Re: training and bets practices for confocal

Posted by Ben Abrams-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589950.html

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Hi,

Regarding the pinhole, I tell my users that 1 AU is almost always a good
place to start, but that there may be some cases where it is worth it to
increase the pinhole size to a degree.  It is still confocal, unless
open to the point where your orthogonal projections look like widefield.
You can get a significant bump in brightness this way ( I'd argue this
/is/ fair) and are still getting a defined optical section thickness, so
3D reconstruction is still possible, just at a lower axial resolution. 
It really depends on the goal of the experiment what the worthwhile
trade-offs are. Users just need to be sure to accurately explain what
they did in the methods section.

I also think offset setting have to be set carefully in a sample/scope
dependent way.  I tell my users to always use a range indicator LUT and
set the offset so they just see a few zero intensity pixels, and have
gain set just below saturation.  This way they are maximizing the
dynamic range of their images.  Samples that are to be compared
quantitatively should use the same settings.  I also talk to them about
the balance of gain and laser power, noise vs. bleaching etc. as Josh
mentioned.

I think it is useful to have this discussion re best practices in
training, thanks.

-Ben

On 10/10/19 9:15 AM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/

--
Benjamin Abrams,  Ph.D.
Director
UCSC Life Sciences Microscopy Center

University of California, Santa Cruz
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