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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 <[log in to unmask]>
To: CONFOCALMICROSCOPY <[log in to unmask]>
Sent: Fri, 11 Oct 2019 12:17
Subject: Re: training and bets practices for confocal

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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: +46 (0) 73 733 5008

LCI website

Follow our microscopy blog!



-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Cammer, Michael
Sent: 10 October 2019 18:16
To: [log in to unmask]
Subject: training and bets practices for confocal



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I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.

Cheers-

Michael





General Confocal Best practices:



  *  The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.

If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.

Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".

  *  Offset. Always use at 0 or 1.

Other numbers are wrong.

  *  Digital gain. The preset is 1. Leave it there.

  *  Use the Range Indicator button to make sure you have no saturated pixels<https://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  [log in to unmask]<mailto:[log in to unmask]<mailto:[log in to unmask]:[log in to unmask]>>

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