Posted by
PEARSON Matthew on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Assessing-phototoxicity-in-live-fluorescence-imaging-tp7587005p7587017.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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.
--
Matt Pearson
Microscopy Facility
MRC Human Genetics Unit
Institute of Genetics and Molecular Medicine (IGMM)
University of Edinburgh
Crewe Road
EH4 2XU
On 19 Jul 2017, at 14:57, Guillermo Marques <
[hidden email]<mailto:
[hidden email]>>
wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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
www.uic.umn.edu
http://uic.umn.edu/content/locationsAny work utilizing UIC equipment or staff support must acknowledge the University Imaging Centers in publications and presentations.
On Jul 18, 2017, at 5:24 PM, Craig Brideau <
[hidden email]> wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.