http://confocal-microscopy-list.275.s1.nabble.com/Re-Assessing-phototoxicity-in-live-fluorescence-imaging-tp7587005p7587014.html
Olympus Fluoview 1000 allows parking of the beam, Nikon A1R does not.
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> On Jul 18, 2017, at 5:24 PM, Craig Brideau <
[hidden email]> wrote:
>
> *****
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>
> I've noted the effect Andreas mentions quite often. I usually set the pixel
> dwell time to the maximum such that the laser will spend the longest time
> possible scanning out a single line, but even then the flyback can disturb
> the reading. The best way is to park the mirrors in the center position,
> although not all systems allow you to do that. Nikon's old C1 platform
> allows for it, and ThorLabs' ThorScanLS has a park button as well. I'm not
> sure about other vendors, but I'm sure others can chime in with their
> experiences.
>
> Craig
>
> On Tue, Jul 18, 2017 at 11:44 AM, Andreas Bruckbauer <
>
[hidden email]> wrote:
>
>> *****
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>>
>> Hi Claire,
>> Confocals usually blank (switch off) the beam on the return and the power
>> meter averages between the on and off phases. Very slow scans are more
>> accurate an I usually use high zoom. Parking the beam is the better option.
>>
>> Best wishes
>>
>> Andreas
>>
>> -----Original Message-----
>> From: "Claire Brown" <
[hidden email]>
>> Sent: 18/07/2017 18:21
>> To: "
[hidden email]" <
[hidden email]>
>> Subject: Re: Assessing phototoxicity in live fluorescence imaging
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> Post images on
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>>
>> Thank you for this great article and pointing to many great resources.
>> I wanted to bring up one issue we have had when trying to work on
>> different microscope and compare light density/exposure.
>>
>> For the CLSM microscopes when we use a power meter at the focal plan the
>> power we measure depends a lot on the scan settings.
>> If we park the beam as a point we get one power. If we go to a 100x100
>> pixel array at zoom 1 with a 10x lens the power is different. if we change
>> the scan speed the power is different again. I suspect this is related to
>> how the power meter integrates the light over time and also how sensitive
>> it is spatially across the sensor. We have decide to just quote our power
>> as the power we measure at the power meter with set conditions and we
>> detail those conditions in our materials and methods section of the paper.
>> We try to use a 10x/0.3 planfluar lens with no phase optics if we can.
>>
>> We have stayed away from trying to calculate the power at the sample
>> because a lot of assumptions have to be made. The assumptions may be
>> different for wide-field versus CLSM versus light sheet versus spinning
>> disk and so on.
>>
>> We ran into these issues when just trying to repeat measurements on two
>> different confocals from two different manufacturers. It can really get
>> quite complex.
>>
>> Does anyone have thoughts on this issue? Any cleaver solutions? It is my
>> thought that comparing relative powers on the same instrument is okay but
>> comparing between systems will be very complex.
>>
>> Ideally, it would be good for the manufacturers to have some kind of laser
>> power measurement in the instrument and software that is always monitored.
>> Even if this is just a relative value to the actual power at the sample it
>> would really improve quantitative microscopy and also help in maintenance
>> and trouble shooting equipment. I'm not sure about others but this kind of
>> a feature would really be a strong selling point for me and the core
>> facilities I manage. In many cases these options are already built into the
>> hardware for the service engineers but are not accessible to the end user.
>>
>>
>> Sincerely,
>>
>> Claire
>>