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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" <[log in to unmask]>
Sent: ‎18/‎07/‎2017 18:21
To: "[log in to unmask]" <[log in to unmask]>
Subject: Re: Assessing phototoxicity in live fluorescence imaging

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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