http://confocal-microscopy-list.275.s1.nabble.com/Spinning-disk-or-new-point-scanning-confocals-for-live-imaging-of-3D-engineered-tissues-tp7590297p7590311.html
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>
> Commercial Response:
>
> The Lattice LightSheet instrument from 3i is most definitely tested at this
> point. Over the past few years, we have delivered a few dozen of these
> instruments around the world. While multi-well plates are not possible with
> this design, multi-site visitation on those same glass coverslips is
> standard practice. From the shared experiences of the large community of
> users, this instrument is the gentlest design in their labs/facilities.
> Quite capable of >200fps, your interest in 100 fps at times is a reasonable
> 3D capture paradigm. With a ~400nm sheet, the instrument illuminates just
> under the objective depth of field with each frame, reducing any out of
> focus illumination/harm. If hyper gentle high-speed 3D capture is your
> primary concern, I cannot suggest another instrument besides a LightSheet
> rig. That being said, pretty much every one of our Lattice LightSheet users
> has a spinning disc confocal to model experimental designs.
>
> Eric Betzig's original Science paper on the topic addressed this particular
> topic with care in his Discussion. (I won't quote him here, but here's the
> link.)
>
>
https://www.ncbi.nlm.nih.gov/pubmed/25342811>
> Cheers,
> -
> Sam
>
>
https://www.intelligent-imaging.com/lattice>
> Samuel Connell
> Director of Sales
>
> Intelligent Imaging Innovations (3i)
> 3509 Ringsby Court
> Denver, CO 80216 USA
> 1-720-437-6926
> www.intelligent-imaging.com
>
>
> On Mon, Dec 16, 2019 at 1:41 AM Francesco Pasqualini <
>
[hidden email]> 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.
> > *****
> >
> > George,
> > thanks for the note re: AausFP1. I will definitively check it out.
> >
> > In a spectrum that goes from (slowest speed / highest 3D SNR) to (highest
> > speed / lowest 3D SNR), my understanding is that MPEF < Scanning
> confocal <
> > 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
> > understand AasusFP1 increased brightness with help SNR and potentially
> > enable faster recordings, but I am not sure how linear that
> > relationship would be given photobleaching...)
> >
> > Another option would be (lattice) light-sheet microscopy but I have not
> > found an implementation that works well on multi-well plates. The
> possible
> > exceptions are the 3i implementations and Luxendo QuVi but they are
> pretty
> > untested at this point. Does anyone have any experience with these
> > platforms?
> >
> > Thanks,
> > Francesco
> >
> >
> > On Mon, Dec 16, 2019 at 3:57 AM George McNamara <
>
[hidden email]
> > >
> > wrote:
> >
> > > 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
> > Visiting Professor University of Pavia
> > Associate Harvard University
> >
> > tel: +39 351-521-7788 (IT)
> > tel: +1 617-401-5243 (USA)
> >
>