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

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Spinning-disk-or-new-point-scanning-confocals-for-live-imaging-of-3D-engineered-tissues-tp7590297p7590304.html

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Francesco,

since >50 um thick, think about multiphoton excitation fluorescence
microscopy ... second harmonic generation (some types of collagen).

Fiber lasers that output single wavelength, typically ~100 femtoseconds,
80 MHz repetition rate, are in the $10K - $60K range, each. If only one
microscope rig, implies point scanning (or TriMscope or similar). That
is, 1 or 3 lasers for potentially a lot less than a tunable Ti:Sapphire
laser (and could start with one fiber laser at most important wavelength
... could be launched into a conventional 1p confocal scanner).

You could have both point scanning (MPEF) and spinning disk confocal on
same microscope. I strongly urge investing in GPU deconvolution
software, optimized for each mode you will use.

FYI - In May 2019 at JHU, Nathan Shaner (UCSD) started his talk on
AausFP1 -- 5x brighter than EGFP -- with a multi-day timelapse video
acquired at 6 min intervals (sped up on playback).

I suggest starting to make AausFP1 constructs, amino acid sequence in
https://www.biorxiv.org/content/10.1101/677344v2 (obligate dimer ... so
tandem dimer and/or monomerization mutation should be installed ... also
codon optimize). Preprint does not discuss Yellow version, should be
doable with one mutation (re: EGFP --> EYFP), so could have two colors.
Tandem dimer of G-->Y should be outstanding for FRET (3rd color).

Also start thinking about optimized filter set(s) and/or confocal/MPEF
capabilities, to get the most of the narrow excitation and emission
peaks of AausFP1(green), AausFP1(Yellow), AausFP1(green-->Yellow).

FYI - our Olympus FV3000RS confocal microscope worked well for GCaMP6,
https://www.physiology.org/doi/abs/10.1152/ajpcell.00351.2018 (our Leica
SP8 confocal would have worked fine, but purchased without 37 C and
environmental control). Here in the U.S., the microscope vendors often
provide a nice trade-in credit, so I encourage asking your new place if
they have an old confocal to trade in toward purchase.

best wishes,

George

p.s. see also current optical clearing methods for fixed specimens, and
expansion microscopy.


On 12/13/2019 12:18 PM, Francesco Pasqualini wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>