Posted by
Arvydas Matiukas on
URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589953.html
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I support very much Phil's approach "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".
However, on the other side I am dissapointed when PI/mentor expects
his/her student become almost a confocal expert after 3hr basic
training, and even suggests to ommit all that physics/optics/electronics
equating that to "rocket science". Quite often I am asked to provide
simple formal training just showing which knobs to turn and buttons to
push because more detail explanation confuses the student with
microscopy advices given by other lab members and lab protocols. My
approach always is that to achieve the best results a researcher of any
level must understand the details of the techniques he/she is using.
Please share your arguments for in depth training.
My General Confocal Best practices (for optimal image):
*fine adjustment of confocal focus
*pinhole at 1AU (exception only when sample is dim)
*gain providing full detector range (few over/under saturated pixels in
ROI ; laser power, offset, digital gain at defaults)
*pixel size matches lens resolution (exception too long acquisition)
*max scan speed
*averaging to reduce noise
*check color
*z-stack interval >= 0.5 optical slice
*save file in lsm format
*quality samples provide quality images
Finally, students often aim for a nice live/acquired image instead of
correct raw image. It is important to obtain raw image with correct
intensities and it can be later processed in many different ways to
produce nice looking final image emphasizing details of interest.\
Best regards,
Arvydas
+++++++++++++++++++++++++++++
Arvydas Matiukas, Ph.D.
Manager of NRB Shared Equipment
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept
>>> "Oshel, Philip Eugene" <
[hidden email]> 10/10/2019 1:00 PM >>>
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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
-----Original Message-----
From: Confocal Microscopy List <
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behalf of "Cammer, Michael" <
[hidden email]>
Reply-To: Confocal Microscopy List <
[hidden email]>
Date: Thursday, 10October, 2019 at 12:16
To: "
[hidden email]"
<
[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<
https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_imagej_saturation_index.html&d=DwIGaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=pjCVoeD77yFjyEe_DZmcKZn4dP-ybv5rCgAwrdbCikc&s=1EDj55zzjOtnvlVuJH4BW5aZzIIuDWCliEAE3QctagM&e=>. 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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