Posted by
Jacqueline Ross on
URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589963.html
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Hi everyone,
I'm late to this discussion but have the following comments. We have both Olympus and Zeiss confocals and previously had Leica as well.
Pinhole setting
I explain the function of the pinhole and recommend that they use Auto/1 AU but I also explain that there are times when you might open or close it. For example, we have people doing live imaging of zebrafish embryos who need to do z stacks at each time point. In this case, we often open up the pinhole a bit to get a thicker optical section so that we don't need to take as small a step size to cover the entire depth. It also means we can get away with less laser power as well as lower voltage, resulting in less noisy images. We always discuss the impact on the resolution, i.e. it's not really confocal but it's still much better than widefield as there's still much less out of focus blur. In fact, this morning I opened the pinhole up to double and we managed to get a good enough image to demonstrate that the staining was probably working but needed to be stronger. Sometimes I close the pinhole down, e.g. for fine structures (if there's plenty of signal) or for reflection imaging (always heaps of signal).
Offset
I do teach people to adjust the offset to make sure that they don't have zero pixel values in their image. One or two zero values (blue) are not a big deal but if they have a lot, then it's not good. If they want to do image analysis (intensity-based), deconvolution, then they should try to ensure no black pixels (or white).
Digital gain
I usually don't use it unless the voltage is very high (resulting in high noise) and I'm unable to increase laser power or slow speed down or do averaging because of the temporal resolution I want or because of photobleaching/phototoxicity.
Range indicator/HiLo/QLUT
I tell users always to use the Look Up Tables used to indicate over and under saturation. This is extremely important and I'm always disappointed if I come into a room and see people setting up their imaging without using these LUTs. The same applies (in my opinion) to widefield systems in regard to saturation. People should use LUTs, pixel saturation indicators or check out the histogram.
Files
We're quite nice and a little disorganised. Ideally all files should be gone from the hard drive after a month at the latest but we don't tidy up files this often. We do have a shared network drive that people can use to transfer files. I do not like any files left on the Desktop though and I will move these into the correct location if I find them there.
File format
For Olympus, I stress that they must always save as .oif or .oib before exporting to TIFF. The same applies for the Zeiss but we also use .lsm for our LSM 710 because I've had issues in the past with the colours coming up incorrectly when using other software with CZI. We now have an LSM 800 and that doesn't allow .lsm so I'm having to change my policy for that system!
Cheers,
Jacqui
Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND
Telephone: Ext 87438; DDI: +64 9 923 7438
Website:
http://www.auckland.ac.nz/biru-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 11 October 2019 5:16 a.m.
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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http://nyulmc.org/micros http://microscopynotes.com/