Re: organoid imaging

Posted by Philipp Tripal on
URL: http://confocal-microscopy-list.275.s1.nabble.com/organoid-imaging-tp7590835p7590838.html

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Dear Mary Ellen,

staining of organoids within matrigel is a challange and results in bad
signal to noise ratios... but if your organoids are expressing GFP or
even better long wavelength fluorophores, imaging can be performed
within the matrigel matrix. A dilution of matrigel with medium works
also well and dilution of up to 1:5 are applicable.

To increase the amount of organoids close to the coverslip, you can
place the dish on an ice pack while you seed the
matrigel-medium-organoid suspensions and keep it on ice for 5 minutes.
Within cold matrigel the organoids sink by gravity and you´ll have more
organoids within the working distance of the lens... I recommend
spinning disc microscopy, due to low photo-toxic stress. Matrigel and
2Photon might be challenging, due to the second harmonics of the
collagen matrix...

Best regards,

Philipp


On 28/04/2020 16:21, Mary Ellen Pease wrote:

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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

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Philipp Tripal
Research Associate
Optical Imaging Centre Erlangen

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