Posted by
Reece, Jeff (NIH/NIDDK) [E]-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589956.html
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For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...
When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU. The sweet spot is usually 0.6-0.7AU.
I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers. It bothers me when I read a paper and all they say is "confocal".
Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly. Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi. We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.
Other things you didn't ask about, but I'll quickly mention:
...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels. Same for z-stack delta which is for sampling in z. These values can be changed somewhat, always exceptions, various reasons. Generally multiply the answer by 1.5-2x if performing decon.
...Use the fastest scan speed possible.
... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly. But thank you for trying, software engineers.
...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).
Cheers,
Jeff
-----Original Message-----
From: Cammer, Michael <
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Sent: Thursday, October 10, 2019 12:16 PM
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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