Posted by
Tim Feinstein on
URL: http://confocal-microscopy-list.275.s1.nabble.com/training-and-bets-practices-for-confocal-tp7589945p7589955.html
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Offset is useful if you are acquiring in 8-bit mode, since your dynamic range is so low that you can't afford background signal, but (in my opinion) people shouldn't acquire in 8-bit mode. Even when someone just needs a snapshot for lab meeting I generally encourage folks to take it in 12-bit and post-process in ImageJ. This seems like a side point, but are there any circumstances where you encourage people to acquire in 8-bit mode? I understand that it's rare for >256 pixels to strike a detector in the space of one pixel, but electron counts can get pretty high so 12-bit seems more appropriate when you're not using photon counting mode.
Re pinholes, I encourage people to know the XY/Z resolution limit of each objective on that system so they can keep the pinhole set to 1 AU when they're near that limit. If resolution (especially in Z) is well below an objective's performance limits then a pinhole of AU=2 shouldn't impact image quality. Mostly I tell people not to touch it since it's the last thing you should try after you've tried everything else.
Best,
T
On 10/10/19, 1:29 PM, "Confocal Microscopy List on behalf of Craig Brideau" <
[hidden email] on behalf of
[hidden email]> wrote:
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On Thu, Oct 10, 2019 at 10:16 AM Cammer, Michael <
[hidden email]> wrote:
> *****
> * 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".
>
I would say it is not 'Airy matched'. It is still optically sectioning,
which is the point of a confocal, and there are foreseeable situations
where a user may be willing to compromise with a thicker optical slice for
additional signal. Live samples, where you are limited in the amount of
laser power you can use, does come to mind in this case.
> * Offset. Always use at 0 or 1.
> Other numbers are wrong.
> * Digital gain. The preset is 1. Leave it there.
>
I strongly agree with the above. Never do anything at acquisition time that
you couldn't do later in post processing if absolutely necessary. By using
offset you are throwing away data. Digital Gain simply multiplies the
captured numbers and is not valid as it does nothing for your
signal-to-noise. If you have to, once you have the complete data loaded in
the software of your choice you can play with offset, etc, but for image
ethics purposes any modifications should be clearly stated, and should only
be for the purposes of clarity.
> * Use the Range Indicator button to make sure you have no saturated
> pixels<
https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&data=02%7C01%7Ctnf8%40PITT.EDU%7C1d89833f5d144db880cb08d74da774de%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637063253921137431&sdata=QcYIH8RK%2BjYKf2QNLCHJ6coQpuXUIlJ3e6KNC9kEq64%3D&reserved=0>. If you
> see red pixels, you need to turn down the Gain or Laser.
> Saving Files
>
I agree that saturation is bad, but there is a subtlety I always have to
emphasize with people who cling too tightly to this mantra: Do not saturate
your *areas of interest* in your sample. Example: your sample exhibits
overstained nuclei when all you care about is microtubules in the
cytoplasm; go ahead and saturate that nucleus if it means you are using
more dynamic range to image the tubulin that you are actually interested in.
> All files should be stored in Drive D:.
> Files left on the desktop, drive C, Pictures folder, etc will be deleted.
>
More generally I tell users to not expect data left on the microscope to
still be there later. I am somewhat flexible on this, but warn users that
if the drive is full beyond a certain threshold I will arbitrarily delete
any stray files to ensure operation of the equipment for the next user. I
also keep a stash of high-capacity thumb drives for users to sign out if
they are desperate for somewhere to temporarily store their data to
transfer it.
Craig