training and bets practices for confocal

[Jeremy Adler-4]

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Laser power is the trickiest choice,
Clearly we need to avoid fluorophore saturation or we relatively increase the photons from out of focus areas.
But how to actually check since - the power will vary between fluorophores, objectives and tissues and mountants,
so needs to be found experimentally.
We can find an in focus object and ramp up the laser  to find the linear region- but this not practicable to do frequently.
But it seems like a feature that could easily be made available in software - is this present in any commercial microscope ?
In practice we mostly suggest sticking to low power - 2% for the diode lasers on our LSM700, but a more rational choice or method of making a choice would be better

Ideas, suggestions.

Jeremy Adler
BioVis
Uppsala

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Mark Cannell
Sent: den 11 oktober 2019 08:37
To: [hidden email]
Subject: Re: training and bets practices for confocal

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Hi George

I'm puzzled as to why you suggest PMTs detectors are non-linear. With proper dynode chain design linearity exists over ~5 orders of magnitude brightness. If the filtering into the A/D converter is too fast for the sampling you could get aliasing effects but that's another story. Photon counting mode is more likely to be non-linear due to pulse pile up at high rates. The intrinsic linearity of PMTs was carefully explored a while ago as they formed the core of all photometry.

Regards Mark

On 10/11/19, 2:17 AM, "Confocal Microscopy List on behalf of George McNamara" <[hidden email] on behalf of [hidden email]> wrote:

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    Hi Jeff,

    Confocal Sweet Spot! I love your phrase!!!

    I posted a visual at linkedin,
    https://www.linkedin.com/posts/georgemcnamara_confocal-sweet-spot-jeff-reese-nih-activity-6588214058011410432-IBV7/


    I suggest a slight simplification to one, easy to remember value: 0.666
    Airy units.

    GPU deconvolve all the data, please!!!

    Best confocal file formats: OIR and LIF (Olympus FV3000RS and Leica SP8)
    ... of course I've retired two Zeiss LSM510's. Good to know that CZI
    supports 60,000x60,000 pixels (another reply in thread).

    PMT gain (HV = High Voltage): my Olympus rep, Jason Brenner, simplified
    this for our FV3000RS: the optimal HV setting for our four GaAsP and two
    GaAs PMTs is 500 (that is, 500 mV). Yes there are times I suggest
    UNoptimal values (usually in steps of 50, but maybe I'll try 666 mV next
    time I am at 0.666 Airy Units), but if these PMTs have an optimum,
    likely everyone's has an optimum. Since the user has control over the
    HV, and the HV controls the amplification of electrons in the PMT (and
    ignoring a lot of PMT details), I'll just claim that very few readers
    (or my) PMT based confocal microscope are really reporting a 2x change
    in fluorophore for intensity level 1000 vs 2000 (after subtracting the
    PMT offset floor from the raw data output). More on quantitation later.

    PMT offset: depends on the detector, mode (FV3000RS galvo mode is 12-bit
    range, resonant scan mode is 10-bit range), I usually teach with Hi-Lo
    LUT enabled, "no blue, no red", while live, no averaging.

    PMT multiplier: 1 ... and a request to confocal companies with this:
    please kill this GUI element!

    Better than PMT: Leica HyD in 'counting mode' counts photons. Yes, there
    is some counting saturation limit (we have 2nd gen, ask your Leica rep
    how much faster counting SMD HyDs are), and other companies have similar
    Hybrid detectors, with their own tweaks (and benefits and price points),
    I believe first with Hybrid was Becker&Hickl (FLIM, now fast FLIM), now
    also ISS, PicoQuant, probably others.

    Disclosure: I am registered for the free (fast) TCSPC workshop hosted by
    Becker&Hickl and Boston Electronics, Oct 31-Nov 1, 2019, Bethesda MD.
    I've also hosted a 2018 Leica FALCON demo, and various microscope demo's
    and talks.

    https://www.becker-hickl.com/events/13th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-science/

    I agree with Jeff: fastest scan speed possible. In particular, Leica SP8
    starts up with default of 400 Hz, I tell all users to use 600 Hz (or
    faster if their objective lens choice and zoom <--> field of view is
    appropriate).

    Best way to make live cells brighter: drop EGFP ---- soooooo 1996 ---
    Nathan Shaner's AausFP1 is 5x brighter and ultra narrow (for FPs)
    emission peak (10x with tandem dimer)
    https://www.biorxiv.org/content/10.1101/677344v2.full Plasmids not in
    addgene yet (its a preprint), sequence in paper and supplemental info
    (and user's might want to use their own codon and/or other expression
    optimizations).

    ***
    Confocal quantitation - some thoughts:

    -- the lasers on confocal microscopes are not perfectly stable, and
    often very unstable (could be the AOTF intensity controller more than
    the laser modules - my recollection is Bob Zucker, US EPA, explained
    this to me ... and no reason to think any of the confocal companies have
    made any serious effort to prevent these fluctiations). So no reason to
    think AM vs PM, or even a few minutes apart, stable (to say nothing of
    corner to corner at low zoom).

    -- I pointed out above there is no reason to think that anyone's PMT
    based confocal microscope value 1000 vs 2000 is really a 2x change in
    fluorophore concentration (per voxel etc). Or that similar values would
    appear if the specimen was scanned AM vs PM (see previous paragraph).

    So: most -- that is PMT point scanner - confocal data is UNquantitative
    with respect to whatever intensity measurement the user thinks they are
    measuring of (a major use of confocal) single cells.

    Photon counting is better (some PMTs can be operated in photon counting,
    APDs are, but often slow, various Hybrid detectors nice behavior,
    trusting Hamamatsu and whoever does the electronics to 'get it right',
    or at least not mess it up too badly), but still subject to laser/AOTF
    intensity fluctuation issues.


    enjoy,
    George
    p.s. one of the other posts in this thread mentioned Leica SP8 pinhole
    value control is hard to find and by default is set to 580 nm: In Leica
    LAS X the drop down is easy to find, the "Airy 1.0" is easy to find, the
    button uses the midpoint of the emission range when a single detector is
    enabled (ex. 510-550 nm range --> 530 nm mid-point). If two detectors
    (we have two HyDs) are on, it uses the midpoitn of those. Easy to turn
    off one detector, click "1" using the other, then turn on both (user
    choice).

    On 10/10/2019 2:46 PM, Reece, Jeff (NIH/NIDDK) [E] 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.
    > *****
    >
    > For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...
    >
    > When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU.  The sweet spot is usually 0.6-0.7AU.
    >
    > I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers.  It bothers me when I read a paper and all they say is "confocal".
    >
    > Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly.  Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi.  We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.
    >
    > Other things you didn't ask about, but I'll quickly mention:
    > ...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
    > ... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
    > ... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels.  Same for z-stack delta which is for sampling in z.  These values can be changed somewhat, always exceptions, various reasons.  Generally multiply the answer by 1.5-2x if performing decon.
    > ...Use the fastest scan speed possible.
    > ... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly.  But thank you for trying, software engineers.
    > ...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).
    >
    > Cheers,
    > Jeff
    >
    > -----Original Message-----
    > From: Cammer, Michael <[hidden email]>
    > Sent: Thursday, October 10, 2019 12:16 PM
    > To: [hidden email]
    > Subject: training and bets practices for confocal
    >
    > *****
    > 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/










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[Sylvie Le Guyader]

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Hi Michael



The question is: why offer a 3h training at all? It gives users/group leaders the false impression that it is possible to be trained in 3h.



In our facility our training takes 15h (5x3h within 1 week, only using the user’s samples and acquiring real data) and this comes after 1h of troubleshooting the sample and experimental design.



We never compromise on the training, even for Master students who will only be at the facility for 3 months. They get the full training as well.

People who come to us in a hurry with ‘a few slides to image before resubmitting their paper’ are told that they will need to collaborate with one of our trained users. The users get to show off their skills and get their name on the paper. The stressed authors get a super collaborator who quickly delivers relevant images. Win/win.



We believe that our role as an imaging facility is not to ensure that our users do not destroy our equipment but to make sure that them not destroying the equipment is a natural consequence of our good training.



This strategy has several beneficial effects:

- our users trust that we know what we are doing to a very level so they come to us to ask questions if they are unsure and therefore we avoid accidents.

- over the years, the level of awareness and education in microscopy in the labs around us has increased very significantly. Group leaders who hear their users explain their points also get educated. Therefore the accuracy and complexity of the scientific questions the group leaders ask is getting higher and higher, making our job super interesting! 😃

I would hate to spend my time putting out fires. Over the past 11 years, I can count on the fingers of my hands the number of times we have had problems because of our users being messy/unaware/careless.



I personally believe that all the parameters should be explained in detail so that the users can choose to correctly match their imaging settings with their sample and scientific question. There is a reason why manufacturers give us the option to adjust the pinhole, the detector offset...



A pinhole of 1 AU is only a compromise between good optical sectioning and brightness. If your samples are very bright, you are much better off setting the pinhole to 0.7 AU (and then you should decide which wavelength is most important to match). But if closing the pinhole means that you then need to push the detector gain so much that SNR is unacceptable or use tons of laser and bleach before the end of your z stack, you need to readjust your first choice.



About the offset, we usually bring it down until we start seeing some 0 value pixels then we bring it one set up. I agree with the suggestion to have no saturation at all in the area of interest: if there is a blog of precipitated antibody outside the cell of interest, it doesn’t matter if it is saturated.



On our webpage<https://ki.se/en/bionut/teaching-material> you can find a document called Typical workflow of how to set a confocal (1.2 AU on a Nikon system corresponds to 1 AU due to some unusual pinhole shape). During the training, we go through every single point, explain what the options are and why, and discuss with the user to find the best objective, settings, strategy… to answer their scientific question reliably. If you look at the document you will see that 15h is no luxury! 😉



If ever you check our videos and Typical workflow and find errors/omission, please let me know! I am very keen on getting feedback!



Med vänlig hälsning / Best regards



Sylvie



@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Blickagången 16,

Room 7362 (lab)/7840 (office)

14157 Huddinge, Sweden

mobile: [phone]

LCI website

Follow our microscopy blog!



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael
Sent: 10 October 2019 18:16
To: [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://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Cb0dbc3e2d7d64d0c937d08d74d9d39de%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063209986890774&amp;sdata=WW8Lb8bYgJPcJ4zR4V%2B40x8t4A57K1n12DH2xmPLLjQ%3D&amp;reserved=0>. 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]<mailto:[hidden email]%3cmailto:[hidden email]>>

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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. <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
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: [hidden email]

http://www.bcm.edu/oivm
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 11:15:46 AM
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=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  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=

[Zdenek Svindrych-2]

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Andreas,

I do just that (convert to ImageJ 32-bit and subtract the offset), but
mostly for camera-based data (widefield, spinning disk). That makes sense,
for the amplitude of the useful signal can in some cases be much lower that
the offset.

Also, HyDs are photon counting. Always. So offset does not make sense here.
There is no real gain either. Only digital gain to scale the photon counts
to the full range (e.g. 16 bit), relative to what Leica assumes is an
adequate range.

This also reminds me the "sweet spot" PMT gain: the sensitivity of a PMT
increases monotonically with gain (the readout noise is made smaller
relative to the signal). But the poisson-limited dynamics range decreases
with increasing gain, that's where the "sweet spot" tradeoff is. In the
extreme case of a photon-counting detector the SNR is 1, that is, you can
only get useful signal after substantial averaging (or summing in this
case)!
So, if there is no other way to boost your signal, increasing the gain will
not hurt (though it may not help either).

Best, zdenek


On Sun, Oct 13, 2019 at 8:59 AM Andreas Bruckbauer <
[hidden email]> 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.
> *****
>
> 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. <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Fri, 11 Oct 2019 0:47
> Subject: Re: training and bets practices for confocal
>
> *****
> 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.
> *****
>
> 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: [hidden email]
>
> http://www.bcm.edu/oivm
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Cammer, Michael <[hidden email]>
> Sent: Thursday, October 10, 2019 11:15:46 AM
> To: [hidden email] <[hidden email]>
> Subject: training and bets practices for confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e=
> 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<
> 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  [hidden email]
> <mailto:[hidden email]>
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Jeremy Adler-4]

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*****

Hi Jason,

Allowing users to adjust the offset at the image acquisition stage is very dangerous.
By editing out the background and any low intensity signal the user dislikes, a diffuse fluorophore can be made to appear to have a much more specific distribution.
It also short circuits the increasing requirement by journals that original images should be made available - the original images are deceptive.
Images should be acquired properly with all intensities on scale - they can subsequently be processed but any processing should described in the methods section of articles.

J. Adler (2005)
Veracity of raw images can also come into question.
Nature, 435, 736.

Jeremy Adler
BioVis
Uppsala Universitet
Sweden

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Andreas Bruckbauer
Sent: den 13 oktober 2019 14:59
To: [hidden email]
Subject: Re: training and bets practices for confocal

*****
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.
*****

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. <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 11 Oct 2019 0:47
Subject: Re: training and bets practices for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

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: [hidden email]

http://www.bcm.edu/oivm
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 11:15:46 AM
To: [hidden email] <[hidden email]>
Subject: training and bets practices for confocal

*****
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e=  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<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  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=








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[Jason M. Kirk]

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*****

Hi Andreas,

I agree that setting the offset too high may necessitate a separate dark current correction in post - however setting this value accurately in the absence of light as I described previously would limit the need for such a correction.

Hi Jeremy,

Allowing users to adjust the offset is just as important (and dangerous) as allowing them to set the gain.  But it is a necessary function that can vary widely between systems.  That was really the point to my original comment on Michael's training tips - the digital offset is not something that should simply be ignored.

You most certainly want to avoid any values being clipped on the low end - however always using an offset value set to 0 relies too heavily on the internal offset calibration done by the vendor to be accurate.  For example, on both of our LSM 780/880 platforms from Zeiss we routinely find the offset for the multi-alkali PMTs to be ~200 (out of 65535) and ~100 for the GaAsP array.  If we just left these at 0 we would get significant clipping on the low end.  

Now I could demand that Zeiss' service recalibrate that offset so they were closer to 0 - but by teaching my users to properly interpret and correct with the offset - they don't have to worry about the precision of the instrument calibration for which they have no control.  And this knowledge carries over onto other systems they might use that will behave slightly differently.

<apologiesfortherant>

Too often we hear people say "just don't touch that setting" without proper context for its use.  Values like offset - and less popular - Digital Gain - have their place and do give the user more control - which is a good thing.

Ultimately the way I look at it is this:  I would much rather have people know how to adjust the image settings so background is above 0 and peak signal is below 65535 (for 16 bit) and then have to make corrections in post rather than have a savvy reviewer throw on a LUT only to ask them why their RAW data is clipped and force them to do the imaging over again.  You wouldn't ever expect a saturated image to pass muster - and the same is true on the low end.  Your instrument has control over this and users should know how to use it.

</ apologiesfortherant>

-------------------------------------------------------------------
Jason M. Kirk
Technical Director, Optical Imaging & Vital Microscopy Core (OiVM)
Baylor College of Medicine
Ph: 713.798.6486
Email: [hidden email]
http://www.bcm.edu/oivm

[Armstrong, Brian]

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*****

Hi, I would have to agree with Jeremy. I am sure all of you Confocal experts are doing it correctly, but this topic started as "what should you teach users in training". The most frightening words in confocal microscopy (and digital imaging for that matter) are "I am just removing the background".

Abuse of the offset is far to common among inexperienced microscopists.

Cheers,

Brian Armstrong PhD
Associate Research Professor
Developmental and Stem Cell Biology
Diabetes and Metabolic Diseases
Director, Light Microscopy Core
Beckman Research Institute, City of Hope



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Monday, October 14, 2019 1:04 AM
To: [hidden email]
Subject: Re: training and bets practices for confocal

[Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.]





*****
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*****

Hi Jason,

Allowing users to adjust the offset at the image acquisition stage is very dangerous.
By editing out the background and any low intensity signal the user dislikes, a diffuse fluorophore can be made to appear to have a much more specific distribution.
It also short circuits the increasing requirement by journals that original images should be made available - the original images are deceptive.
Images should be acquired properly with all intensities on scale - they can subsequently be processed but any processing should described in the methods section of articles.

J. Adler (2005)
Veracity of raw images can also come into question.
Nature, 435, 736.

Jeremy Adler
BioVis
Uppsala Universitet
Sweden

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Andreas Bruckbauer
Sent: den 13 oktober 2019 14:59
To: [hidden email]
Subject: Re: training and bets practices for confocal

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

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. <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 11 Oct 2019 0:47
Subject: Re: training and bets practices for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

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: [hidden email]

http://www.bcm.edu/oivm
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 11:15:46 AM
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=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  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=








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[Jacqueline Ross]

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*****

Hi Brian/Jeremy,

I think what most people, including me, are saying is that we take care not to remove any information, i.e. we do not recommend making the image "blacker" but the opposite, make sure no zero value pixels are in the image.

In my experience, there are times when the image would have a lot of "black" zero value pixels if the offset was not adjusted to make sure all values were above zero. Increasing voltage/laser power in these cases may just result in over-saturation.

We make sure all of our users understand that offset should not be used to remove what they perceive is background...and sometimes it isn't background!

Cheers,

Jacqui
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Brian Armstrong [[hidden email]]
Sent: 15 October 2019 04:30
To: [hidden email]
Subject: Re: training and bets practices for confocal

*****
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*****

Hi, I would have to agree with Jeremy. I am sure all of you Confocal experts are doing it correctly, but this topic started as "what should you teach users in training". The most frightening words in confocal microscopy (and digital imaging for that matter) are "I am just removing the background".

Abuse of the offset is far to common among inexperienced microscopists.

Cheers,

Brian Armstrong PhD
Associate Research Professor
Developmental and Stem Cell Biology
Diabetes and Metabolic Diseases
Director, Light Microscopy Core
Beckman Research Institute, City of Hope



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Monday, October 14, 2019 1:04 AM
To: [hidden email]
Subject: Re: training and bets practices for confocal

[Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.]





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*****

Hi Jason,

Allowing users to adjust the offset at the image acquisition stage is very dangerous.
By editing out the background and any low intensity signal the user dislikes, a diffuse fluorophore can be made to appear to have a much more specific distribution.
It also short circuits the increasing requirement by journals that original images should be made available - the original images are deceptive.
Images should be acquired properly with all intensities on scale - they can subsequently be processed but any processing should described in the methods section of articles.

J. Adler (2005)
Veracity of raw images can also come into question.
Nature, 435, 736.

Jeremy Adler
BioVis
Uppsala Universitet
Sweden

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Andreas Bruckbauer
Sent: den 13 oktober 2019 14:59
To: [hidden email]
Subject: Re: training and bets practices for confocal

*****
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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. <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 11 Oct 2019 0:47
Subject: Re: training and bets practices for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
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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: [hidden email]

http://www.bcm.edu/oivm
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 11:15:46 AM
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=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  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=








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[Praju Vikas Anekal]

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*****

Hi all,



*first time poster*



I wanted to thank all the contributors for an 'illuminating' discussion (pun incidental).



In the training I typically conduct for confocal, there is one more topic I like to cover: Nyquist sampling

Since the Image capturing process is essential an 'Analog to Digital' conversion, I try to explain to the users the importance of achieving Nyquist sampling. (or slight oversampling to 3X in most cases)

I also explain that the Nyquist applies not only to digital pixel resolution X/Y and Z (compared to the optical resolution) but also to the image bit rate or to temporal sampling rate.

Finally I finish that topic by saying that the image acquisition is only the first part that will lead into quantitative image analysis. And this part requires a useful sample size for high statistical power in the analysis.

The take home message being to "Optimise the acquisition to the information you want to analyse"





Yours sincerely,



Praju Vikas Anekal. Ph.D.

Biomed Imaging Microscopist, Biomedical Imaging Research Unit.

Faculty of Medical and Health Sciences, The University of Auckland.

E-Mail : [hidden email] , Ext : 87831





>>>-----Original Message-----

>>>From: Confocal Microscopy List

>>><[hidden email]> On Behalf Of Brian

>>>Armstrong

>>>Sent: Tuesday, 15 October 2019 4:31 AM

>>>To: [hidden email]

>>>Subject: Re: training and bets practices for confocal

>>>

>>>*****

>>>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.

>>>*****

>>>

>>>Hi, I would have to agree with Jeremy. I am sure all of you Confocal experts are

>>>doing it correctly, but this topic started as "what should you teach users in training".

>>>The most frightening words in confocal microscopy (and digital imaging for that

>>>matter) are "I am just removing the background".

>>>

>>>Abuse of the offset is far to common among inexperienced microscopists.

>>>

>>>Cheers,

>>>

>>>Brian Armstrong PhD

>>>Associate Research Professor

>>>Developmental and Stem Cell Biology

>>>Diabetes and Metabolic Diseases

>>>Director, Light Microscopy Core

>>>Beckman Research Institute, City of Hope

>>>

>>>

>>>

>>>-----Original Message-----

>>>From: Confocal Microscopy List

>>>[mailto:[hidden email]] On Behalf Of Jeremy

>>>Adler

>>>Sent: Monday, October 14, 2019 1:04 AM

>>>To: [hidden email]<mailto:[hidden email]>

>>>Subject: Re: training and bets practices for confocal

>>>

>>>[Attention: This email came from an external source. Do not open attachments or

>>>click on links from unknown senders or unexpected emails.]

>>>

>>>

>>>

>>>

>>>

>>>*****

>>>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.

>>>*****

>>>

>>>Hi Jason,

>>>

>>>Allowing users to adjust the offset at the image acquisition stage is very dangerous.

>>>By editing out the background and any low intensity signal the user dislikes, a diffuse

>>>fluorophore can be made to appear to have a much more specific distribution.

>>>It also short circuits the increasing requirement by journals that original images

>>>should be made available - the original images are deceptive.

>>>Images should be acquired properly with all intensities on scale - they can

>>>subsequently be processed but any processing should described in the methods

>>>section of articles.

>>>

>>>J. Adler (2005)

>>>Veracity of raw images can also come into question.

>>>Nature, 435, 736.

>>>

>>>Jeremy Adler

>>>BioVis

>>>Uppsala Universitet

>>>Sweden

>>>

>>>-----Original Message-----

>>>From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>>

>>>On Behalf Of Andreas Bruckbauer

>>>Sent: den 13 oktober 2019 14:59

>>>To: [hidden email]<mailto:[hidden email]>

>>>Subject: Re: training and bets practices for confocal

>>>

>>>*****

>>>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.

>>>*****

>>>

>>>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. <[hidden email]<mailto:[hidden email]>>

>>>To: CONFOCALMICROSCOPY

>>><[hidden email]<mailto:[hidden email]>>

>>>Sent: Fri, 11 Oct 2019 0:47

>>>Subject: Re: training and bets practices for confocal

>>>

>>>*****

>>>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.

>>>*****

>>>

>>>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: [hidden email]<mailto:[hidden email]>

>>>

>>>http://www.bcm.edu/oivm

>>>________________________________

>>>From: Confocal Microscopy List

>>><[hidden email]<mailto:[hidden email]>> on behalf of Cammer,

>>>Michael <[hidden email]<mailto:[hidden email]>>

>>>Sent: Thursday, October 10, 2019 11:15:46 AM

>>>To: [hidden email]<mailto:[hidden email]>

>>><[hidden email]<mailto:[hidden email]>>

>>>Subject: training and bets practices for confocal

>>>

>>>*****

>>>To join, leave or search the confocal microscopy listserv, go to:

>>>https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-<https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e>

>>>2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-<https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e>

>>>KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8<https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e>

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>>>Post images on https://urldefense.proofpoint.com/v2/url?u=http-<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e>

>>>3A__www.imgur.com&d=DwIFAg&c=ZQs-<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e>

>>>KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e>

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>>>IUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e>=  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<https://urldefense.proofpoint.com/v2/url?u=http-<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=%20>

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>>>wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=w53VhO<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=%20>

>>>V7aYHc2JbvneV55tVHLwioHN59G6XB36pQTxs&e= <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=%20> >. 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]<mailto:[hidden email]%3cmailto:[hidden email]>>

>>>https://urldefense.proofpoint.com/v2/url?u=http-<https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e>

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>>>https://urldefense.proofpoint.com/v2/url?u=http-<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e>

>>>3A__microscopynotes.com_&d=DwIFAg&c=ZQs-<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e>

>>>KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e>

>>>wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e>

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>>>

>>>

>>>

>>>

>>>

>>>

>>>

>>>

>>>När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi

>>>behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här:

>>>http://www.uu.se/om-uu/dataskydd-personuppgifter/

>>>

>>>E-mailing Uppsala University means that we will process your personal data. For

>>>more information on how this is performed, please read here:

>>>http://www.uu.se/en/about-uu/data-protection-policy

>>>

>>>------------------------------------------------------------

>>>-SECURITY/CONFIDENTIALITY WARNING-

>>>

>>>This message and any attachments are intended solely for the individual or entity to

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[0000001ed7f52e4a-dmarc-request]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Sylvie, Do you do your 15 hours training with a group of trainees or 1:1? best wishes Andreas  -----Original Message-----
From: Sylvie Le Guyader <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 11 Oct 2019 12:17
Subject: Re: training and bets practices for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
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Hi Michael



The question is: why offer a 3h training at all? It gives users/group leaders the false impression that it is possible to be trained in 3h.



In our facility our training takes 15h (5x3h within 1 week, only using the user’s samples and acquiring real data) and this comes after 1h of troubleshooting the sample and experimental design.



We never compromise on the training, even for Master students who will only be at the facility for 3 months. They get the full training as well.

People who come to us in a hurry with ‘a few slides to image before resubmitting their paper’ are told that they will need to collaborate with one of our trained users. The users get to show off their skills and get their name on the paper. The stressed authors get a super collaborator who quickly delivers relevant images. Win/win.



We believe that our role as an imaging facility is not to ensure that our users do not destroy our equipment but to make sure that them not destroying the equipment is a natural consequence of our good training.



This strategy has several beneficial effects:

- our users trust that we know what we are doing to a very level so they come to us to ask questions if they are unsure and therefore we avoid accidents.

- over the years, the level of awareness and education in microscopy in the labs around us has increased very significantly. Group leaders who hear their users explain their points also get educated. Therefore the accuracy and complexity of the scientific questions the group leaders ask is getting higher and higher, making our job super interesting! 😃

I would hate to spend my time putting out fires. Over the past 11 years, I can count on the fingers of my hands the number of times we have had problems because of our users being messy/unaware/careless.



I personally believe that all the parameters should be explained in detail so that the users can choose to correctly match their imaging settings with their sample and scientific question. There is a reason why manufacturers give us the option to adjust the pinhole, the detector offset...



A pinhole of 1 AU is only a compromise between good optical sectioning and brightness. If your samples are very bright, you are much better off setting the pinhole to 0.7 AU (and then you should decide which wavelength is most important to match). But if closing the pinhole means that you then need to push the detector gain so much that SNR is unacceptable or use tons of laser and bleach before the end of your z stack, you need to readjust your first choice.



About the offset, we usually bring it down until we start seeing some 0 value pixels then we bring it one set up. I agree with the suggestion to have no saturation at all in the area of interest: if there is a blog of precipitated antibody outside the cell of interest, it doesn’t matter if it is saturated.



On our webpage<https://ki.se/en/bionut/teaching-material> you can find a document called Typical workflow of how to set a confocal (1.2 AU on a Nikon system corresponds to 1 AU due to some unusual pinhole shape). During the training, we go through every single point, explain what the options are and why, and discuss with the user to find the best objective, settings, strategy… to answer their scientific question reliably. If you look at the document you will see that 15h is no luxury! 😉



If ever you check our videos and Typical workflow and find errors/omission, please let me know! I am very keen on getting feedback!



Med vänlig hälsning / Best regards



Sylvie



@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Blickagången 16,

Room 7362 (lab)/7840 (office)

14157 Huddinge, Sweden

mobile: [phone]

LCI website

Follow our microscopy blog!



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael
Sent: 10 October 2019 18:16
To: [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://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Cb0dbc3e2d7d64d0c937d08d74d9d39de%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637063209986890774&sdata=WW8Lb8bYgJPcJ4zR4V%2B40x8t4A57K1n12DH2xmPLLjQ%3D&reserved=0>. 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]<mailto:[hidden email]%3cmailto:[hidden email]>>

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[Reece, Jeff (NIH/NIDDK) [E]-2]

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Hi George, Everyone,

I share the exuberance of having another catchy phrase in light microscopy, to help us all remember the nitty gritty.  However, I certainly cannot take credit for the concept of such a "sweet spot".  The best illustration for this concept that I have found is on the following web page from Leica, Figure 1 specifically, which I use when training people in confocal principles. 
https://www.leica-microsystems.com/science-lab/hyvolution-super-resolution-imaging-with-a-confocal-microscope/

Leica references where the black lines come from in that figure, Tony Wilson's 1990 book "Confocal Microscopy".
Tony revived some of that material (which I don't have in front of me right now) in a more recent attempt to clarify and improve "in a particularly simple fashion" ;-):
https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2818.2011.03549.x
 
Cheers,
Jeff

----------------------------------------------------------------
Subject: Re: training and bets practices for confocal
From: George McNamara <mailto:[hidden email]>
Reply-To: Confocal Microscopy List <mailto:[hidden email]>
Date: Thu, 10 Oct 2019 21:01:25 -0400

Hi Jeff,

Confocal Sweet Spot! I love your phrase!!!

I posted a visual at linkedin,
https://www.linkedin.com/posts/georgemcnamara_confocal-sweet-spot-jeff-reese-nih-activity-6588214058011410432-IBV7/ 


I suggest a slight simplification to one, easy to remember value: 0.666
Airy units.

GPU deconvolve all the data, please!!!

Best confocal file formats: OIR and LIF (Olympus FV3000RS and Leica SP8)
... of course I've retired two Zeiss LSM510's. Good to know that CZI
supports 60,000x60,000 pixels (another reply in thread).

PMT gain (HV = High Voltage): my Olympus rep, Jason Brenner, simplified
this for our FV3000RS: the optimal HV setting for our four GaAsP and two
GaAs PMTs is 500 (that is, 500 mV). Yes there are times I suggest
UNoptimal values (usually in steps of 50, but maybe I'll try 666 mV next
time I am at 0.666 Airy Units), but if these PMTs have an optimum,
likely everyone's has an optimum. Since the user has control over the
HV, and the HV controls the amplification of electrons in the PMT (and
ignoring a lot of PMT details), I'll just claim that very few readers
(or my) PMT based confocal microscope are really reporting a 2x change
in fluorophore for intensity level 1000 vs 2000 (after subtracting the
PMT offset floor from the raw data output). More on quantitation later.

PMT offset: depends on the detector, mode (FV3000RS galvo mode is 12-bit
range, resonant scan mode is 10-bit range), I usually teach with Hi-Lo
LUT enabled, "no blue, no red", while live, no averaging.

PMT multiplier: 1 ... and a request to confocal companies with this:
please kill this GUI element!

Better than PMT: Leica HyD in 'counting mode' counts photons. Yes, there
is some counting saturation limit (we have 2nd gen, ask your Leica rep
how much faster counting SMD HyDs are), and other companies have similar
Hybrid detectors, with their own tweaks (and benefits and price points),
I believe first with Hybrid was Becker&Hickl (FLIM, now fast FLIM), now
also ISS, PicoQuant, probably others.

Disclosure: I am registered for the free (fast) TCSPC workshop hosted by
Becker&Hickl and Boston Electronics, Oct 31-Nov 1, 2019, Bethesda MD.
I've also hosted a 2018 Leica FALCON demo, and various microscope demo's
and talks.

https://www.becker-hickl.com/events/13th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-science/

I agree with Jeff: fastest scan speed possible. In particular, Leica SP8
starts up with default of 400 Hz, I tell all users to use 600 Hz (or
faster if their objective lens choice and zoom <--> field of view is
appropriate).

Best way to make live cells brighter: drop EGFP ---- soooooo 1996 ---
Nathan Shaner's AausFP1 is 5x brighter and ultra narrow (for FPs)
emission peak (10x with tandem dimer)
https://www.biorxiv.org/content/10.1101/677344v2.full Plasmids not in
addgene yet (its a preprint), sequence in paper and supplemental info
(and user's might want to use their own codon and/or other expression
optimizations).

***
Confocal quantitation - some thoughts:

-- the lasers on confocal microscopes are not perfectly stable, and
often very unstable (could be the AOTF intensity controller more than
the laser modules - my recollection is Bob Zucker, US EPA, explained
this to me ... and no reason to think any of the confocal companies have
made any serious effort to prevent these fluctiations). So no reason to
think AM vs PM, or even a few minutes apart, stable (to say nothing of
corner to corner at low zoom).

-- I pointed out above there is no reason to think that anyone's PMT
based confocal microscope value 1000 vs 2000 is really a 2x change in
fluorophore concentration (per voxel etc). Or that similar values would
appear if the specimen was scanned AM vs PM (see previous paragraph).

So: most -- that is PMT point scanner - confocal data is UNquantitative
with respect to whatever intensity measurement the user thinks they are
measuring of (a major use of confocal) single cells.

Photon counting is better (some PMTs can be operated in photon counting,
APDs are, but often slow, various Hybrid detectors nice behavior,
trusting Hamamatsu and whoever does the electronics to 'get it right',
or at least not mess it up too badly), but still subject to laser/AOTF
intensity fluctuation issues.


enjoy,
George
p.s. one of the other posts in this thread mentioned Leica SP8 pinhole
value control is hard to find and by default is set to 580 nm: In Leica
LAS X the drop down is easy to find, the "Airy 1.0" is easy to find, the
button uses the midpoint of the emission range when a single detector is
enabled (ex. 510-550 nm range --> 530 nm mid-point). If two detectors
(we have two HyDs) are on, it uses the midpoitn of those. Easy to turn
off one detector, click "1" using the other, then turn on both (user
choice).

On 10/10/2019 2:46 PM, Reece, Jeff (NIH/NIDDK) [E] 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.
> *****
>
> For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...
>
> When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU.  The sweet spot is usually 0.6-0.7AU.
>
> I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers.  It bothers me when I read a paper and all they say is "confocal".
>
> Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly.  Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi.  We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.
>
> Other things you didn't ask about, but I'll quickly mention:
> ...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
> ... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
> ... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels.  Same for z-stack delta which is for sampling in z.  These values can be changed somewhat, always exceptions, various reasons.  Generally multiply the answer by 1.5-2x if performing decon.
> ...Use the fastest scan speed possible.
> ... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly.  But thank you for trying, software engineers.
> ...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).
>
> Cheers,
> Jeff
>
> -----Original Message-----
> From: Cammer, Michael <mailto:[hidden email]>
> Sent: Thursday, October 10, 2019 12:16 PM
> To: mailto:[hidden email]
> Subject: training and bets practices for confocal
>
> *****
> 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  mailto:[hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/

[0000001ed7f52e4a-dmarc-request]

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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi  Zenek,

>HyDs are photon counting. Always. So offset does not make sense here.
But somehow they have to discriminate photon induced avalanche effects from dark-current initiated effects, while there is no user changeable offset parameter, this is built in or calibrated by the manufacturer. When I compare  low signals e.g. from the Argolight slide between GaAsP PMTs in normal mode with HyDs, it looks as if the HyD cuts off some of the signal. 

> In the extreme case of a photon-counting detector the SNR is 1, You would adjust the  pixel dwell time to collect enough photons, the main contribution to noise is then given by Poisson statistics and the SNR is given by the square root of the number of photons, e.g. 10 for 100 photons. 

When I measure SNR as a function of gain using a Chroma slide, it is almost constant, no sweet spot there. When I average more, the mean intensity stays the same but the noise distribution is narrower (visible in the image as less noise). But now I am further away from saturation than in the noisy case and can e.g. increase the laser power before the first pixels reach saturation and get an even better image, but it takes more time. The same works by increasing the laser power and reducing gain at the risk of bleaching. 

best wishes

Andreas

-----Original Message-----
From: Zdenek Svindrych <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Mon, 14 Oct 2019 12:53
Subject: Re: training and bets practices for confocal

*****
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*****

Andreas,

I do just that (convert to ImageJ 32-bit and subtract the offset), but
mostly for camera-based data (widefield, spinning disk). That makes sense,
for the amplitude of the useful signal can in some cases be much lower that
the offset.

Also, HyDs are photon counting. Always. So offset does not make sense here.
There is no real gain either. Only digital gain to scale the photon counts
to the full range (e.g. 16 bit), relative to what Leica assumes is an
adequate range.

This also reminds me the "sweet spot" PMT gain: the sensitivity of a PMT
increases monotonically with gain (the readout noise is made smaller
relative to the signal). But the poisson-limited dynamics range decreases
with increasing gain, that's where the "sweet spot" tradeoff is. In the
extreme case of a photon-counting detector the SNR is 1, that is, you can
only get useful signal after substantial averaging (or summing in this
case)!
So, if there is no other way to boost your signal, increasing the gain will
not hurt (though it may not help either).

Best, zdenek


On Sun, Oct 13, 2019 at 8:59 AM Andreas Bruckbauer <
[hidden email]> 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.
> *****
>
> 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. <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Fri, 11 Oct 2019 0:47
> Subject: Re: training and bets practices for confocal
>
> *****
> 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.
> *****
>
> 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: [hidden email]
>
> http://www.bcm.edu/oivm
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Cammer, Michael <[hidden email]>
> Sent: Thursday, October 10, 2019 11:15:46 AM
> To: [hidden email] <[hidden email]>
> Subject: training and bets practices for confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e=
> 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<
> 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  [hidden email]
> <mailto:[hidden email]>
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Zdenek Svindrych-2]

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*****

Hi Andreas,

yes, the manufacturer sets the detector gain and discriminator threshold to
find the balance between photon counting efficiency and dark count rate.
But there is no way to differentiate between dark counts and photon-induced
counts. There is no way to subtract the dark counts. Note, in the case of
hybrid detectors, the dark counts are quite low, much less than 1000 cps,
that is less than 0.001 counts per pixel in a typical image.

I'm not a lucky owner of one of the test slides, but when you are comparing
the integrating (PMT) and photon counting (HyD) images, are you making sure
that the excitation intensity, dwell times, averaging, and everything else
is identical (or perhaps adjusted to account for different photocathode
quantum efficiency)? Because it's quite possible that the low intensity
details in your HyD images are lost in the photon noise...

And to the PMT gain - the "sweet spot" will begin to emerge when you try to
utilize the full range of your detector (for given gain). This range (in
photons/s) will be much smaller with high gain, hence lower SNR.

Best, zdenek

On Wed, Oct 16, 2019 at 5:48 PM Andreas Bruckbauer <
[hidden email]> 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.
> *****
>
> Hi  Zenek,
>
> >HyDs are photon counting. Always. So offset does not make sense here.
> But somehow they have to discriminate photon induced avalanche effects
> from dark-current initiated effects, while there is no user changeable
> offset parameter, this is built in or calibrated by the manufacturer. When
> I compare  low signals e.g. from the Argolight slide between GaAsP PMTs in
> normal mode with HyDs, it looks as if the HyD cuts off some of the signal.
>
> > In the extreme case of a photon-counting detector the SNR is 1, You
> would adjust the  pixel dwell time to collect enough photons, the main
> contribution to noise is then given by Poisson statistics and the SNR is
> given by the square root of the number of photons, e.g. 10 for 100 photons.
>
> When I measure SNR as a function of gain using a Chroma slide, it is
> almost constant, no sweet spot there. When I average more, the mean
> intensity stays the same but the noise distribution is narrower (visible in
> the image as less noise). But now I am further away from saturation than in
> the noisy case and can e.g. increase the laser power before the first
> pixels reach saturation and get an even better image, but it takes more
> time. The same works by increasing the laser power and reducing gain at the
> risk of bleaching.
>
> best wishes
>
> Andreas
>
> -----Original Message-----
> From: Zdenek Svindrych <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Mon, 14 Oct 2019 12:53
> Subject: Re: training and bets practices for confocal
>
> *****
> 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.
> *****
>
> Andreas,
>
> I do just that (convert to ImageJ 32-bit and subtract the offset), but
> mostly for camera-based data (widefield, spinning disk). That makes sense,
> for the amplitude of the useful signal can in some cases be much lower that
> the offset.
>
> Also, HyDs are photon counting. Always. So offset does not make sense here.
> There is no real gain either. Only digital gain to scale the photon counts
> to the full range (e.g. 16 bit), relative to what Leica assumes is an
> adequate range.
>
> This also reminds me the "sweet spot" PMT gain: the sensitivity of a PMT
> increases monotonically with gain (the readout noise is made smaller
> relative to the signal). But the poisson-limited dynamics range decreases
> with increasing gain, that's where the "sweet spot" tradeoff is. In the
> extreme case of a photon-counting detector the SNR is 1, that is, you can
> only get useful signal after substantial averaging (or summing in this
> case)!
> So, if there is no other way to boost your signal, increasing the gain will
> not hurt (though it may not help either).
>
> Best, zdenek
>
>
> On Sun, Oct 13, 2019 at 8:59 AM Andreas Bruckbauer <
> [hidden email]> 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.
> > *****
> >
> > 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. <[hidden email]>
> > To: CONFOCALMICROSCOPY <[hidden email]>
> > Sent: Fri, 11 Oct 2019 0:47
> > Subject: Re: training and bets practices for confocal
> >
> > *****
> > 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.
> > *****
> >
> > 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: [hidden email]
> >
> > http://www.bcm.edu/oivm
> > ________________________________
> > From: Confocal Microscopy List <[hidden email]> on
> > behalf of Cammer, Michael <[hidden email]>
> > Sent: Thursday, October 10, 2019 11:15:46 AM
> > To: [hidden email] <[hidden email]>
> > Subject: training and bets practices for confocal
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e=
> > 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<
> >
> 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  [hidden email]
> > <mailto:[hidden email]>
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=
> >
>
>
> --
> --
> Zdenek Svindrych, Ph.D.
> Research Associate - Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[0000001ed7f52e4a-dmarc-request]

*****
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.
*****

Hi zdenek,>There is no way to subtract the dark counts
Just measure without laser and subtract :-)  >the dark counts are quite low, much less than 1000 cps,In photon counting mode on a HyD detector I indeed measure something like 0.006 counts in 2.4 us dwell time (2500 cps), in normal mode with '100% gain' it is then about 100 times higher (no surprise).  >Argolight slideYes, I measured laser power on both systems, tried to set everything similar, I think the photocathodes are the same between HyD and GaAsP PMTbut for some reason Leica claims a little higher QE?When you put the laser power too high, the slides loses fluorescence (but recovers afterwards), sample drift becomes a problem when scanning too slow sample (I like to acquire z-stacks), the spectral settings should be narrow enough e.g. 500 - 550 nm) as otherwise the resolution will be worse, so it is quite at the limit of what I can image, especially with smaller pinhole sizes. Whereas with the GaAsP detector on an older LSM 780, I had no problem. The HyD was on a brand new SP8 and I even had Leica over to have a look.   Maybe it is a case of noise actually helping to discriminate useful signal an idea Toshio Yanagida promotes for biological systems.  best wishes  Andreas  -----Original Message-----
From: Zdenek Svindrych <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 17 Oct 2019 14:15
Subject: Re: training and bets practices for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Andreas,

yes, the manufacturer sets the detector gain and discriminator threshold to
find the balance between photon counting efficiency and dark count rate.
But there is no way to differentiate between dark counts and photon-induced
counts. There is no way to subtract the dark counts. Note, in the case of
hybrid detectors, the dark counts are quite low, much less than 1000 cps,
that is less than 0.001 counts per pixel in a typical image.

I'm not a lucky owner of one of the test slides, but when you are comparing
the integrating (PMT) and photon counting (HyD) images, are you making sure
that the excitation intensity, dwell times, averaging, and everything else
is identical (or perhaps adjusted to account for different photocathode
quantum efficiency)? Because it's quite possible that the low intensity
details in your HyD images are lost in the photon noise...

And to the PMT gain - the "sweet spot" will begin to emerge when you try to
utilize the full range of your detector (for given gain). This range (in
photons/s) will be much smaller with high gain, hence lower SNR.

Best, zdenek

On Wed, Oct 16, 2019 at 5:48 PM Andreas Bruckbauer <
[hidden email]> 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.
> *****
>
> Hi  Zenek,
>
> >HyDs are photon counting. Always. So offset does not make sense here.
> But somehow they have to discriminate photon induced avalanche effects
> from dark-current initiated effects, while there is no user changeable
> offset parameter, this is built in or calibrated by the manufacturer. When
> I compare  low signals e.g. from the Argolight slide between GaAsP PMTs in
> normal mode with HyDs, it looks as if the HyD cuts off some of the signal.
>
> > In the extreme case of a photon-counting detector the SNR is 1, You
> would adjust the  pixel dwell time to collect enough photons, the main
> contribution to noise is then given by Poisson statistics and the SNR is
> given by the square root of the number of photons, e.g. 10 for 100 photons.
>
> When I measure SNR as a function of gain using a Chroma slide, it is
> almost constant, no sweet spot there. When I average more, the mean
> intensity stays the same but the noise distribution is narrower (visible in
> the image as less noise). But now I am further away from saturation than in
> the noisy case and can e.g. increase the laser power before the first
> pixels reach saturation and get an even better image, but it takes more
> time. The same works by increasing the laser power and reducing gain at the
> risk of bleaching.
>
> best wishes
>
> Andreas
>
> -----Original Message-----
> From: Zdenek Svindrych <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Mon, 14 Oct 2019 12:53
> Subject: Re: training and bets practices for confocal
>
> *****
> 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.
> *****
>
> Andreas,
>
> I do just that (convert to ImageJ 32-bit and subtract the offset), but
> mostly for camera-based data (widefield, spinning disk). That makes sense,
> for the amplitude of the useful signal can in some cases be much lower that
> the offset.
>
> Also, HyDs are photon counting. Always. So offset does not make sense here.
> There is no real gain either. Only digital gain to scale the photon counts
> to the full range (e.g. 16 bit), relative to what Leica assumes is an
> adequate range.
>
> This also reminds me the "sweet spot" PMT gain: the sensitivity of a PMT
> increases monotonically with gain (the readout noise is made smaller
> relative to the signal). But the poisson-limited dynamics range decreases
> with increasing gain, that's where the "sweet spot" tradeoff is. In the
> extreme case of a photon-counting detector the SNR is 1, that is, you can
> only get useful signal after substantial averaging (or summing in this
> case)!
> So, if there is no other way to boost your signal, increasing the gain will
> not hurt (though it may not help either).
>
> Best, zdenek
>
>
> On Sun, Oct 13, 2019 at 8:59 AM Andreas Bruckbauer <
> [hidden email]> 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.
> > *****
> >
> > 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. <[hidden email]>
> > To: CONFOCALMICROSCOPY <[hidden email]>
> > Sent: Fri, 11 Oct 2019 0:47
> > Subject: Re: training and bets practices for confocal
> >
> > *****
> > 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.
> > *****
> >
> > 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: [hidden email]
> >
> > http://www.bcm.edu/oivm
> > ________________________________
> > From: Confocal Microscopy List <[hidden email]> on
> > behalf of Cammer, Michael <[hidden email]>
> > Sent: Thursday, October 10, 2019 11:15:46 AM
> > To: [hidden email] <[hidden email]>
> > Subject: training and bets practices for confocal
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=Tvz9pGlXjmwEpOQI_BnTSYoiV7hGgUkSq1JfCGWa_mc&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=gvt97LEIUTCFT8E2hL45VGBhEIaCDr9pLu0OUeAaOvc&e=
> > 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<
> >
> 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  [hidden email]
> > <mailto:[hidden email]>
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=M7JzVTNBPr_XJct6rKm7rI4KjJaXxj3L4l4GBte2PMk&e=
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=7O3sO8Sxov-n6Lwylr3_wJWjm4zrmf_5l0iTcQvUFgE&e=
> >
>
>
> --
> --
> Zdenek Svindrych, Ph.D.
> Research Associate - Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Steffen Dietzel]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Am 14.10.2019 um 22:17 schrieb Praju Vikas Anekal:
> Since the Image capturing process is essential an 'Analog to Digital' conversion, I try to explain to the users the importance of achieving Nyquist sampling. (or slight oversampling to 3X in most cases)

Hi Praju,

coming from the cell nucleus architecture field where the confocal
resolution was just barely good enough, I also emphasized this quite a
bit. It took me some time to understand that some people and their
samples just don't need best possible resolution. Some questions like
where are the labeled cells in a tissue can nicely be answered with e.g.
1 µm sized pixels, but the sample might still profit from oil immersion
for the better NA and brightness.

Yes, users should know about Nyquist sampling, but it is not an absolute.

And welcome to the list :-)

Steffen

> --

------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Nicholas Condon]

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Hello List,

The discussion on training regimes has been a very interesting topic to follow along to.

Here at the Institute for Molecular Bioscience, (at the University of Queensland Australia) in our core facility (https://imb.uq.edu.au/microscopy) we have 22 systems between two staff (but we are currently looking for more! (Senior Microscopist)

The way we conduct the training of our researchers and staff follows a two tiered approach. First all new users must be trained on a basic system (stereo or basic wide-field microscope) and then complete 10 hours of usage before they can request training on more 'advanced' systems (live-cell imaging wide-fields and confocals; LSM & spinning disk). Trainings are always machine specific as well.

Using the booking software PPMS we can monitor user's actual usage on the microscopes (which unsurprisingly is often much less than their booked time). This also lets us limit 'novice' users to only access facility equipment in staffed hours where support can be immediately provided. After 10 hours, at the users request, outside of staffed hours access can be granted on a case-by-case basis.

Providing the basic training up front means users understand basic principles (Kohler, Nyquist imaging requirements, signal: noise, 3D imaging with correct spacing, etc) before undergoing their 'advanced' system trainings, freeing up time more machine specific training (confocal pinholes, laser safety, detector types, gain and offsets).

A typical training session lasts 2 hours, and begins using standardised facility slides, and ends with the users own sample if one is provided. This ensures the user knows what to expect from the system with optimal samples, and no distractions during the important steps. Lastly if provided they know what to expect with their own samples; this is often a time to discuss sample preparation techniques if required.

More advanced trainings on machine specific modalities is performed post the initial training, this includes STED, FLIM, Airyscan, Jobs/HC, 2P which are extra additions to a basic confocal.

We find our method is a nice balance between whats required for safe operation & preventing user damage and what PIs will both pay for and accept.

Our trainings are $62 AUD per hour, which covers our staff time. To request training users fill in a questionnaire about their imaging experiments (Dyes, objectives, DIC/Phase, live/fixed samples etc) which helps us triage which equipment is best for them. Training requests have a usual maximum wait time of 7 days from the time of questionnaire submission.

Thanks,
Nick (& James Springfield, Facility Manager)

Dr Nicholas Condon
Light Microscopy Officer, ACRF: Cancer Biology Imaging Facility
Institute for Molecular Bioscience
The University of Queensland
Brisbane Qld 4072 Australia

T [phone] M 0400 909 510

E [hidden email]
CRICOS code: 00025B