Re: training and bets practices for confocal

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URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589958.html

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Hi Arvydas, Michael and others,

We switched to czi when using the Elyra super-resolution microscope, as the lsm format did not handle the small pixel sizes properly. As I understand czi has other advantages such as handling very large images, I mean not only large data sets but single images with e.g. 60,000 x 60,000 pixels which exceeds the 2^31 limit. Maybe not so common in confocal microscopy, but happens when you image a whole coverslip with 300 nm resolution.

A Zeiss application specialist once told me that for the newer Zeiss microscopes (710 onwards?) the digital gain does more than just multiplying by a factor, not sure what exactly, but it seems to me important where the multiplication happens, if it is before the A/D conversion, it might actually help to reduce digitalisation noise. I found it helpful in multi-photon microscopy when the signal is low. 

For the pinhole setting, on the Leica microscopes there is a wavelength setting to which the 1 AU refers (usually hidden), the preset seems to be 580 nm, so you might want to change it for green or blue dyes. They also automatically resets to 1 AU when you change the objective which helps the inexperienced user, but can be very annoying when working with other pinhole sizes. When the signal is very bright we sometimes close the pinhole to increase resolution, easier than working with silly low laser powers like 0.03%

best wishes

Andreas


-----Original Message-----
From: Arvydas Matiukas <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 10 Oct 2019 19:44
Subject: Re: training and bets practices for confocal

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Michael,

Thanks for bringing up for the discussion  general confocal  training principles. It was very useful.
 
I wonder how  .czi image format is better than  .lsm . I have been having problems importing .czi into ImageJ so I advise users on .lsm . Did I miss late development in this situation?


Thanks,
Arvydas
>>> "Cammer, Michael" <[hidden email]> 10/10/2019 12:15 PM >>>
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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=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=IBOH5Kp8DzC98-n5vrmelr6womyIubyxQ4_5lZlO17A&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  [hidden email]<mailto:[hidden email]>
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