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Hi Jason,
>set the offset so all pixels are just above 0 (showing no blue - for Zeiss or green - for Leica)
While I do something similar, this method necessarily leads to an above zero background signal. When the signal fluctuates around an offset with a certain noise distribution and you set the instrument offset to allow no (or just a few) zero pixels, your mean signal is probably 3 or more standard deviations above zero. So best would be to do this is post-processing, convert to a format which allows negative values (32 bit in Fiji) and subtract the background, so that it is actually zero, but who does this? For a strong signal this does not matter much, but for noisy signal it might make quite a difference.
For my liking, the fixed offset setting on the Leica HyD detectors is a bit too high, it seems to cut off some very low signal, whereas this does not happen on a GaAsP PMT.
best wishes
Andreas
-----Original Message-----
From: Kirk, Jason M. <[log in to unmask]>
To: CONFOCALMICROSCOPY <[log in to unmask]>
Sent: Fri, 11 Oct 2019 0:47
Subject: Re: training and bets practices for confocal
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Hi Michael,
A note on the offset item:
“ * Offset. Always use at 0 or 1. Other numbers are wrong.”
This offset value is highly dependent on the calibration vendors do behind the scenes. So YMMV when using their defaults such as 0 or 1.
Ideally you just don’t want to clip the low end values in the final image.
What I typically recommend is when using your range indicator LUT, scan the sample with no laser active (AOTF off) and set the offset so all pixels are just above 0 (showing no blue - for Zeiss or green - for Leica).
I find that on a given system - the values wind up being fairly consistent - but there may be some variation between instruments.
Hope this helps!
-Jason
--------------------------------------------
Jason M. Kirk
Technical Director, Optical Imaging & Vital Microscopy Core (OiVM)
Baylor College of Medicine
Ph: 713.798.6486
Email: [log in to unmask]
http://www.bcm.edu/oivm
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From: Confocal Microscopy List <[log in to unmask]> on behalf of Cammer, Michael <[log in to unmask]>
Sent: Thursday, October 10, 2019 11:15:46 AM
To: [log in to unmask] <[log in to unmask]>
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=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=w53VhOV7aYHc2JbvneV55tVHLwioHN59G6XB36pQTxs&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 [log in to unmask]<mailto:[log in to unmask]>
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