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

[Francesco Pasqualini]

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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

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

tel: [phone] (IT)
tel: [phone] (USA)

[George McNamara]

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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 Pasqualini]

*****
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: [phone] (IT)
tel: [phone] (USA)

[samuel connell]

*****
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.
*****

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: [phone] (IT)
> tel: [phone] (USA)
>

[lechristophe]

*****
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.
*****

There are signs that the upcoming Zeiss lattice-light sheet will allow
standard vessels like 35-mm glass bottom dishes, in an "inverted"
configuration
https://www.electronic-supply.dk/procurement/view/74024/inverted_lattice_light_sheet_microscope

Christophe

On Fri, 13 Dec 2019 at 18:29, 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.
> *****
>
> 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: [phone] (IT)
> tel: [phone] (USA)
>

[Francesco Pasqualini]

*****
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.
*****

Sam,
Agreed on the uniqueness of the 3i approach and I'm in contact with Nicole
@ 3i to discuss options.

In my earlier message, I meant the system isn't necessarily tested on the
type of applications I am interested in: fluorescent bead-embedding,
engineered hydrogels for cell culture.

For that, i will demo as many systems as I can early next year - but I am
curious of other users' experience.

Best
Francesco




On Mon, Dec 16, 2019, 5:26 PM samuel connell <[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.
> *****
>
> 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: [phone] (IT)
> > tel: [phone] (USA)
> >
>

[Arvydas Matiukas]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hello List,

The HP 820 workstation that controls LSM780 is over 5 years old and no longer supported under service contract, and Windows 7 support ends in January 2020. Please share your latest experience of:
1) upgrading/not upgrading to Windows 10
2) upgrading to a (cheaper) new custom built computer.

One computer geek explained me that termination of Microscoft support for current OS is not a big deal - businesses still are using computers running on Windows XP and even 2000. It is a good and up-to-date antivirus software that provides the protection.

Regarding computer upgrade I  looked up what imaging workstations HP is offering. HP Z2 Tower G4 Workstation (i5 8500 6 cores 3GHz/Nvidia Quadro P620 4GB DDR5/8GB DDR4/256GB PCIe SSD/DVD writer/500W power/supply/Windows 10 Pro 64  at $1099 seems like a real bargain and can be easily upgraded with 16GB RAM and 2TB HDD.
To build custom computer from parts (especially high end ones as George McNamara suggested in earlier posts) would cost even more though it will run much faster as well that may be important if ,e.g, online deconvolution  is/will be performed.

Any ideas, feedback, suggestions are appreciated.


Merry Christmas (2nd day European Tradition) and Happy New Year,
Arvydas

+++++++++++++++++++++++++++++
Arvydas Matiukas, Ph.D.
Manager of NRB Shared  Research Equipment
Director of Advanced Microscopy Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept

Email: [hidden email]


>>> Francesco Pasqualini <[hidden email]> 12/16/2019 4:40 AM >>>
*****
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=mggAMhnKbnPe2ZdbN-8CjPUuE8RUMwNtFynTTmgt63Q&e=  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://urldefense.proofpoint.com/v2/url?u=https-3A__www.biorxiv.org_content_10.1101_677344v2&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=_jzLWYEbanIKBXFqUswseorsYDNhxaZEGUfZvquDgyg&e=  (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://urldefense.proofpoint.com/v2/url?u=https-3A__www.physiology.org_doi_abs_10.1152_ajpcell.00351.2018&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=ET9N1EbSlzPvMprIjdbbjKfvVSDG0Q9_gZmoGixrSlo&e=  (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:
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=AJjv8jedDfsaIj6mFFgKL-FINkuWS7WNz9z1rbZvgjU&e= 

> > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=mggAMhnKbnPe2ZdbN-8CjPUuE8RUMwNtFynTTmgt63Q&e=  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: [phone] (IT)
tel: [phone] (USA)

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Arvydas,

A couple of quick thoughts:

1. clone the current drive (probably an HDD) onto a same or larger
capacity SATA-6 SSD (i.e. Silicon Power), unplug the old drive, plug in
the new drive, upgrade that to Windows 10.

2. Can at your and your computer geek(s) leisure, build or buy a new
computer, pop the new Win10 SSD into that. If you go with a new PC with
an NVMe SSD as C: you can (try to) clone the SATA-6 SSD contents onto
that. New PC would also need to get whatever special cards Zeiss uses to
drive your LSM780 (and hopefully have win10 drivers).

Notes:

* in our (work ratio: my computer geek's 99% perspiration, my 1%
inspiration) experience, sometimes "takes" Win 10 a couple of tries to
install onto a drive.

* the ASUS Hyper M.2 PCIe card + (I suggest four 2Tb Silicon Power) NVMe
combination (~$1050) is not expected to be usable as a boot drive. Also
needs BIOS that supports x16 --> x4x4x4x4 bifurcation (5 year old PC
BIOS probably not, but worth testing). One NVMe SSD in the top position
of the Hyper M.2 card (top = away from PCIe slot) can be recognized in
conventional x16 slot (other NVMe's would not be seen), so $57 Hyper +
$244 (Silicon Power 2TB NVMe) could be useful in older PCs.

happy holidays,

George


On 12/26/2019 11:28 AM, Arvydas Matiukas 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.
> *****
>
> Hello List,
>
> The HP 820 workstation that controls LSM780 is over 5 years old and no longer supported under service contract, and Windows 7 support ends in January 2020. Please share your latest experience of:
> 1) upgrading/not upgrading to Windows 10
> 2) upgrading to a (cheaper) new custom built computer.
>
> One computer geek explained me that termination of Microscoft support for current OS is not a big deal - businesses still are using computers running on Windows XP and even 2000. It is a good and up-to-date antivirus software that provides the protection.
>
> Regarding computer upgrade I  looked up what imaging workstations HP is offering. HP Z2 Tower G4 Workstation (i5 8500 6 cores 3GHz/Nvidia Quadro P620 4GB DDR5/8GB DDR4/256GB PCIe SSD/DVD writer/500W power/supply/Windows 10 Pro 64  at $1099 seems like a real bargain and can be easily upgraded with 16GB RAM and 2TB HDD.
> To build custom computer from parts (especially high end ones as George McNamara suggested in earlier posts) would cost even more though it will run much faster as well that may be important if ,e.g, online deconvolution  is/will be performed.
>
> Any ideas, feedback, suggestions are appreciated.
>
>
> Merry Christmas (2nd day European Tradition) and Happy New Year,
> Arvydas
>
> +++++++++++++++++++++++++++++
> Arvydas Matiukas, Ph.D.
> Manager of NRB Shared  Research Equipment
> Director of Advanced Microscopy Core
> SUNY Upstate Medical University
> Neuroscience & Physiology Dept
>
> Email: [hidden email]
>
>
>>>> Francesco Pasqualini <[hidden email]> 12/16/2019 4:40 AM >>>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=AJjv8jedDfsaIj6mFFgKL-FINkuWS7WNz9z1rbZvgjU&e=
>
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=mggAMhnKbnPe2ZdbN-8CjPUuE8RUMwNtFynTTmgt63Q&e=  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://urldefense.proofpoint.com/v2/url?u=https-3A__www.biorxiv.org_content_10.1101_677344v2&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=_jzLWYEbanIKBXFqUswseorsYDNhxaZEGUfZvquDgyg&e=  (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://urldefense.proofpoint.com/v2/url?u=https-3A__www.physiology.org_doi_abs_10.1152_ajpcell.00351.2018&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=ET9N1EbSlzPvMprIjdbbjKfvVSDG0Q9_gZmoGixrSlo&e=  (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:
>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=AJjv8jedDfsaIj6mFFgKL-FINkuWS7WNz9z1rbZvgjU&e=
>>> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=5O_UqzQCsaTecA6gtZOvYZ0L387YferRuYhXvzpJlyA&s=mggAMhnKbnPe2ZdbN-8CjPUuE8RUMwNtFynTTmgt63Q&e=  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
>>>
>

[RJ3]

*****
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*****

George's advice is good. I use Clonezilla (it's free) to create perfect images of disks. They can be handy in case things go sour and you just want to go back to your old setup (nevermind the fact that the data should be backed up elsewhere).

Windows upgrades don't go so well in my experience. You might want to consider a fresh Win10 install. Personally, I can't stand any version of Win10 other than the LTSC/LTSB versions which have cortana and the store removed; but you may have a difficult time finding those versions with your licensing scheme.

Your IT guy is right, you can leave older versions of windows around longer if you need to. Best bet is to disconnect it from the internet though.

[VERMEREN Matthieu]

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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?

*****
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: [phone] (IT)
tel: [phone] (USA)
The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.

[Sylvie Le Guyader]

*****
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*****

Hi Mattieu

I am curious to know why it would not be possible to use JOBS with the silicon objective. To my knowledge you can use JOBS with any objectives.
Do you perhaps mean that you cannot image a multiwell plate with an oil objective? If this is the case, I can say that it is possible since we do it at our facility. You need an oil dispenser for your objective. We bought one quite a long time ago from EMBLEM, the commercial side of EMBL (https://embl-em.de/). It works without problem.

Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
LCI microscopy blog

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of VERMEREN Matthieu
Sent: Sunday, 5 January, 2020 13:55
To: [hidden email]
Subject: Re: Spinning disk or (new) point scanning confocals for live imaging of 3D engineered tissues?

*****
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Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916080122&amp;sdata=am3dgas9TvzghJ%2BaNk2zd8BpYAanylijfCPzI8z0MQ4%3D&amp;reserved=0 and include the link in your posting.
*****

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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Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916090122&amp;sdata=9xFFxI0njDr%2B7nH3owxD%2FSlALKPchC09wMTk1PKGouM%3D&amp;reserved=0 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: [phone] (IT)
tel: [phone] (USA)
The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.


När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.


Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

[Sylvie Le Guyader]

*****
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*****

Hi Francesco

Are there any single point scanning microscopes (i.e. not spinning disk confocals) that scan 2048x2048 pixels at Nyquist pixel size for the 60x objective at 100 fps?
I do not think so, not even with a resonance scanner cannot. You can go very fast with a point scanning confocal but you need to compromise the FOV or pixel size (resolution). And that would be even worse for lower magnification objectives. You need a camera based system.

For your applications, I would definitely go for a spinning disk with bypass to go widefield which you might need for your calcium imaging on monolayers.

Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
LCI microscopy blog


-----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?

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916090122&amp;sdata=9xFFxI0njDr%2B7nH3owxD%2FSlALKPchC09wMTk1PKGouM%3D&amp;reserved=0 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: [phone] (IT)
tel: [phone] (USA)
The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.


När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.


Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

[ICIT]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

[Commercial post]
Dear Francesco,

for applications you describe a spinning disk system would very likely be the only choice: as Sylvie mentioned with a point scanning system you would end up making compromises.

In the past two years we have installed multiple laser-free structured illumination spinning disk systems from Aurox (Clarity: not to mix up with the tissue clearing method). The application range is pretty wide and runs from localization-based super resolution (SRRF) to fast imaging (at least 100 Hz is possible) to whole organism imaging.

As the system is camera-based and collects both in-focus and out of focus light it is extremely photon efficient, it allows the laser-free operation which is of course gentle to your samples.

Since Clarity is an economic add-on module that can be fitted to almost any microscope frame it has a very high investment value. Certainly worth having a look:

www.aurox.co.uk

Best regards,

Mika

[Francesco Pasqualini]

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*****

Thanks for the feedback, and definitively let us know how the FRET/Ca2+
imaging experiments turn out. Francesco


On Sun, Jan 5, 2020 at 2:18 PM VERMEREN Matthieu <[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.
> *****
>
> 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?
>
> *****
> 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: [phone] (IT)
> tel: [phone] (USA)
> The University of Edinburgh is a charitable body, registered in Scotland,
> with registration number SC005336.
>

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

tel: [phone] (IT)
tel: [phone] (USA)

[VERMEREN Matthieu]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Sylvie,

I was not aware of a dispenser for oil or silicone, thanks for the tip :-)

How do you avoid spillage?

Sincerely,

Matthieu

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Sunday, 05 January, 2020 13:25
To: [hidden email]
Subject: Re: Spinning disk or (new) point scanning confocals for live imaging of 3D engineered tissues?

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Mattieu

I am curious to know why it would not be possible to use JOBS with the silicon objective. To my knowledge you can use JOBS with any objectives.
Do you perhaps mean that you cannot image a multiwell plate with an oil objective? If this is the case, I can say that it is possible since we do it at our facility. You need an oil dispenser for your objective. We bought one quite a long time ago from EMBLEM, the commercial side of EMBL (https://embl-em.de/). It works without problem.

Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
LCI microscopy blog

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of VERMEREN Matthieu
Sent: Sunday, 5 January, 2020 13:55
To: [hidden email]
Subject: Re: Spinning disk or (new) point scanning confocals for live imaging of 3D engineered tissues?

*****
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Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916080122&amp;sdata=am3dgas9TvzghJ%2BaNk2zd8BpYAanylijfCPzI8z0MQ4%3D&amp;reserved=0 and include the link in your posting.
*****

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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Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916090122&amp;sdata=9xFFxI0njDr%2B7nH3owxD%2FSlALKPchC09wMTk1PKGouM%3D&amp;reserved=0 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: [phone] (IT)
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[Francesco Pasqualini]

*****
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*****

Hi Sylvie - I am also very interested but I don't seem to be able to find
the product at https://embl-em.de/ - do you have any tips on how to get the
device? Thanks, Francesco


On Wed, Jan 8, 2020 at 12:57 PM VERMEREN Matthieu <
[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.
> *****
>
> Hi Sylvie,
>
> I was not aware of a dispenser for oil or silicone, thanks for the tip :-)
>
> How do you avoid spillage?
>
> Sincerely,
>
> Matthieu
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Sylvie Le Guyader
> Sent: Sunday, 05 January, 2020 13:25
> To: [hidden email]
> Subject: Re: Spinning disk or (new) point scanning confocals for live
> imaging of 3D engineered tissues?
>
> *****
> 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 Mattieu
>
> I am curious to know why it would not be possible to use JOBS with the
> silicon objective. To my knowledge you can use JOBS with any objectives.
> Do you perhaps mean that you cannot image a multiwell plate with an oil
> objective? If this is the case, I can say that it is possible since we do
> it at our facility. You need an oil dispenser for your objective. We bought
> one quite a long time ago from EMBLEM, the commercial side of EMBL (
> https://embl-em.de/). It works without problem.
>
> Med vänlig hälsning / Best regards
> Sylvie
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Blickagången 16,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> LCI microscopy blog
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of VERMEREN Matthieu
> Sent: Sunday, 5 January, 2020 13:55
> To: [hidden email]
> Subject: Re: Spinning disk or (new) point scanning confocals for live
> imaging of 3D engineered tissues?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916080122&amp;sdata=QhP9C5P28OGIPkTfW2fQhHK%2FCIjgVN43KgQgF%2BHLgak%3D&amp;reserved=0
> Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916080122&amp;sdata=am3dgas9TvzghJ%2BaNk2zd8BpYAanylijfCPzI8z0MQ4%3D&amp;reserved=0
> and include the link in your posting.
> *****
>
> 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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916080122&amp;sdata=QhP9C5P28OGIPkTfW2fQhHK%2FCIjgVN43KgQgF%2BHLgak%3D&amp;reserved=0
> Post images on
> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Caab63e63cdef4257be8808d791e1b4ff%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637138270916090122&amp;sdata=9xFFxI0njDr%2B7nH3owxD%2FSlALKPchC09wMTk1PKGouM%3D&amp;reserved=0
> 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: [phone] (IT)
> tel: [phone] (USA)
> The University of Edinburgh is a charitable body, registered in Scotland,
> with registration number SC005336.
>
>
> När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI
> kommer att behandla dina personuppgifter. Här finns information om hur KI
> behandlar personuppgifter<
> https://ki.se/medarbetare/integritetsskyddspolicy>.
>
>
> Sending email to Karolinska Institutet (KI) will result in KI processing
> your personal data. You can read more about KI’s processing of personal
> data here<https://ki.se/en/staff/data-protection-policy>.
>

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

tel: [phone] (IT)
tel: [phone] (USA)

[Steffen Dietzel]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Dear all,

I am currently to update a microscope system from Win 7 pro to Win 10
Enterprise 2016 LTSB. The latter version is what Leica is using on their
newest machines, so the OS should be relatively safe for their software.

Since the direct update is not allowed, the plan is to update first to
2015 LTSB and then to 2016 LTSB. Done the first step, but the second
fails: The option "keep personal files and apps" is grayed out and it
says that the editions of Windows do not match. I suspect the reason is
the different language: the 2015 version is en-gb, the 2016 en-us.

en-us is fine, that is what Leica is distributing (in Germany at least).

What I have are these files:

SW_DVD5_WIN_ENT_LTSB_10_2015_64BIT_Eng_Intl_MLF_X20-26801.ISO
SW_DVD5_WIN_ENT_N_LTSB_2016_64BIT_English_MLF_X21-07487.ISO

It appears that "English" stands for en-us and Eng_Int for en-gb. So I
would need an iso file WIN_ENT_LTSB_10_2015_64BIT with "English", not
Eng_Intl. (And I will have to go back to win 7 first)

Does anybody know a place where this could be downloaded? Private mails
welcome.

Note: I found some hints that the language of an existing installation
can be changed with a dism command. This also should work. But it only
works if you boot into a WinPE environment from USB, and when I do that
my hard disk is not recognized, so I would have to inject drivers for
the HD into PE, which seems to me more complicated than to use the
correct language version)

Best

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Steffen Dietzel]

*****
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.
*****

got an en-us version of LTSB 2015, still the same problem: ".. because
you're installing an edition of Windows that's different than the one
you're currently using".

If somebody has an idea what the problem is, I am all ears

Steffen

Am 08.01.2020 um 14:29 schrieb Steffen Dietzel:

> *****
> 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.
> *****
>
> Dear all,
>
> I am currently to update a microscope system from Win 7 pro to Win 10
> Enterprise 2016 LTSB. The latter version is what Leica is using on
> their newest machines, so the OS should be relatively safe for their
> software.
>
> Since the direct update is not allowed, the plan is to update first to
> 2015 LTSB and then to 2016 LTSB. Done the first step, but the second
> fails: The option "keep personal files and apps" is grayed out and it
> says that the editions of Windows do not match. I suspect the reason
> is the different language: the 2015 version is en-gb, the 2016 en-us.
>
> en-us is fine, that is what Leica is distributing (in Germany at least).
>
> What I have are these files:
>
> SW_DVD5_WIN_ENT_LTSB_10_2015_64BIT_Eng_Intl_MLF_X20-26801.ISO
> SW_DVD5_WIN_ENT_N_LTSB_2016_64BIT_English_MLF_X21-07487.ISO
>
> It appears that "English" stands for en-us and Eng_Int for en-gb. So I
> would need an iso file WIN_ENT_LTSB_10_2015_64BIT with "English", not
> Eng_Intl. (And I will have to go back to win 7 first)
>
> Does anybody know a place where this could be downloaded? Private
> mails welcome.
>
> Note: I found some hints that the language of an existing installation
> can be changed with a dism command. This also should work. But it only
> works if you boot into a WinPE environment from USB, and when I do
> that my hard disk is not recognized, so I would have to inject drivers
> for the HD into PE, which seems to me more complicated than to use the
> correct language version)
>
> Best
>
> Steffen
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de