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In cases when people are interested in relative brightness, I advise them first to image the "most positive" control and adjust the brightness such that there are no or few saturated pixels in the areas of interest. And then to keep the same settings for all other images that need to be compared, even if they look too dark
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From: Confocal Microscopy List <
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Sent: Thursday, October 10, 2019 1:00 PM
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Subject: Re: training and bets practices for confocal
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Generally I would agree, but:
Only Siths deal in absolutes. (Which is an absolute...) So, "Always use" and the like is not true. Sometimes one has to do something against the rules to get the best results. So you title "General Best Practices" should be understood as that, not hard rules.
* "No saturated pixels" should be "One or a few saturated and one or a few undersaturated pixels." If there isn't that "one or a few", then the user can't know if they are actually using the full dynamic range available, and therefore could be losing data.
But *only* one or a few.
* There is nothing wrong with saving files as TIFF (or other uncompressed file format), *but* also save the image/experiment in the confocal system's native file format (e.g. nd2 for Nikon).
I would add that initial training MUST include not just how to use the microscopy and software but WHY the various controls and software settings are being used when and as they are.
Meaning, training must include some physics and chemistry as well as electronics. What exactly are the Gain and Offset (Black level) adjusting and in what part of the system. What does varying laser power *really* adjust? The laser itself? a neutral density filter? an ATOF? Why and when would one turn up the Gain instead of the Laser. Etc. and so on.
Easy enough to cover in a class, but these can still be covered during a training session.
My explanation when training is the user is looking at the image, and wants to achieve a certain effect: clean up an image (and what "clean up' means - reduce noise or something else?), get a brighter signal, or whatever. The user should be able to look at the image and go to the correct control(s) and make the correct adjustment(s) without having to sit and think or page through notes or whatever and bleach or kill their sample. They can't do this if they don't understand the whys of the system, not only the hows.
Other than that, I just point out the things they can do that are Bad and Expensive and They Will Get To Pay For It. That works well.
Phil
-------------
Philip Oshel
Imaging Facility Director
Biology Department
1304 Biosciences
1455 Calumet Ct.
Central Michigan University
Mt. Pleasant, MI 48859
989 774-3576 office
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Date: Thursday, 10October, 2019 at 12:16
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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<
https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&data=02%7C01%7Cmmodel%40KENT.EDU%7C836b4364d165408312ed08d74da37a43%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637063236839198887&sdata=Uq06XIU1GeU8V%2FmVDdIxXfJSBPfeJT9ehHAxV6AjWEk%3D&reserved=0>. 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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