COVID Core Facilities Training

Hi All,

I posted this to the confocal listserv already - so my apologies if you have read this already!

As we continue to grapple with the long term effects of COVID-19 - those of us who operate shared instrumentation facilities have been finding ways to address the problem of how to provide the same high level of instruction while maintaining our commitment to social distancing and limited interpersonal contact. While there is no one-size-fits-all solution – I wanted to share the route we have chosen in the hopes that it will provide some benefit to others.

Confocal microscopes are the bread and butter of our core – and we have created video-based training that so far has been doing a consistent job replicating our traditional confocal training. The key for us has been creating high quality video content that replicates every aspect of our in-person training session. The main issue I’ve noticed with much of the video based training publicly available is that it is nowhere near as in-depth as we traditionally get during our one-on-one sessions. Creating detailed content that is specific to our instruments has allowed us to almost completely remove ourselves from the initial training.

We’ve been requiring users to watch an introductory video as well as the full training video before arriving for their ‘in-person’ events. When they arrive we give them a quick overview of the core and then have them go through the full resolution video on a monitor right next to the instrument. This way they can follow along and start/stop the instruction to move at their pace. A second session is then used to gauge how well the user is absorbing the material.

It has been quite well received – and I’ve posted the full 4K videos for our first instrument (Zeiss LSM 880 with Airyscan) to Youtube here:

We will be adding videos as we expand training for our other instruments – as well as more advanced techniques as our users request them.

Of course these videos are specific to our instruments – but I hope they can be useful to others looking to do similar things.

Cheers!

-Jason


Jason M. Kirk
Technical Director, Optical Imaging & Vital Microscopy Core (OiVM)
Baylor College of Medicine
Ph: 713.798.6486
jason.kirk@bcm.edu
http://www.bcm.edu/oivm

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Thanks for sharing Jason!

This was really great, thanks so much for putting it together! I will definitely be making use of the AiryScan video to help explain how that works to our users :slight_smile:

I thought I remembered (in one of the videos) something about simultaneous acquisition with the 3 confocal detectors and using the Airyscan detector for the fourth channel… but I cannot seem to get that to work. The closest I can do is simultaneous acquisition with 3 channels (±TPMT) and then the AS detector on a separate track in Line mode.

I also do not have a filter to send the DAPI signal to the AS detector, so that is another issue, but the video did make me realize that it would be easy enough to use the AS detector for my far red, where it is much more sensitive than the CH3 detector. I had occasionally been sending the Far red detection, when weak, to the CH2 detector (no Meta, just GaAsP), but that was causing some interesting cross talk with the CH1 detector (it was crazy troubleshooting THAT).

Added question, since your core seems invested in the LSM880s… have you ever noticed that shifting a track up or down in the track list changes the relative laser power in ASFast mode? Not the original laser power, but the split changes slightly based on the position. That weirded me out since I figured the tracks were purely programmatic constructs. Never did get a response from Zeiss about why that happened.

For simultaneous acquisition - 4 colors are not possible. Using the Airyscan as a 4th detector is only available using sequential imaging. Our videos discuss sequential imaging only.

I just took a look at our system after your comment about changing the track order in FAST mode and I don’t see any changes to the relative laser power when moving the track order. Maybe this is something that is addressed in a software hotfix? Not sure what hotfix you are currently using - but we are using ZEN 2.3 SP1 HF23.

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Ah, ok, that matches with what I was able to do! Was wondering if I missed something, but I must have simply misunderstood.

As for the ASFast, this is an example of what I would see.


Left and right are two different tracks, but both using the same laser (405), and, in this case, different initial laser power (7.5 and 7.0), yet they have the same final laser power. Not something that is terribly important since there is not normally much reason to swap tracks around, but it did make me curious :slight_smile:
As for the version, it looks like we are running 2.3 SP1 FP3. I honestly do not know how that relates to the hotfixes, but it would have been updated within the last several months.

I checked again a few minutes ago, this time with the 561 line and still saw the same result. Swapping the positions of the tracks swaps the relative laser powers, even though all of the settings are the same in the imaging setup! Fun.

That is quite interesting. I checked our instrument again with these variations and unfortunately I do not see the same response. Our values remain the same regardless of track changes.

One question though - are you loading these configurations from the Experiment Manager or from Reuse? If so - you may want to set up the tracks manually to see if you get the same effect. I have had issues in the past where configurations or images created in older versions create little gremlins like this in newer updates. Recreating the configurations in the new version solved a lot of that.

To find out what hotfix you have - look at the Help > About tab under Version. The 3rd decimal place will tell you which hotfix you have. In my case - HF23.

about_zen

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Generally from experiment manager, though in this particular case I took one of the default confocal setups and changed the tracks around manually for testing. I will give it a try from scratch.

It looks like we are on hotfix 22, so I will try and see if an update changes anything.

Hmm, it looks like hotfix 22 is the most recent one I can find. I’ll try to contact Zeiss directly, maybe that one has not been released or tested for stability yet?

Zeiss recently put their software behind a registration wall.

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Ah, I may give the hotfix upgrade a try as the multiposition DF offset support sounds like something a few people could make use of. It sounds like that hotfix is a bit more involved than some of the others since it involves a firmware upgrade.

I started from scratch (still on HF22) and created 4 of the same AiryscanFast setting. There is still the swap in laser power between the 2nd and 3rd track positions. The top two track positions are the same (in terms of laser power estimation), and the bottom two are the same.

Laser splitting by position in the track list for the 561
2.1 - 0.25
2.1 - 0.25
2.1 - 0.32
2.1 - 0.32

Maybe the hotfix will take care of things, or maybe it is something particular about our system modifications for 2P or not having a meta detector.

Thank you for taking the time to check @jasonkirk, and sorry to have bothered you with this as your system is not seeing the same issues!

And, as I have derailed this thread quite a bit (and do not have the privileges to fix it on this forum :slight_smile: ), @jennifer feel free to split these posts to a different thread if you want!

It turns out that whatever the issue is, it is not related to the hotfix. Even with HotFix23 and starting from scratch, the laser power distribution changes between tracks 1,2 and 3,4.

Maybe someday someone will come across this with an answer!