http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5043157.html
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
a 10x lens. Below I added a link to a strip FRAP protocol that one of
W.A. (Gert) van Cappellen
> 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:
>>
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>