http://confocal-microscopy-list.275.s1.nabble.com/Re-Assessing-phototoxicity-in-live-fluorescence-imaging-tp7587005p7587018.html
Correct. You can select a point ROI for photostimulation. But you cannot image that point. For measuring the laser power it may work.
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> On Jul 19, 2017, at 9:11 AM, PEARSON Matthew <
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> I'm sure i've parked the beam on our Nikon A1, you can set a single pixel as an ROI and only image at that point, thus not scanning with the galvo's. Its in the simple ROI editor and is a crosshair icon, can't remember what its called, bleach point or something like that.
>
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> Matt Pearson
> Microscopy Facility
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> On 19 Jul 2017, at 14:57, Guillermo Marques <
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> Olympus Fluoview 1000 allows parking of the beam, Nikon A1R does not.
> Guillermo Marqués
> University of Minnesota
> Twin Cities Campus
> University Imaging Centers
> Nikon Center of Excellence
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> On Jul 18, 2017, at 5:24 PM, Craig Brideau <
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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 <
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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" <
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> Sent: 18/07/2017 18:21
> To: "
[hidden email]" <
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> 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
>
>
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