issue.  This, of course depends on what your
experiment is.  If you are trying to observe FRAP of a protein or
mechanism that transports on microfilaments, then depolymerising the actin
won't work.

John.



Dear All,

Thanks you very much for all of your input (comments, suggestions,
protocols
etc ) in regard to the photobleaching in our Zeiss510. The good news is
that
we got some progress on this but bad news is that a new question needs to
be
addressed: the movement of the bleaching subject/organelle.

We are using transiently GFP-expressed tobacco leaves and want to bleach
whole or partial nucleus. However the nucleus seems to move at lease about
1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
the plane of focus is still an issue (so only saw partial bleach). Zeiss
specialist suggest to stick the leaves on the slide with krazy glue and
surrounded by something to avoid the leave floating/vibrating (we are
using
inverted microscope). We tried but the movement was still noticeable. Have
some of you meet a similar problem and how did it solved?

Meantime I am want to install a macro that could track the cell movement
(developed by Ellenberg). Any experience on it?

Thank you in advance.
--
View this message in context:
http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5051254.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.



*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site
(http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)

Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)

Oxford Brookes Master's in Bioimaging with Molecular Technology
(http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)



*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site
(http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)

Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)

Oxford Brookes Master's in Bioimaging with Molecular Technology
(http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)