laser lines for CFP/YFP FRET

[Knecht, David]

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The multiline argon laser on our Nikon A1R confocal died the other day and we have been considering what to do.  It seems that gas lasers are disappearing and replaced by solid state.  The only reason we have the multiline argon is so that we can do CFP/YFP FRET with the 457 and 514 (or 488) lines of the gas laser.  However, looking at the excitation/emission profiles it appears taht we could do as well or better using the 405 for CFP and 488 for YFP.  We should get slightly less CFP signal with 405 but much less YFP non-FRET signal coming from 457 excitation.  So I would anticipate the ratios to be at least as good or better. Has anyone directly compared these two scenarios?  If 405 works fine for CFP, then we only need to get a solid state 488 which would simplify the repair.  Thanks- Dave

David Knecht, Ph.D.
Professor and Head of Core Microscopy Facility
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Kurt Thorn]

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We have never been able to get good CFP imaging with 405 nm excitation.  
I think you need a 440 nm diode if you really want to image CFP.  
Alternatively, just switch to Clover / mRuby2 for your FRET experiments;
that's what I've been encouraging users here to do.

Kurt

On 12/6/2013 9:44 AM, Knecht, David wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The multiline argon laser on our Nikon A1R confocal died the other day and we have been considering what to do.  It seems that gas lasers are disappearing and replaced by solid state.  The only reason we have the multiline argon is so that we can do CFP/YFP FRET with the 457 and 514 (or 488) lines of the gas laser.  However, looking at the excitation/emission profiles it appears taht we could do as well or better using the 405 for CFP and 488 for YFP.  We should get slightly less CFP signal with 405 but much less YFP non-FRET signal coming from 457 excitation.  So I would anticipate the ratios to be at least as good or better. Has anyone directly compared these two scenarios?  If 405 works fine for CFP, then we only need to get a solid state 488 which would simplify the repair.  Thanks- Dave
>
> David Knecht, Ph.D.
> Professor and Head of Core Microscopy Facility
> Department of Molecular and Cell Biology
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>
>

[Feinstein, Timothy]

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Hi David,

My postdoctoral group has extensive experience with FRET on an A1s.  You
may find these tips helpful.

1. Do you absolutely need to do FRET using the confocal?  The
non-linearity of PMT detection makes it significantly more difficult to do
the corrections that are essential to properly-controlled FRET
experiments.  We have found spectral confocal useful for multiplexing
different FRET biosensors (whether anyone has published a convincing
non-spectral multiplexed FRET assay is a topic for another dayŠ) but
otherwise we tried to stick exclusively to FRET detection by widefield,
mostly using a DualView2.  I cannot recommend that highly enough.

2. FRET by confocal and TIRF has one major advantage in that 442 nm lasers
barely excite YFP at all.  We found its cross-talk excitation to be so low
that we left it out of most of our correction calculations.  If you can
use a 442 nm laser with TIRF (as we often did) or with spinning disc then
you get the best both worlds, or at least a good chunk of it.

3. On the plus side for scanning lasers, the ability to bleach YFP with a
514 nm ROI is very nice for acceptor photobleach experiments.

4. Other laser lines kind of suck.  You will have a grand old time with
the 405 nm looking for a power level high enough to excite CFP but low
enough not to bleach.  In my opinion 457 excites YFP too well but given no
other choice it will do the job.  Just take care with your cross-talk
corrections.  

5. Installing a 442 nm laser like we did carries some non-trivial caveats.
 One, they are expensive and fragile.  Keep up your service contract.  We
did not replace ours in five years but a 442 did fritz out during our
scope demo.  Two, for some reason you can have either a 442 nm or a 405 nm
on your laser launch but not both.  I never understood exactly why,
something to do with optics that the laser launch uses to select lasers,
but the 405 nm has more uses than the 442 nm line if you are not doing a
lot of FRET.  So choose carefully.

Hope this helps!

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 12/6/13, 12:44 PM, "Knecht, David" <[hidden email]> wrote:

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>oN92tyw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>The multiline argon laser on our Nikon A1R confocal died the other day
>and we have been considering what to do.  It seems that gas lasers are
>disappearing and replaced by solid state.  The only reason we have the
>multiline argon is so that we can do CFP/YFP FRET with the 457 and 514
>(or 488) lines of the gas laser.  However, looking at the
>excitation/emission profiles it appears taht we could do as well or
>better using the 405 for CFP and 488 for YFP.  We should get slightly
>less CFP signal with 405 but much less YFP non-FRET signal coming from
>457 excitation.  So I would anticipate the ratios to be at least as good
>or better. Has anyone directly compared these two scenarios?  If 405
>works fine for CFP, then we only need to get a solid state 488 which
>would simplify the repair.  Thanks- Dave
>
>David Knecht, Ph.D.
>Professor and Head of Core Microscopy Facility
>Department of Molecular and Cell Biology
>U-3125
>91 N. Eagleville Rd.
>University of Connecticut
>Storrs, CT 06269
>860-486-2200
>860-486-4331 (fax)

[Mark Cannell-2]

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Hang on a moment. PMT’s are extremely linear when operating in conventional current mode.
I’ve heard this comment before but I assume it comes from folks who use photon counting where the max count rate is limited. The latter is not the way *most* confocals work.

Cheers Mark

On 6/12/2013, at 7:05 pm, Feinstein, Timothy <[hidden email]> wrote:

>
>
> 1. Do you absolutely need to do FRET using the confocal?  The
> non-linearity of PMT detection makes it significantly more difficult to do
> the corrections that are essential to properly-controlled FRET
> experiments.  We have found spectral confocal useful for multiplexing
> different FRET biosensors (whether anyone has published a convincing
> non-spectral multiplexed FRET assay is a topic for another dayŠ) but
> otherwise we tried to stick exclusively to FRET detection by widefield,
> mostly using a DualView2.  I cannot recommend that highly enough.

[Feinstein, Timothy]

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Hi Mark,

That is an interesting point.  I see that this has come up before.

http://lists.umn.edu/cgi-bin/wa?A2=ind0810&L=CONFOCALMICROSCOPY&T=0&F=&S=&P
=7843

Just last month I checked for myself using a Mol Probes FocalCheck slide.
Our A1-RS did show more of a threshold relative to our ORCA2.8 sCMOS
camera in detecting beads of 10% max intensity and lower.  However, I
would gladly admit to 'winging it' as far as protocol and it may be that
the sCMOS gain was relatively low and the PMT software hv set relatively
high, as the lower intensity beads did tend to disappear into background
noise.  

As concerns this thread though, that thing about noise is a significant
point.  When possible I prefer to measure FRET live in order to use
sensitized emission to measure response kinetics after perturbing steady
state.  That almost always means working with short acquisition times, low
laser power, moderate to high detector gain and a decent amount of
background noise.  

The higher QE of a binned CCD was a factor in my suggestion, but I also
had the idea that the linear range of detection reaches farther down into
the lower intensities for a CCD relative to a PMT.  However I am hardly
married to the idea.  My job would get easier should it be wrong.

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 12/7/13, 5:48 AM, "Mark Cannell" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://scanmail.trustwave.com/?c=129&d=-_-i0tjggD94ZSbcKKoMoMq0va_oQOcRIMC
>q_QhjtA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>Hang on a moment. PMT's are extremely linear when operating in
>conventional current mode.
>I've heard this comment before but I assume it comes from folks who use
>photon counting where the max count rate is limited. The latter is not
>the way *most* confocals work.
>
>Cheers Mark
>
>On 6/12/2013, at 7:05 pm, Feinstein, Timothy <[hidden email]>
>wrote:
>>
>>
>> 1. Do you absolutely need to do FRET using the confocal?  The
>> non-linearity of PMT detection makes it significantly more difficult to
>>do
>> the corrections that are essential to properly-controlled FRET
>> experiments.  We have found spectral confocal useful for multiplexing
>> different FRET biosensors (whether anyone has published a convincing
>> non-spectral multiplexed FRET assay is a topic for another dayŠ) but
>> otherwise we tried to stick exclusively to FRET detection by widefield,
>> mostly using a DualView2.  I cannot recommend that highly enough.

[Knecht, David]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks for all the useful information.  The discussion has made me think of a related question.  We have both widefield scopes and confocal scopes set up for CFP/YFP FRET.  The widefield systems use the ~440 wavelength cited as optimal for C/Y FRET, whereas the confocal system is using the 457 argon line.  We could move toward a 440 laser, but is there much to be gained with confocal FRET vs. widefield FRET?  We are mostly doing intramolecular FRET with live cells over time and have not directly compared the two systems.  Thanks- Dave

On Dec 8, 2013, at 9:07 PM, Feinstein, Timothy <[hidden email]<mailto:[hidden email]>> wrote:

*****
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*****

Hi Mark,

That is an interesting point.  I see that this has come up before.

http://lists.umn.edu/cgi-bin/wa?A2=ind0810&L=CONFOCALMICROSCOPY&T=0&F=&S=&P
=7843

Just last month I checked for myself using a Mol Probes FocalCheck slide.
Our A1-RS did show more of a threshold relative to our ORCA2.8 sCMOS
camera in detecting beads of 10% max intensity and lower.  However, I
would gladly admit to 'winging it' as far as protocol and it may be that
the sCMOS gain was relatively low and the PMT software hv set relatively
high, as the lower intensity beads did tend to disappear into background
noise.

As concerns this thread though, that thing about noise is a significant
point.  When possible I prefer to measure FRET live in order to use
sensitized emission to measure response kinetics after perturbing steady
state.  That almost always means working with short acquisition times, low
laser power, moderate to high detector gain and a decent amount of
background noise.

The higher QE of a binned CCD was a factor in my suggestion, but I also
had the idea that the linear range of detection reaches farther down into
the lower intensities for a CCD relative to a PMT.  However I am hardly
married to the idea.  My job would get easier should it be wrong.

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 12/7/13, 5:48 AM, "Mark Cannell" <[hidden email]> wrote:

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q_QhjtA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
roscopy
*****

Hang on a moment. PMT's are extremely linear when operating in
conventional current mode.
I've heard this comment before but I assume it comes from folks who use
photon counting where the max count rate is limited. The latter is not
the way *most* confocals work.

Cheers Mark

On 6/12/2013, at 7:05 pm, Feinstein, Timothy <[hidden email]>
wrote:


1. Do you absolutely need to do FRET using the confocal?  The
non-linearity of PMT detection makes it significantly more difficult to
do
the corrections that are essential to properly-controlled FRET
experiments.  We have found spectral confocal useful for multiplexing
different FRET biosensors (whether anyone has published a convincing
non-spectral multiplexed FRET assay is a topic for another dayŠ) but
otherwise we tried to stick exclusively to FRET detection by widefield,
mostly using a DualView2.  I cannot recommend that highly enough.

David Knecht, Ph.D.
Professor and Head of Core Microscopy Facility
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Feinstein, Timothy]

*****
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*****

Hi David,

For the most part I would choose confocal to do FRET for the same reasons
I'd  choose confocal in general.  Choose it when you need spatial
resolution, especially in Z.  The following advantages are also
non-trivial.  

* As stated 442 nm excitation causes almost zero YFP cross-talk
excitation.  This can be quite valuable depending on your experiment.
This also makes TIRF-FRET an appealing option whenever your interaction of
interest involves the basal plasma membrane.  I cannot say whether 440
nm-ish LED exciters have the same property, but all conventional light
sources that I have tried cause significant x-talk excitation.  But don't
forget that 442 nm lasers prevent you from having a 405 on your laser
launch.  

* Selective ROI bleaching can be useful for acceptor photobleach
experiments.  

* Resonant scanning is great for speed and less bleaching when working
with modest expression levels, which should be done whenever possible.

* Some people use spectral detection to skip bleed-through corrections.
Combined with the benefits of a 442 nm laser spectral unmixing lets you
get CFP and FRET intensities quantified right out of the scope.  On the
other hand, keep in mind that spectral detectors always lose a little
light to the extra optics and do not incorporate newer hybrid PMT's.

* I am not sure why folks use a white light laser such as Leica sells for
regular confocal use, but some people do it.  Those form the basis for
Leica's STED and time-resolved FRET, so if you have one you might as well
add at least one of those modules.  TR-FRET is pretty awesome.

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 12/9/13, 11:34 AM, "Knecht, David" <[hidden email]> wrote:

>*****
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>http://scanmail.trustwave.com/?c=129&d=l_Gl0ooSyGnT7_PY1TBmg9tqK5qoVZMsKI4
>Okfkv4A&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>Thanks for all the useful information.  The discussion has made me think
>of a related question.  We have both widefield scopes and confocal scopes
>set up for CFP/YFP FRET.  The widefield systems use the ~440 wavelength
>cited as optimal for C/Y FRET, whereas the confocal system is using the
>457 argon line.  We could move toward a 440 laser, but is there much to
>be gained with confocal FRET vs. widefield FRET?  We are mostly doing
>intramolecular FRET with live cells over time and have not directly
>compared the two systems.  Thanks- Dave
>
>On Dec 8, 2013, at 9:07 PM, Feinstein, Timothy
><[hidden email]<mailto:[hidden email]>> wrote:
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://scanmail.trustwave.com/?c=129&d=l_Gl0ooSyGnT7_PY1TBmg9tqK5qoVZMsKI4
>Okfkv4A&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>Hi Mark,
>
>That is an interesting point.  I see that this has come up before.
>
>http://scanmail.trustwave.com/?c=129&d=l_Gl0ooSyGnT7_PY1TBmg9tqK5qoVZMsKNs
>LwaIitA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA2%3dind0810%26L
>%3dCONFOCALMICROSCOPY%26T%3d0%26F%3d%26S%3d%26P
>=7843
>
>Just last month I checked for myself using a Mol Probes FocalCheck slide.
>Our A1-RS did show more of a threshold relative to our ORCA2.8 sCMOS
>camera in detecting beads of 10% max intensity and lower.  However, I
>would gladly admit to 'winging it' as far as protocol and it may be that
>the sCMOS gain was relatively low and the PMT software hv set relatively
>high, as the lower intensity beads did tend to disappear into background
>noise.
>
>As concerns this thread though, that thing about noise is a significant
>point.  When possible I prefer to measure FRET live in order to use
>sensitized emission to measure response kinetics after perturbing steady
>state.  That almost always means working with short acquisition times, low
>laser power, moderate to high detector gain and a decent amount of
>background noise.
>
>The higher QE of a binned CCD was a factor in my suggestion, but I also
>had the idea that the linear range of detection reaches farther down into
>the lower intensities for a CCD relative to a PMT.  However I am hardly
>married to the idea.  My job would get easier should it be wrong.
>
>Cheers,
>
>
>TF
>
>Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
>333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
>Phone: [phone] | Email: [hidden email]
>
>
>
>
>
>
>
>On 12/7/13, 5:48 AM, "Mark Cannell" <[hidden email]> wrote:
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://scanmail.trustwave.com/?c=129&d=-_-i0tjggD94ZSbcKKoMoMq0va_oQOcRIMC
>q_QhjtA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>Hang on a moment. PMT's are extremely linear when operating in
>conventional current mode.
>I've heard this comment before but I assume it comes from folks who use
>photon counting where the max count rate is limited. The latter is not
>the way *most* confocals work.
>
>Cheers Mark
>
>On 6/12/2013, at 7:05 pm, Feinstein, Timothy <[hidden email]>
>wrote:
>
>
>1. Do you absolutely need to do FRET using the confocal?  The
>non-linearity of PMT detection makes it significantly more difficult to
>do
>the corrections that are essential to properly-controlled FRET
>experiments.  We have found spectral confocal useful for multiplexing
>different FRET biosensors (whether anyone has published a convincing
>non-spectral multiplexed FRET assay is a topic for another dayŠ) but
>otherwise we tried to stick exclusively to FRET detection by widefield,
>mostly using a DualView2.  I cannot recommend that highly enough.
>
>David Knecht, Ph.D.
>Professor and Head of Core Microscopy Facility
>Department of Molecular and Cell Biology
>U-3125
>91 N. Eagleville Rd.
>University of Connecticut
>Storrs, CT 06269
>860-486-2200
>860-486-4331 (fax)

[Adam Hoppe]

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*****

Dear David,

FRET in a confocal can be somewhat complicated owing to the large parameter space that must be controlled (gain setting, pinhole sizes etc).  
However, for highly localized FRET signals embedded in thicker specimens confocal or spectral/spatial deconvolution methods (complicated) may be the only way.

The use/misuse of spectral unmixing for FRET is also a problem.   Often 'changes in signal' can be recovered from biosensors, but quantitative analysis (particularly for intermolecular FRET) requires a bit more care.

Two papers that speak to these issues are:

http://www.cell.com/biophysj/abstract/S0006-3495(08)70313-8

and

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0064760

The 440 nm laser or diode is a must for quantitative intermolecular FRET imaging.   405 nm or even 457 nm are tough going to much autofluoresence w/405 nm and 457 nm barely hits CFP.

Best Regards,
Adam Hoppe



On Dec 9, 2013, at 10:34 AM, "Knecht, David" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks for all the useful information.  The discussion has made me think of a related question.  We have both widefield scopes and confocal scopes set up for CFP/YFP FRET.  The widefield systems use the ~440 wavelength cited as optimal for C/Y FRET, whereas the confocal system is using the 457 argon line.  We could move toward a 440 laser, but is there much to be gained with confocal FRET vs. widefield FRET?  We are mostly doing intramolecular FRET with live cells over time and have not directly compared the two systems.  Thanks- Dave
>
> On Dec 8, 2013, at 9:07 PM, Feinstein, Timothy <[hidden email]<mailto:[hidden email]>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mark,
>
> That is an interesting point.  I see that this has come up before.
>
> http://lists.umn.edu/cgi-bin/wa?A2=ind0810&L=CONFOCALMICROSCOPY&T=0&F=&S=&P
> =7843
>
> Just last month I checked for myself using a Mol Probes FocalCheck slide.
> Our A1-RS did show more of a threshold relative to our ORCA2.8 sCMOS
> camera in detecting beads of 10% max intensity and lower.  However, I
> would gladly admit to 'winging it' as far as protocol and it may be that
> the sCMOS gain was relatively low and the PMT software hv set relatively
> high, as the lower intensity beads did tend to disappear into background
> noise.
>
> As concerns this thread though, that thing about noise is a significant
> point.  When possible I prefer to measure FRET live in order to use
> sensitized emission to measure response kinetics after perturbing steady
> state.  That almost always means working with short acquisition times, low
> laser power, moderate to high detector gain and a decent amount of
> background noise.
>
> The higher QE of a binned CCD was a factor in my suggestion, but I also
> had the idea that the linear range of detection reaches farther down into
> the lower intensities for a CCD relative to a PMT.  However I am hardly
> married to the idea.  My job would get easier should it be wrong.
>
> Cheers,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
> Phone: [phone] | Email: [hidden email]
>
>
>
>
>
>
>
> On 12/7/13, 5:48 AM, "Mark Cannell" <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://scanmail.trustwave.com/?c=129&d=-_-i0tjggD94ZSbcKKoMoMq0va_oQOcRIMC
> q_QhjtA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
> roscopy
> *****
>
> Hang on a moment. PMT's are extremely linear when operating in
> conventional current mode.
> I've heard this comment before but I assume it comes from folks who use
> photon counting where the max count rate is limited. The latter is not
> the way *most* confocals work.
>
> Cheers Mark
>
> On 6/12/2013, at 7:05 pm, Feinstein, Timothy <[hidden email]>
> wrote:
>
>
> 1. Do you absolutely need to do FRET using the confocal?  The
> non-linearity of PMT detection makes it significantly more difficult to
> do
> the corrections that are essential to properly-controlled FRET
> experiments.  We have found spectral confocal useful for multiplexing
> different FRET biosensors (whether anyone has published a convincing
> non-spectral multiplexed FRET assay is a topic for another dayŠ) but
> otherwise we tried to stick exclusively to FRET detection by widefield,
> mostly using a DualView2.  I cannot recommend that highly enough.
>
> David Knecht, Ph.D.
> Professor and Head of Core Microscopy Facility
> Department of Molecular and Cell Biology
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)