Advice on equipment for FRET in live cells

[Pedro Camello]

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Hi all,

a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.

The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)

Thanks in advance

[Craig Brideau]

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If widefield is sufficient for their imaging, then why not simply use a two
camera solution with appropriate filters? If cell viability due to light
exposure is a problem then I do agree spinning disk would be gentler, but
certainly more expensive.

Craig

On Thu, Mar 28, 2019, 4:42 AM Pedro Camello <[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 all,
>
> a colleague is trying to set up a system to make FRET in living cells. A
> cheap option is to upgrade an existing imaging with a spininng disk.
>
> The alternative is to go for a new system. In this case, I would
> appreciate opinions/experiences regarding systems (or technologies
> -spininng, point scanning,...-)
>
> Thanks in advance
>

[Darran Clements]

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If you are considering certain spinning disk systems, perhaps, as an alternative, the Leica SP8 Falcon would be in the mix? Not as fast, or as kind, but would offer fully integrated spectral FLIM-FRET, not just filter based FRET imaging.
BTW it's been many years since I was employed by Leica, this was just thinking out loud based on capability of systems.


Get Outlook for Android<https://aka.ms/ghei36>

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Pedro Camello <[hidden email]>
Sent: Thursday, March 28, 2019 10:21:03 AM
To: [hidden email]
Subject: Advice on equipment for FRET in live cells

*****
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*****

Hi all,

a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.

The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)

Thanks in advance

[Tim Feinstein]

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Hi Pedro,

Please provide details about your experiment.  Do you plan to combine FRET with localization information, or are you using a biosensor for which whole-cells measurement is ok?  Do you plan to measure time kinetics with sensitized emission, or do single measurements with either SE or acceptor photobleach?  If you're measuring time kinetics, then what sample rate do you need?  Is photobleaching or photodamage a concern?  Will you do intramolecular FRET?  Antibody-antibody FRET?  Transgenic labels?  

I'd start with a widefield system and one camera.  If you need faster measurements then get an emission splitter like a DualView.  If you need high speed AND the full camera frame, use a more expensive emission splitter that mounts two cameras.  If you need high sensitivity localization information and speed is not quite as important, then use a confocal.  However if doing that you need to have an appropriate laser such as a 442nm line for CFP/YFP FRET.  

Feel free to contact me with any other questions,


TF

Timothy Feinstein, Ph.D.
Research Scientist
Department of Developmental Biology
University of Pittsburgh

 


On 3/28/19, 6:43 AM, "Confocal Microscopy List on behalf of Pedro Camello" <[hidden email] on behalf of [hidden email]> wrote:

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    *****
   
    Hi all,
   
    a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.
   
    The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)
   
    Thanks in advance
   

[Kevin Dean]

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Most live-cell FRET measurements that I have been a part of have used wide-field illumination and detection, and avoid electron multiplying gain.  The multiple camera configuration is nice, but requires accurate registration of the cameras.  The most versatile option is a single camera, with an emission filter wheel.  The choice of emission filters is important, and we regularly also correct for spectral cross-talk, bleaching, camera noise, etc.

Frequency-domain FLIM is great, but less frequently used.

Best,
Kevin

Kevin Dean
Assistant Professor
Department of Cell Biology
UT Southwestern Medical Center
6000 Harry Hines Blvd, NL5.116A
Dallas, TX 75390.
(P) 214-648-4833
(C) 303-681-8863
(E) [hidden email]<mailto:[hidden email]>

On Mar 28, 2019, at 9:26 AM, Darran Clements <[hidden email]<mailto:[hidden email]>> wrote:

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*****

If you are considering certain spinning disk systems, perhaps, as an alternative, the Leica SP8 Falcon would be in the mix? Not as fast, or as kind, but would offer fully integrated spectral FLIM-FRET, not just filter based FRET imaging.
BTW it's been many years since I was employed by Leica, this was just thinking out loud based on capability of systems.


Get Outlook for Android<https://aka.ms/ghei36>

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Pedro Camello <[hidden email]>
Sent: Thursday, March 28, 2019 10:21:03 AM
To: [hidden email]
Subject: Advice on equipment for FRET in live cells

*****
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 all,

a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.

The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)

Thanks in advance


________________________________

UT Southwestern


Medical Center



The future of medicine, today.

[Periasamy, Ammasi (ap3t)-2]

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*****

Hello,
Not sure what type of system you would like to upgrade??
Assuming that you have a wide-field system, Yes, spinning disk is an ideal option. Not sure you are looking for laser based spinning disk or arc lamp based spinning disk.
If you are trying to upgrade wide field fluorescence microscopy with camera to collect FRET images, you may need excitation and emission filter wheels. For example, for cerulean and Venus pair, you may have to use the same dichroic mirror to collect two emissions (donor and acceptor) with donor excitation wavelength.
Use high quantum efficiency camera and that will reduce your collection time, reduce the photobleaching.
As you know the wide-field not good for looking FRET signal inside the cells. The confocal will help you to collect FRET signal at various optical sections.
Also you should remember that there will be lot of spectral bleedthrough in the FRET channel, either you sue spinning disk or confocal. You should correct the contamination in the FRET signal. Look at this paper, if you wanted to write your own code, Cytometry A. 83A(9): 780-793, 2013.

If you have the money, you should consider to purchase FLIM imaging system, no contamination correction is required in the lifetime imaging system. Princeton has the camera based FLIM. Just purchase the camera and integrate with the wide-field microscope.
For confocal or 2photon FLIM- Becker-Hickl, Leica FALCON system, Pico Quant, ISS, Lambert, etc
Hope this helps. Contact me directly, I am happy to help.
Good luck,
Ammasi

Dr. Ammasi Periasamy
Center Director, WM Keck Center for Cellular Imaging,
Prof. of Biology & Biomed. Eng., University of Virginia,
Physical & Life Sciences Building (PLSB 005)
90 Geldard Dr., Charlottesville, VA 22904, USA.

http://www.kcci.virginia.edu/people/profile/ap3t
Phone: [phone] or 982-4869
Fax: [phone]
E-mail: [hidden email]

FRET/FLIM Workshop-March 9-13, 2020: http://www.kcci.virginia.edu/workshop 



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Pedro Camello <[hidden email]>
Sent: Thursday, March 28, 2019 10:21:03 AM
To: [hidden email]
Subject: Advice on equipment for FRET in live cells

*****
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 all,

a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.

The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)

Thanks in advance


________________________________

UT Southwestern


Medical Center



The future of medicine, today.

[Gerhard Holst]

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*****

Dear all,

I am afraid it'll be a commercial post this time, but only in the sense that there are also other camera based FLIM imaging camera systems, like our pco.flim. It works nicely with FRET measurements at a widefield microscope.

And also other not mentioned systems (Lambert Instruments, Photon Score)

Best regards,

Gerhard Holst

PCO AG
Head of Research Department
Donaupark 11
93309 Kelheim, Germany

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Periasamy, Ammasi (ap3t)
Gesendet: Donnerstag, 28. März 2019 20:12
An: [hidden email]
Betreff: Re: Advice on equipment for FRET in live cells


*****
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,
Not sure what type of system you would like to upgrade??
Assuming that you have a wide-field system, Yes, spinning disk is an ideal option. Not sure you are looking for laser based spinning disk or arc lamp based spinning disk.
If you are trying to upgrade wide field fluorescence microscopy with camera to collect FRET images, you may need excitation and emission filter wheels. For example, for cerulean and Venus pair, you may have to use the same dichroic mirror to collect two emissions (donor and acceptor) with donor excitation wavelength.
Use high quantum efficiency camera and that will reduce your collection time, reduce the photobleaching.
As you know the wide-field not good for looking FRET signal inside the cells. The confocal will help you to collect FRET signal at various optical sections.
Also you should remember that there will be lot of spectral bleedthrough in the FRET channel, either you sue spinning disk or confocal. You should correct the contamination in the FRET signal. Look at this paper, if you wanted to write your own code, Cytometry A. 83A(9): 780-793, 2013.

If you have the money, you should consider to purchase FLIM imaging system, no contamination correction is required in the lifetime imaging system. Princeton has the camera based FLIM. Just purchase the camera and integrate with the wide-field microscope.
For confocal or 2photon FLIM- Becker-Hickl, Leica FALCON system, Pico Quant, ISS, Lambert, etc
Hope this helps. Contact me directly, I am happy to help.
Good luck,
Ammasi

Dr. Ammasi Periasamy
Center Director, WM Keck Center for Cellular Imaging,
Prof. of Biology & Biomed. Eng., University of Virginia,
Physical & Life Sciences Building (PLSB 005)
90 Geldard Dr., Charlottesville, VA 22904, USA.

http://www.kcci.virginia.edu/people/profile/ap3t
Phone: [phone] or 982-4869
Fax: [phone]
E-mail: [hidden email]

FRET/FLIM Workshop-March 9-13, 2020: http://www.kcci.virginia.edu/workshop 



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Pedro Camello <[hidden email]>
Sent: Thursday, March 28, 2019 10:21:03 AM
To: [hidden email]
Subject: Advice on equipment for FRET in live cells

*****
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 all,

a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.

The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)

Thanks in advance


________________________________

UT Southwestern


Medical Center



The future of medicine, today.

[John Pediani]

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*****

We have successfully used a dual view 2 image splitting device to record the donor and fret channelimages simultaneously 


-----Original Message-----
From: Kevin Dean <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, Mar 28, 2019 04:41 PM
Subject: Re: Advice on equipment for FRET in live cells


*****
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.
*****

Most live-cell FRET measurements that I have been a part of have used wide-field illumination and detection, and avoid electron multiplying gain.  The multiple camera configuration is nice, but requires accurate registration of the cameras.  The Formost versatile option is a single camera, with an emission filter wheel.  The choice of emission filters is important, and we regularly also correct for spectral cross-talk, bleaching, camera noise, etc.

Frequency-domain FLIM is great, but less frequently used.

Best,
Kevin

Kevin Dean
Assistant Professor
Department of Cell Biology
UT Southwestern Medical Center
6000 Harry Hines Blvd, NL5.116A
Dallas, TX 75390.
(P) 214-648-4833
(C) 303-681-8863
(E) [hidden email]<mailto:[hidden email]>

On Mar 28, 2019, at 9:26 AM, Darran Clements <[hidden email]<mailto:[hidden email]>> wrote:

*****
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.
*****

If you are considering certain spinning disk systems, perhaps, as an alternative, the Leica SP8 Falcon would be in the mix? Not as fast, or as kind, but would offer fully integrated spectral FLIM-FRET, not just filter based FRET imaging.
BTW it's been many years since I was employed by Leica, this was just thinking out loud based on capability of systems.


Get Outlook for Android<https://aka.ms/ghei36>

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Pedro Camello <[hidden email]>
Sent: Thursday, March 28, 2019 10:21:03 AM
To: [hidden email]
Subject: Advice on equipment for FRET in live cells

*****
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 all,

a colleague is trying to set up a system to make FRET in living cells. A cheap option is to upgrade an existing imaging with a spininng disk.

The alternative is to go for a new system. In this case, I would appreciate opinions/experiences regarding systems (or technologies -spininng, point scanning,...-)

Thanks in advance


________________________________

UT Southwestern


Medical Center



The future of medicine, today.

[Zdenek Svindrych-2]

*****
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*****

Hi Gerhard,

I was always curious how those dual-pixel cameras compare to
intensifier-based FD-FLIM cameras? The QE (I guess the stated 39% includes
fill factor and other losses, but at what wavelength?) is comparable to
GaAsP intensifiers, but how does the readout noise (48 electrons RMS for
pco.FLIM) compare with the excess noise of an intensifier?
Your paper (https://doi.org/10.1002/jemt.22587) show some interesting
details about the camera, but the piece of information I'm missing badly
is: What cerulean-venus FRET standard did you use? What was the linker?
What is the consensus FRET efficiency of that standard? Are the polar plots
showing the difference between 0% and 20% FRET? Or 0$ and 50% FRET?

Thanks for your any insights!

Best, zdenek


On Thu, Mar 28, 2019 at 3:57 PM Gerhard Holst <[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.
> *****
>
> Dear all,
>
> I am afraid it'll be a commercial post this time, but only in the sense
> that there are also other camera based FLIM imaging camera systems, like
> our pco.flim. It works nicely with FRET measurements at a widefield
> microscope.
>
> And also other not mentioned systems (Lambert Instruments, Photon Score)
>
> Best regards,
>
> Gerhard Holst
>
> PCO AG
> Head of Research Department
> Donaupark 11
> 93309 Kelheim, Germany
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]]
> Im Auftrag von Periasamy, Ammasi (ap3t)
> Gesendet: Donnerstag, 28. März 2019 20:12
> An: [hidden email]
> Betreff: Re: Advice on equipment for FRET in live cells
>
>
> *****
> 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,
> Not sure what type of system you would like to upgrade??
> Assuming that you have a wide-field system, Yes, spinning disk is an ideal
> option. Not sure you are looking for laser based spinning disk or arc lamp
> based spinning disk.
> If you are trying to upgrade wide field fluorescence microscopy with
> camera to collect FRET images, you may need excitation and emission filter
> wheels. For example, for cerulean and Venus pair, you may have to use the
> same dichroic mirror to collect two emissions (donor and acceptor) with
> donor excitation wavelength.
> Use high quantum efficiency camera and that will reduce your collection
> time, reduce the photobleaching.
> As you know the wide-field not good for looking FRET signal inside the
> cells. The confocal will help you to collect FRET signal at various optical
> sections.
> Also you should remember that there will be lot of spectral bleedthrough
> in the FRET channel, either you sue spinning disk or confocal. You should
> correct the contamination in the FRET signal. Look at this paper, if you
> wanted to write your own code, Cytometry A. 83A(9): 780-793, 2013.
>
> If you have the money, you should consider to purchase FLIM imaging
> system, no contamination correction is required in the lifetime imaging
> system. Princeton has the camera based FLIM. Just purchase the camera and
> integrate with the wide-field microscope.
> For confocal or 2photon FLIM- Becker-Hickl, Leica FALCON system, Pico
> Quant, ISS, Lambert, etc
> Hope this helps. Contact me directly, I am happy to help.
> Good luck,
> Ammasi
>
> Dr. Ammasi Periasamy
> Center Director, WM Keck Center for Cellular Imaging,
> Prof. of Biology & Biomed. Eng., University of Virginia,
> Physical & Life Sciences Building (PLSB 005)
> 90 Geldard Dr., Charlottesville, VA 22904, USA.
>
> http://www.kcci.virginia.edu/people/profile/ap3t
> Phone: [phone] or 982-4869
> Fax: [phone]
> E-mail: [hidden email]
>
> FRET/FLIM Workshop-March 9-13, 2020: http://www.kcci.virginia.edu/workshop
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Pedro Camello <[hidden email]>
> Sent: Thursday, March 28, 2019 10:21:03 AM
> To: [hidden email]
> Subject: Advice on equipment for FRET in live cells
>
> *****
> 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 all,
>
> a colleague is trying to set up a system to make FRET in living cells. A
> cheap option is to upgrade an existing imaging with a spininng disk.
>
> The alternative is to go for a new system. In this case, I would
> appreciate opinions/experiences regarding systems (or technologies
> -spininng, point scanning,...-)
>
> Thanks in advance
>
>
> ________________________________
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Philippe clemenceau]

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

Hi All,

It looks like the new TOGGEL  CCD camera for frequency domain FLIM made by Lambert Instruments was forgotten in the discussion below.
https://www.lambertinstruments.com/toggel#toggel-introduction <https://www.lambertinstruments.com/toggel#toggel-introduction>

We distribute Lambert Instruments in North America and anyone is welcome to contact me if you have questions.

Regards,

Philippe Clémenceau,
Co-Founder, MS in Optical Science
Axiom Optics, Excellence in advanced imaging and optical metrology
 
Axiom Optics/Imagine Optic Inc
@Greentown Labs
444 Somerville ave
Somerville, MA 02143, USA
 
Phone:  [phone]
Mobile: [phone]
www.axiomoptics.com <http://www.axiomoptics.com/>  

Meet us at the NALM microscopy workshop <http://www.axiomoptics.com/events/new-approaches-in-light-microscopy-workshop-2019/>, June 4-6, 2019 at Harvard University, Cambridge, MA , USA

Check out ARGO-Power <http://www.axiomoptics.com/alm/argo-slides/>, a new solution for fluorescence microscopy quality assessment

> On Mar 29, 2019, at 1:54 PM, Zdenek Svindrych <[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 Gerhard,
>
> I was always curious how those dual-pixel cameras compare to
> intensifier-based FD-FLIM cameras? The QE (I guess the stated 39% includes
> fill factor and other losses, but at what wavelength?) is comparable to
> GaAsP intensifiers, but how does the readout noise (48 electrons RMS for
> pco.FLIM) compare with the excess noise of an intensifier?
> Your paper (https://doi.org/10.1002/jemt.22587) show some interesting
> details about the camera, but the piece of information I'm missing badly
> is: What cerulean-venus FRET standard did you use? What was the linker?
> What is the consensus FRET efficiency of that standard? Are the polar plots
> showing the difference between 0% and 20% FRET? Or 0$ and 50% FRET?
>
> Thanks for your any insights!
>
> Best, zdenek
>
>
> On Thu, Mar 28, 2019 at 3:57 PM Gerhard Holst <[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.
>> *****
>>
>> Dear all,
>>
>> I am afraid it'll be a commercial post this time, but only in the sense
>> that there are also other camera based FLIM imaging camera systems, like
>> our pco.flim. It works nicely with FRET measurements at a widefield
>> microscope.
>>
>> And also other not mentioned systems (Lambert Instruments, Photon Score)
>>
>> Best regards,
>>
>> Gerhard Holst
>>
>> PCO AG
>> Head of Research Department
>> Donaupark 11
>> 93309 Kelheim, Germany
>>
>> -----Ursprüngliche Nachricht-----
>> Von: Confocal Microscopy List [mailto:[hidden email]]
>> Im Auftrag von Periasamy, Ammasi (ap3t)
>> Gesendet: Donnerstag, 28. März 2019 20:12
>> An: [hidden email]
>> Betreff: Re: Advice on equipment for FRET in live cells
>>
>>
>> *****
>> 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,
>> Not sure what type of system you would like to upgrade??
>> Assuming that you have a wide-field system, Yes, spinning disk is an ideal
>> option. Not sure you are looking for laser based spinning disk or arc lamp
>> based spinning disk.
>> If you are trying to upgrade wide field fluorescence microscopy with
>> camera to collect FRET images, you may need excitation and emission filter
>> wheels. For example, for cerulean and Venus pair, you may have to use the
>> same dichroic mirror to collect two emissions (donor and acceptor) with
>> donor excitation wavelength.
>> Use high quantum efficiency camera and that will reduce your collection
>> time, reduce the photobleaching.
>> As you know the wide-field not good for looking FRET signal inside the
>> cells. The confocal will help you to collect FRET signal at various optical
>> sections.
>> Also you should remember that there will be lot of spectral bleedthrough
>> in the FRET channel, either you sue spinning disk or confocal. You should
>> correct the contamination in the FRET signal. Look at this paper, if you
>> wanted to write your own code, Cytometry A. 83A(9): 780-793, 2013.
>>
>> If you have the money, you should consider to purchase FLIM imaging
>> system, no contamination correction is required in the lifetime imaging
>> system. Princeton has the camera based FLIM. Just purchase the camera and
>> integrate with the wide-field microscope.
>> For confocal or 2photon FLIM- Becker-Hickl, Leica FALCON system, Pico
>> Quant, ISS, Lambert, etc
>> Hope this helps. Contact me directly, I am happy to help.
>> Good luck,
>> Ammasi
>>
>> Dr. Ammasi Periasamy
>> Center Director, WM Keck Center for Cellular Imaging,
>> Prof. of Biology & Biomed. Eng., University of Virginia,
>> Physical & Life Sciences Building (PLSB 005)
>> 90 Geldard Dr., Charlottesville, VA 22904, USA.
>>
>> http://www.kcci.virginia.edu/people/profile/ap3t
>> Phone: [phone] or 982-4869
>> Fax: [phone]
>> E-mail: [hidden email]
>>
>> FRET/FLIM Workshop-March 9-13, 2020: http://www.kcci.virginia.edu/workshop
>>
>>
>>
>> ________________________________
>> From: Confocal Microscopy List <[hidden email]> on
>> behalf of Pedro Camello <[hidden email]>
>> Sent: Thursday, March 28, 2019 10:21:03 AM
>> To: [hidden email]
>> Subject: Advice on equipment for FRET in live cells
>>
>> *****
>> 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 all,
>>
>> a colleague is trying to set up a system to make FRET in living cells. A
>> cheap option is to upgrade an existing imaging with a spininng disk.
>>
>> The alternative is to go for a new system. In this case, I would
>> appreciate opinions/experiences regarding systems (or technologies
>> -spininng, point scanning,...-)
>>
>> Thanks in advance
>>
>>
>> ________________________________
>>
>> UT Southwestern
>>
>>
>> Medical Center
>>
>>
>>
>> The future of medicine, today.
>>
>
>
> --
> --
> Zdenek Svindrych, Ph.D.
> Research Associate - Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>

[Gerhard Holst]

*****
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 Zdenek,

the given peak QE is in the green, on our website you will find a QE curve (https://www.pco.de/flim-camera/pcoflim/ ). (and yes, the QE values include fill factor, front glass and everything, it was measured with the camera).

Unfortunately I have no experience with the image intensifier based systems, because in past times I used time domain and CCD cameras. Certainly the camera in terms of noise and sensitivity is not comparable to sCMOS. But meanwhile it has been successfully applied to a variety of different applications from:
Optical chemical measurements: https://pubs.acs.org/doi/10.1021/acs.analchem.8b02534 and https://pubs.acs.org/doi/10.1021/acs.analchem.8b05869 to indicator driven experiments: https://www.ncbi.nlm.nih.gov/pubmed/30636030 and it has been applied to FRET and endogenous fluorescence (still to be published).

My best guess is, for very low signals, the image intensifier still gives you a better response.

About the FRET experiments, which Hongtao and Prof. Gratton did, I am afraid, you will have to ask them. I did together with Fred Wouters and Gertrude Bunt FRET measurements with CFP/YFP and used homodyne detection. As reference we used from Starna Scientific their SFG which by measurement had a lifetime of 3.75 ns.

with best regards,

Gerhard


Dr. Gerhard Holst
Head of Research & Science
Business Development
[phone]

PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
Vorstand / Managing Board: Dr. Emil Ott (Chairman), Alexander Grünig, Luitpold Kaspar
Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Johann Plöb
USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court: Amtsgericht Regensburg HRB 9157
Sitz der Gesellschaft / Registered Office: Kelheim

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Zdenek Svindrych
Gesendet: Freitag, 29. März 2019 18:54
An: [hidden email]
Betreff: Re: Advice on equipment for FRET in live cells


*****
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 Gerhard,

I was always curious how those dual-pixel cameras compare to
intensifier-based FD-FLIM cameras? The QE (I guess the stated 39% includes
fill factor and other losses, but at what wavelength?) is comparable to
GaAsP intensifiers, but how does the readout noise (48 electrons RMS for
pco.FLIM) compare with the excess noise of an intensifier?
Your paper (https://doi.org/10.1002/jemt.22587) show some interesting
details about the camera, but the piece of information I'm missing badly
is: What cerulean-venus FRET standard did you use? What was the linker?
What is the consensus FRET efficiency of that standard? Are the polar plots
showing the difference between 0% and 20% FRET? Or 0$ and 50% FRET?

Thanks for your any insights!

Best, zdenek


On Thu, Mar 28, 2019 at 3:57 PM Gerhard Holst <[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.
> *****
>
> Dear all,
>
> I am afraid it'll be a commercial post this time, but only in the sense
> that there are also other camera based FLIM imaging camera systems, like
> our pco.flim. It works nicely with FRET measurements at a widefield
> microscope.
>
> And also other not mentioned systems (Lambert Instruments, Photon Score)
>
> Best regards,
>
> Gerhard Holst
>
> PCO AG
> Head of Research Department
> Donaupark 11
> 93309 Kelheim, Germany
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]]
> Im Auftrag von Periasamy, Ammasi (ap3t)
> Gesendet: Donnerstag, 28. März 2019 20:12
> An: [hidden email]
> Betreff: Re: Advice on equipment for FRET in live cells
>
>
> *****
> 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,
> Not sure what type of system you would like to upgrade??
> Assuming that you have a wide-field system, Yes, spinning disk is an ideal
> option. Not sure you are looking for laser based spinning disk or arc lamp
> based spinning disk.
> If you are trying to upgrade wide field fluorescence microscopy with
> camera to collect FRET images, you may need excitation and emission filter
> wheels. For example, for cerulean and Venus pair, you may have to use the
> same dichroic mirror to collect two emissions (donor and acceptor) with
> donor excitation wavelength.
> Use high quantum efficiency camera and that will reduce your collection
> time, reduce the photobleaching.
> As you know the wide-field not good for looking FRET signal inside the
> cells. The confocal will help you to collect FRET signal at various optical
> sections.
> Also you should remember that there will be lot of spectral bleedthrough
> in the FRET channel, either you sue spinning disk or confocal. You should
> correct the contamination in the FRET signal. Look at this paper, if you
> wanted to write your own code, Cytometry A. 83A(9): 780-793, 2013.
>
> If you have the money, you should consider to purchase FLIM imaging
> system, no contamination correction is required in the lifetime imaging
> system. Princeton has the camera based FLIM. Just purchase the camera and
> integrate with the wide-field microscope.
> For confocal or 2photon FLIM- Becker-Hickl, Leica FALCON system, Pico
> Quant, ISS, Lambert, etc
> Hope this helps. Contact me directly, I am happy to help.
> Good luck,
> Ammasi
>
> Dr. Ammasi Periasamy
> Center Director, WM Keck Center for Cellular Imaging,
> Prof. of Biology & Biomed. Eng., University of Virginia,
> Physical & Life Sciences Building (PLSB 005)
> 90 Geldard Dr., Charlottesville, VA 22904, USA.
>
> http://www.kcci.virginia.edu/people/profile/ap3t
> Phone: [phone] or 982-4869
> Fax: [phone]
> E-mail: [hidden email]
>
> FRET/FLIM Workshop-March 9-13, 2020: http://www.kcci.virginia.edu/workshop
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Pedro Camello <[hidden email]>
> Sent: Thursday, March 28, 2019 10:21:03 AM
> To: [hidden email]
> Subject: Advice on equipment for FRET in live cells
>
> *****
> 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 all,
>
> a colleague is trying to set up a system to make FRET in living cells. A
> cheap option is to upgrade an existing imaging with a spininng disk.
>
> The alternative is to go for a new system. In this case, I would
> appreciate opinions/experiences regarding systems (or technologies
> -spininng, point scanning,...-)
>
> Thanks in advance
>
>
> ________________________________
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth