Posted by
Mark Scott-3 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589961.html
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Hi Michael,
I have always tended to teach the offset to be one of either two things, low enough so you just get a few "zero" pixels, or the next step up that results in no zero pixels - my thought is that ensuring the offset is high enough reduces potential for lost information, however part of me always worries about "zero" values when doing any quantification.
Most settings like pinhole size and how much you go into it (along with the reasoning) tends to stem from the user you are training as well. Often for novice users it is just easier to get them started by claiming 1AU is the optimal confocal setting, especially those who are just interested in getting their images and getting out of there. I do normally run through what the pinhole actually does to cut out out-of-focus light and the bigger the pinhole the thicker the slice and the more out of focus light they will see, then if they are trying out things like Airyscan, Hyvolution then I will explain more.
I'm with you on not touching Digital Gain, no matter how much vendors try and convince me that it is fine to use these days - in my view, if you need to raise this to see your fluorescence then you probably should be going back to re-optimise your antibody labelling.
Cheers
Mark
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From: Confocal Microscopy List <
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Sent: Friday, 11 October 2019 2:16 AM
To:
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Subject: training and bets practices for confocal
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I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity. But other people have been disagreeing with all three points. Interested whether there is a consensus. Does anyone disagree with the guidelines below? Any comments welcome.
Cheers-
Michael
General Confocal Best practices:
* The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
* Offset. Always use at 0 or 1.
Other numbers are wrong.
* Digital gain. The preset is 1. Leave it there.
* Use the Range Indicator button to make sure you have no saturated pixels<
http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.
Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
Office: 646-501-0567 Cell: 914-309-3270
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