Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/organoid-imaging-tp7590835p7590840.html
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Hi Mary Allen,
Jawad Allen -- now Dr. Jawad Allen -- in Cindy Sears lab did long term
(weekend) imaging on our Ross Bldg (9th floor) Olympus FV3000RS
confocal inverted (IX83) microscope (OkoLab stage top + plexiglas shroud
incubator, 37 C, tage top humidifier & optional 5% CO2 ... 10x/0.40 WD
3.1 mm or 20x/0.75 WD 0.6 mm, if I recall correctly ... standard lenses,
so equivalent on your LSM710MP would work fine),
http://confocal.jhu.edu/current-equipment/fv3000/(I believe) embedded in matrigel, similar to Philipp Tripal's reply. You
should be able to get full details from Cindy (i.e. manuscript in prep).
Yes, Jawad brought his SBS plates between buildings (in an appropriate
container). I strongly recommend the research lab have a quarantine
incubator for plates that leave the lab and return.
Your user is welcome to try out our FV3000RS for comparison - once JHU
SOM re-opens. Full set of laser lines from 405-730 nm. We'll probably be
busy all summer catching up, but should be able to squeeze your user in.
I encourage use of thin bottom SBS plates or 35 mm imaging dish. Mattek
(and some others) offer #0 coverglass bottom, which provides an extra
~70 um working distance compared to #1.5 coverglass (nominally 170 um;
ibidi's bottoms are typically their image quality plastic, ~180 um).
George
p.s. my JHU email handle is gmcnama2
On 4/28/2020 10:21 AM, Mary Ellen Pease wrote:
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>
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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