Posted by
Richard Lisle on
URL: http://confocal-microscopy-list.275.s1.nabble.com/organoid-imaging-tp7590835p7590858.html
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Hi Mary Ellen
We regularly image organoids embedded in matrigel on an LSM710 (invert) for up to 4 days.
We've imaged GFP, Venus, mBanana, RFP and mCherry, using the standard single photon laser lines. The organoids have been embedded in a matrigel cylinder around 3mm in diameter x 5mm deep, attached to the bottom of a 24 well plate with media filling the well. This has kept multiple organoids per matrigel cylinder viable for the full timecourse. Running non-imaged controls alongside as not shown any noticeable phototoxicity either.
We've been taking Z stacks up to approx. 1.5mm from the plate bottom without any problems or needing to increase gain or laser power through the stack, so we're not finding the matrigel to be a problem.
The caveat here is that we've been able to get away with using a 10x/0.45 objective for our needs, but as long as you have the working distance in the objective you need, it should be worth a try.
Best wishes
Richard
Ludwig Institute for Cancer Research, University of Oxford, Nuffield Department of Medicine, Old Road Campus Research Building
Roosevelt Drive, Oxford
-----Original Message-----
From: Confocal Microscopy List <
[hidden email]> On Behalf Of Mary Ellen Pease
Sent: 28 April 2020 15:22
To:
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Subject: organoid imaging
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Hi all,
I have a user who would like to image live organoids via confocal/multiphoton without jeopardizing the sample. Has anyone figured out a material in which the organoid could be placed that would eliminate movement/drift during imaging, without damaging it so the sample could go back into culture afterwards? So far, the papers I've looked at on this topic cryoembed them at a certain time point for immunohistochemistry. My user's lab has samples which are expressing GFP and RFP, and would like to look at them in vivo. Someone in our department suggested putting them in a mix of matrigel and culture media, which could slow them down temporarily, but in my experience, matrigel attenuates the signal. We have a multiphoton system on an inverted Zeiss platform (LSM 710). we can image using an objective inverter and dipping lenses, or from the bottom through a coverglass dish. Since we are working remotely at this point in time, I was hoping to get some direction from this group.
Best,
Mary Ellen Pease
Manager, Wilmer Microscopy and Imaging Core Facility (MICF) Johns Hopkins SOM, Baltimore MD