flash 4.0v2 for single molecule imaging

[Jeff Spector]

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Greetings,
 We recently acquired a Flash4.0v2 scmos camera and have been quite happy
with it for use on our spinning disk. We are now trying to do some single
molecule imaging and have so far been unimpressed by our results. I was just
wondering if anyone has experience using this camera for single molecule (TIRF)
imaging. We are trying to look at molecules move on a surface but we don't
need incredible hight frame rates.Any advice on combinations of objective mag,
camera binning, and frame rates that will work would be most helpful..

-Jeff

[Gerhard Holst]

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Hi Jeff,

do you have any idea of the number of photons you are looking at? Binning in sCMOS does improve the signal-to-noise a little, but it is always a digital summation (that's the same effect if you reduce resolution by summing up pixel groups afterwards in the result image), therefore I doubt that it'll bring a big improvement. Bute the "binned" image will look nicer due to increased contrast.

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Jeff Spector
Gesendet: Donnerstag, 17. Juli 2014 01:58
An: [hidden email]
Betreff: flash 4.0v2 for single molecule imaging

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*****

Greetings,
 We recently acquired a Flash4.0v2 scmos camera and have been quite happy
with it for use on our spinning disk. We are now trying to do some single
molecule imaging and have so far been unimpressed by our results. I was just
wondering if anyone has experience using this camera for single molecule (TIRF)
imaging. We are trying to look at molecules move on a surface but we don't
need incredible hight frame rates.Any advice on combinations of objective mag,
camera binning, and frame rates that will work would be most helpful..

-Jeff

[Colin Coates]

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**commercial response**
Hi Jeff,
Bottom line, if you happen to have access to an EMCCD that's generally
what I'd recommend for dynamic single molecule TIRF. After a few years of
our single molecule imaging users comparing both our EMCCD and sCMOS
cameras, the feedback has been pretty convincing. Its generalising, but the
light levels involved in single molecule tend to be below the practical cross
over point in the SNR vs photon flux curve, whereby an EMCCD has the
advantage. Localisation super-resolution microscopy can be the exception,
since the approach requires you to drive up photon levels to be successful.

Having said that, if you are trying to adapt an sCMOS to dynamic single
molecule, one option is to at least bring the effective pixel size closer to
what you are used to with a larger pixel EMCCD. The best way is to use
between 1.5x to 2x demagnification before the camera (depends on the
objective as to what you can get away with and still oversample).
Alternatively, 2x2 binning of the 6.5 um pixel (yielding a 13um binned
pixel) can also be effective. The photons per pixel will quadruple, but be
aware the read noise will also double - however, there's still a net gain in
SNR.

Best,
Colin

Dr Colin Coates
Product Manager - Imaging
Andor Technology

[Jeff Spector]

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Thanks for the replies so far.
  We do have access to EMCCD cameras and that is what we use most of the
time for single molecule dynamics. Most of what I have looked at says that
sCMOs is better than EMCCD but the crossover point seem to be right around
the S/N you get when doing single molecule imaging. I guess we thought that
since you can do single molecule localization you could do dynamics but I
guess in the localization case you really are pumping a lot more photons
out of the fluorophore (which I guess means you image it for a shorter
time?).
thanks...
-Jeff


On Thu, Jul 17, 2014 at 4:59 AM, Colin Coates <[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**
> Hi Jeff,
> Bottom line, if you happen to have access to an EMCCD that's generally
> what I'd recommend for dynamic single molecule TIRF. After a few years of
> our single molecule imaging users comparing both our EMCCD and sCMOS
> cameras, the feedback has been pretty convincing. Its generalising, but the
> light levels involved in single molecule tend to be below the practical
> cross
> over point in the SNR vs photon flux curve, whereby an EMCCD has the
> advantage. Localisation super-resolution microscopy can be the exception,
> since the approach requires you to drive up photon levels to be successful.
>
> Having said that, if you are trying to adapt an sCMOS to dynamic single
> molecule, one option is to at least bring the effective pixel size closer
> to
> what you are used to with a larger pixel EMCCD. The best way is to use
> between 1.5x to 2x demagnification before the camera (depends on the
> objective as to what you can get away with and still oversample).
> Alternatively, 2x2 binning of the 6.5 um pixel (yielding a 13um binned
> pixel) can also be effective. The photons per pixel will quadruple, but be
> aware the read noise will also double - however, there's still a net gain
> in
> SNR.
>
> Best,
> Colin
>
> Dr Colin Coates
> Product Manager - Imaging
> Andor Technology
>

[Gerhard Holst]

Hi Jeff,

That was the reason, why I asked you about an estimation about the number of photons we are talking about. As far as my experiences go with localization events, there are quite a few photons involved at short exposure times (range 40 - 100 photons).
When you mention dynamics, are you referring to timely dynamic behavior of the sample (e.g. processes that are ongoing) or is it about dynamics in the image?

If the signal is in the range of 1 - 10 or 1 - 15 photons, then I agree with Colin, an emCCD would be the better choice.

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Jeff Spector
Gesendet: Donnerstag, 17. Juli 2014 17:13
An: [hidden email]
Betreff: Re: flash 4.0v2 for single molecule imaging

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

Thanks for the replies so far.
  We do have access to EMCCD cameras and that is what we use most of the
time for single molecule dynamics. Most of what I have looked at says that
sCMOs is better than EMCCD but the crossover point seem to be right around
the S/N you get when doing single molecule imaging. I guess we thought that
since you can do single molecule localization you could do dynamics but I
guess in the localization case you really are pumping a lot more photons
out of the fluorophore (which I guess means you image it for a shorter
time?).
thanks...
-Jeff


On Thu, Jul 17, 2014 at 4:59 AM, Colin Coates <[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**
> Hi Jeff,
> Bottom line, if you happen to have access to an EMCCD that's generally
> what I'd recommend for dynamic single molecule TIRF. After a few years of
> our single molecule imaging users comparing both our EMCCD and sCMOS
> cameras, the feedback has been pretty convincing. Its generalising, but the
> light levels involved in single molecule tend to be below the practical
> cross
> over point in the SNR vs photon flux curve, whereby an EMCCD has the
> advantage. Localisation super-resolution microscopy can be the exception,
> since the approach requires you to drive up photon levels to be successful.
>
> Having said that, if you are trying to adapt an sCMOS to dynamic single
> molecule, one option is to at least bring the effective pixel size closer
> to
> what you are used to with a larger pixel EMCCD. The best way is to use
> between 1.5x to 2x demagnification before the camera (depends on the
> objective as to what you can get away with and still oversample).
> Alternatively, 2x2 binning of the 6.5 um pixel (yielding a 13um binned
> pixel) can also be effective. The photons per pixel will quadruple, but be
> aware the read noise will also double - however, there's still a net gain
> in
> SNR.
>
> Best,
> Colin
>
> Dr Colin Coates
> Product Manager - Imaging
> Andor Technology
>