Re: Spinning disk or (new) point scanning confocals for live imaging of 3D engineered tissues?

Posted by VERMEREN Matthieu on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Spinning-disk-or-new-point-scanning-confocals-for-live-imaging-of-3D-engineered-tissues-tp7590297p7590345.html

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Hi Francesco,

We have a Nikon A1R on Ti2 on demo, resonant scanning combined with Nikon's new silicone lenses (long working distance, better match to live system) means we get excellent images on thick immunostained samples  (organoids in matrix, tissue slices, young embryos).  An organoid that would take ~30 minutes to image on a standard system is done in less than 6 minutes and with very little noise...  meaning some experiments that were going to be too long and too expensive are now considered do-able by a fair few post docs and PIs.  The advantage is that we can also use Nikon's JOB for multiwell system (sadly not with the silicone lenses, but water lenses are an option).

I'm hoping to test it on Ca++ imaging or on FRET.

Sincerely,

Matthieu

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Francesco Pasqualini
Sent: 13 December 2019 17:18
To: [hidden email]
Subject: Spinning disk or (new) point scanning confocals for live imaging of 3D engineered tissues?

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Hi,
I will start my ERC-funded lab in Italy in 2020, which means I am fortunate to be shopping for a confocal instrument right about now.

Since I do mostly live experiments (more details below) I was going to get a spinning disk system. But, I realized that the new scanning confocals are also relatively fast and gentle. The question is how much (if at all) slower and harsher are they with respect to spinning disks?

More details:
- I do a lot of live experiments (traction force microscopy and voltage/calcium-sensitive dyes) on engineered cells (300x300 um FOV) and tissues (600x600 um FOV) in 2D or 3D (<300 um thick) that I complement with immunostainings after fixation. Needed acquisition rates go from <1 fps to 100s depending on the application.

- Originally, I was oriented towards a spinning disk confocal (Yokogawa/SORA, Crest) and was looking at ways to deal with the issues of confocality on the 3D tissue (600x600x300 um volumes) case. But, I realized all vendors have new point scanning confocal (980+Airyscan2, FV3000, A1R) with resonant scanners that acquire full frames/ROIs at 10s/100s of fps

- Of course, structured illumination (Elyra-7, N-SIM) and light-sheet microscopes (QuVi) are also appealing but relatively untested in my applications of interests....

The application specialists from all vendors in my area have been great to work with but since my lab is not up and running, yet, I can't demo these systems directly. Therefore, I could use help and feedback, especially from people who have had related experiences in the recent past.

Thanks,
Francesco

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Francesco S. Pasqualini
Visiting Professor University of Pavia
Associate Harvard University

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