http://confocal-microscopy-list.275.s1.nabble.com/Spinning-disk-or-new-point-scanning-confocals-for-live-imaging-of-3D-engineered-tissues-tp7590297p7590305.html
thanks for the note re: AausFP1. I will definitively check it out.
Spinning confocal. That is, one has to trade 3D SNR for speed and I need to
be able to hit 100s fps in certain important applications. Am I wrong? (I
found an implementation that works well on multi-well plates. The possible
untested at this point. Does anyone have any experience with these
> 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
> >
>
Francesco S. Pasqualini