http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589957.html
manage the file. This is especially true if the question of interest is one
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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 <
[hidden email]>
> Sent: Thursday, October 10, 2019 12:16 PM
> To:
[hidden email]
> 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
[hidden email]
> <mailto:
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>
http://nyulmc.org/micros http://microscopynotes.com/>