Re: Photobleach (FRAP) problem in Zeiss 510Meta

Posted by Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038264.html

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

-- 

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