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FLIM using Cy3 as a donor ?

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Jean-Pierre CLAMME-2

FLIM using Cy3 as a donor ?

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Hi,

I was wondering if anyone had done or tried FLIM experiments using Cy3 (or
Dy547) as a donor. I have seen a few FLIM publications were Cy3 is the
acceptor for GFP but none were Cy3 is the donor.
I don't have a FLIM system handy so I can't just go a try it. So I'm
looking for this information to determine if what I want to do is feasible
before I go and find a Lab were I could do the experiment.

Thank you in advance

JP

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Chief Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: +760.435.7065

yuansheng sun

Re: FLIM using Cy3 as a donor ?

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

Dear Jean-Pierre,

I guess you are trying to do FLIM-FRET with Cy3 as the FRET donor. I have
not personally used Cy3 as the FRET donor. But I think Cy3 can be a FRET
donor, depending upon what is the acceptor. There is a paper (given below)
from Taekjip Ha group, where they used Cy3 as the FRET donor.

Single-Molecule Three-Color FRET. Biophys J. 2004 August; 87(2): 1328–1337

There are other considerations - Would you use 1p or 2p for FLIM? If you
will use 2p, you need to make sure that you can excite Cy3 efficiently with
your 2p laser. In FLIM-FRET analysis, you want your donor-alone (Cy3)
decay to be simple - ideally mono-exponential. It is worth searching if
anybody has measured the lifetime of Cy3, since you do not have a system on
site. However, it is always important to know what is the lifetime of Cy3
in your sample.

I hope it helps. And good luck.

Sheng
Yuansheng Sun, Ph.D.
Keck Center for Cellular Imaging
University of Virginia

We held a workshop on FRET Microscopy
http://www.kcci.virginia.edu/workshop/workshop2013/index.php

On Mon, Mar 19, 2012 at 8:47 PM, Jean-Pierre CLAMME <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I was wondering if anyone had done or tried FLIM experiments using Cy3 (or
> Dy547) as a donor. I have seen a few FLIM publications were Cy3 is the
> acceptor for GFP but none were Cy3 is the donor.
> I don't have a FLIM system handy so I can't just go a try it. So I'm
> looking for this information to determine if what I want to do is feasible
> before I go and find a Lab were I could do the experiment.
>
> Thank you in advance
>
> JP
>
> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> Jean-Pierre CLAMME, PhD
> Chief Scientist
> Nitto Denko Technical
> 501 Via Del Monte
> Oceanside, CA 92058
> E-mail: [hidden email]
> Phone: +760.435.7065
>

Jean-Pierre CLAMME-2

Re: FLIM using Cy3 as a donor ?

Dear Sheng,

I also have used Cy3 as a donor in experiment before. However I read it
has a short lifetime compared to GFP for example. And so I was wondering
if anyone was successful using it as a donor in a FLIM experiment.

Thank you

JP

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Chief Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: +760.435.7065

Confocal Microscopy List <[hidden email]> wrote on
03/20/2012 09:59:04 AM:

> yuansheng sun <[hidden email]>
> Sent by: Confocal Microscopy List <[hidden email]>
>
> 03/20/2012 09:59 AM
>
> Please respond to
> Confocal Microscopy List <[hidden email]>
>
> To
>
> [hidden email]
>
> cc
>
> Subject
>
> Re: FLIM using Cy3 as a donor ?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****

> Dear Jean-Pierre,

> I guess you are trying to do FLIM-FRET with Cy3 as the FRET donor. I
have
> not personally used Cy3 as the FRET donor. But I think Cy3 can be a
FRET
> donor, depending upon what is the acceptor. There is a paper (given
below)
> from Taekjip Ha group, where they used Cy3 as the FRET donor.

> Single-Molecule Three-Color FRET. Biophys J. 2004 August; 87(2):
1328–1337

> There are other considerations - Would you use 1p or 2p for FLIM? If
you
> will use 2p, you need to make sure that you can excite Cy3 efficiently
with
> your 2p laser. In FLIM-FRET analysis, you want your donor-alone (Cy3)
> decay to be simple - ideally mono-exponential. It is worth searching if
> anybody has measured the lifetime of Cy3, since you do not have a system
on
> site. However, it is always important to know what is the lifetime of
Cy3
> in your sample.

> I hope it helps. And good luck.

> Sheng
> Yuansheng Sun, Ph.D.
> Keck Center for Cellular Imaging
> University of Virginia

> We held a workshop on FRET Microscopy
> http://www.kcci.virginia.edu/workshop/workshop2013/index.php

>
> On Mon, Mar 19, 2012 at 8:47 PM, Jean-Pierre CLAMME <
> [hidden email]> wrote:

> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > I was wondering if anyone had done or tried FLIM experiments using Cy3
(or
> > Dy547) as a donor. I have seen a few FLIM publications were Cy3 is
the
> > acceptor for GFP but none were Cy3 is the donor.
> > I don't have a FLIM system handy so I can't just go a try it. So I'm
> > looking for this information to determine if what I want to do is
feasible

> > before I go and find a Lab were I could do the experiment.
> >
> > Thank you in advance
> >
> > JP
> >
> > - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> > Jean-Pierre CLAMME, PhD
> > Chief Scientist
> > Nitto Denko Technical
> > 501 Via Del Monte
> > Oceanside, CA 92058
> > E-mail: [hidden email]
> > Phone: +760.435.7065
> >

Simon Ameer-Beg

Re: FLIM using Cy3 as a donor ?

In reply to this post by Jean-Pierre CLAMME-2

Hi Jean-Pierre,

Cy3 has been used in FRET FLIM as a donor. You will find some data in Ng et al, Science 1999.

http://m.sciencemag.org/content/283/5410/2085.short

From memory, Cy3 was used as a donor to Cy5. Yes, the lifetime is short (on conjugation may also be biphasic) and I personally would not recommend this pairing for FLIM using TCSPC. Tony's paper (above) was frequency domain FLIM of course. We have had reasonable success with Alexa 546 as donor in antibody based studies. Lifetime around 2.4-2.6 ns under our standard fixation and mounting procedures.

Best wishes
Simon

Sent from my HTC

----- Reply message -----
From: "Jean-Pierre CLAMME" <[hidden email]>
To: "[hidden email]" <[hidden email]>
Subject: [CONFOCALMICROSCOPY] FLIM using Cy3 as a donor ?
Date: Tue, Mar 20, 2012 5:16 pm

Dear Sheng,

I also have used Cy3 as a donor in experiment before. However I read it
has a short lifetime compared to GFP for example. And so I was wondering
if anyone was successful using it as a donor in a FLIM experiment.

Thank you

JP

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Chief Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: +760.435.7065

Confocal Microscopy List <[hidden email]> wrote on
03/20/2012 09:59:04 AM:

Unruh, Jay

Re: FLIM using Cy3 as a donor ?

You can easily convert TCSPC FLIM into a frequency domain experiment using Enrico Gratton's techniques. Cy3 has an excited state photoisomerisation that gives it a viscosity and interaction dependent lifetime (high viscosity=longer lifetime). The lifetime in solution can be as short as 200 picoseconds--difficult to measure with even the best FLIM systems. Cy3 labeled to proteins typically has a longer lifetime, but this is difficult to predict. This can be even more difficult when the interaction you measure with FRET actually changes the environment of the donor. Single molecule folks (like Ha group) like Cy3 because it is very lives a long time before photobleaching but they are typically using ratiometric FRET for the measurement, not FLIM.

Jay Unruh
Stowers Insitute for Medical Research

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ameer-Beg, Simon
Sent: Tuesday, March 20, 2012 1:49 PM
To: [hidden email]
Subject: Re: FLIM using Cy3 as a donor ?

Hi Jean-Pierre,

Cy3 has been used in FRET FLIM as a donor. You will find some data in Ng et al, Science 1999.

http://m.sciencemag.org/content/283/5410/2085.short

From memory, Cy3 was used as a donor to Cy5. Yes, the lifetime is short (on conjugation may also be biphasic) and I personally would not recommend this pairing for FLIM using TCSPC. Tony's paper (above) was frequency domain FLIM of course. We have had reasonable success with Alexa 546 as donor in antibody based studies. Lifetime around 2.4-2.6 ns under our standard fixation and mounting procedures.

Best wishes
Simon

Sent from my HTC

----- Reply message -----
From: "Jean-Pierre CLAMME" <[hidden email]>
To: "[hidden email]" <[hidden email]>
Subject: [CONFOCALMICROSCOPY] FLIM using Cy3 as a donor ?
Date: Tue, Mar 20, 2012 5:16 pm

Dear Sheng,

I also have used Cy3 as a donor in experiment before. However I read it has a short lifetime compared to GFP for example. And so I was wondering if anyone was successful using it as a donor in a FLIM experiment.

Thank you

JP

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jean-Pierre CLAMME, PhD Chief Scientist Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: +760.435.7065

Confocal Microscopy List <[hidden email]> wrote on
03/20/2012 09:59:04 AM:

> yuansheng sun <[hidden email]> Sent by: Confocal Microscopy
> List <[hidden email]>
>
> 03/20/2012 09:59 AM
>
> Please respond to
> Confocal Microscopy List <[hidden email]>
>
> To
>
> [hidden email]
>
> cc
>
> Subject
>
> Re: FLIM using Cy3 as a donor ?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****

> Dear Jean-Pierre,

> I guess you are trying to do FLIM-FRET with Cy3 as the FRET donor. I
have
> not personally used Cy3 as the FRET donor. But I think Cy3 can be a
FRET
> donor, depending upon what is the acceptor. There is a paper (given
below)
> from Taekjip Ha group, where they used Cy3 as the FRET donor.

> Single-Molecule Three-Color FRET. Biophys J. 2004 August; 87(2):
1328–1337

> There are other considerations - Would you use 1p or 2p for FLIM? If
you
> will use 2p, you need to make sure that you can excite Cy3 efficiently
with
> your 2p laser. In FLIM-FRET analysis, you want your donor-alone (Cy3)
> decay to be simple - ideally mono-exponential. It is worth searching
> if anybody has measured the lifetime of Cy3, since you do not have a
> system
on
> site. However, it is always important to know what is the lifetime of
Cy3
> in your sample.

> I hope it helps. And good luck.

> Sheng
> Yuansheng Sun, Ph.D.
> Keck Center for Cellular Imaging
> University of Virginia

> We held a workshop on FRET Microscopy
> http://www.kcci.virginia.edu/workshop/workshop2013/index.php

>
> On Mon, Mar 19, 2012 at 8:47 PM, Jean-Pierre CLAMME <
> [hidden email]> wrote:

> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > I was wondering if anyone had done or tried FLIM experiments using
> > Cy3
(or
> > Dy547) as a donor. I have seen a few FLIM publications were Cy3 is
the
> > acceptor for GFP but none were Cy3 is the donor.
> > I don't have a FLIM system handy so I can't just go a try it. So I'm
> > looking for this information to determine if what I want to do is
feasible

> > before I go and find a Lab were I could do the experiment.
> >
> > Thank you in advance
> >
> > JP
> >
> > - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> > Jean-Pierre CLAMME, PhD Chief Scientist Nitto Denko Technical
> > 501 Via Del Monte
> > Oceanside, CA 92058
> > E-mail: [hidden email]
> > Phone: +760.435.7065
> >

Jean-Pierre CLAMME-2

Re: FLIM using Cy3 as a donor ?

Thank you for the information,

In my experiment Cy3 (actually Dy547) is conjugated to an oligonucleotide
so it's life time might go up.

Confocal Microscopy List <[hidden email]> wrote on
03/20/2012 02:00:53 PM:

> "Unruh, Jay" <[hidden email]>
> Sent by: Confocal Microscopy List <[hidden email]>
>
> 03/20/2012 02:06 PM
>
> Please respond to
> Confocal Microscopy List <[hidden email]>
>
> To
>
> [hidden email]
>
> cc
>
> Subject
>
> Re: FLIM using Cy3 as a donor ?
>
> You can easily convert TCSPC FLIM into a frequency domain experiment
> using Enrico Gratton's techniques. Cy3 has an excited state
> photoisomerisation that gives it a viscosity and interaction
> dependent lifetime (high viscosity=longer lifetime). The lifetime
> in solution can be as short as 200 picoseconds--difficult to measure
> with even the best FLIM systems. Cy3 labeled to proteins typically
> has a longer lifetime, but this is difficult to predict. This can
> be even more difficult when the interaction you measure with FRET
> actually changes the environment of the donor. Single molecule
> folks (like Ha group) like Cy3 because it is very lives a long time
> before photobleaching but they are typically using ratiometric FRET
> for the measurement, not FLIM.

> Jay Unruh
> Stowers Insitute for Medical Research

> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Ameer-Beg, Simon
> Sent: Tuesday, March 20, 2012 1:49 PM
> To: [hidden email]
> Subject: Re: FLIM using Cy3 as a donor ?

> Hi Jean-Pierre,

> Cy3 has been used in FRET FLIM as a donor. You will find some data
> in Ng et al, Science 1999.

> http://m.sciencemag.org/content/283/5410/2085.short

> From memory, Cy3 was used as a donor to Cy5. Yes, the lifetime is
> short (on conjugation may also be biphasic) and I personally would
> not recommend this pairing for FLIM using TCSPC. Tony's paper
> (above) was frequency domain FLIM of course. We have had reasonable
> success with Alexa 546 as donor in antibody based studies. Lifetime
> around 2.4-2.6 ns under our standard fixation and mounting procedures.

> Best wishes
> Simon

>
> Sent from my HTC

> ----- Reply message -----
> From: "Jean-Pierre CLAMME" <[hidden email]>
> To: "[hidden email]"
<[hidden email]>
> Subject: [CONFOCALMICROSCOPY] FLIM using Cy3 as a donor ?
> Date: Tue, Mar 20, 2012 5:16 pm

>
> Dear Sheng,

> I also have used Cy3 as a donor in experiment before. However I read
> it has a short lifetime compared to GFP for example. And so I was
> wondering if anyone was successful using it as a donor in a FLIM
experiment.

> Thank you

> JP

>
> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> Jean-Pierre CLAMME, PhD Chief Scientist Nitto Denko Technical
> 501 Via Del Monte
> Oceanside, CA 92058
> E-mail: [hidden email]
> Phone: +760.435.7065

>
> Confocal Microscopy List <[hidden email]> wrote on
> 03/20/2012 09:59:04 AM:

> > yuansheng sun <[hidden email]> Sent by: Confocal Microscopy
> > List <[hidden email]>
> >
> > 03/20/2012 09:59 AM
> >
> > Please respond to
> > Confocal Microscopy List <[hidden email]>
> >
> > To
> >
> > [hidden email]
> >
> > cc
> >
> > Subject
> >
> > Re: FLIM using Cy3 as a donor ?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****

> > Dear Jean-Pierre,

> > I guess you are trying to do FLIM-FRET with Cy3 as the FRET donor. I
> have
> > not personally used Cy3 as the FRET donor. But I think Cy3 can be a
> FRET
> > donor, depending upon what is the acceptor. There is a paper (given
> below)
> > from Taekjip Ha group, where they used Cy3 as the FRET donor.

> > Single-Molecule Three-Color FRET. Biophys J. 2004 August; 87(2):
> 1328–1337

> > There are other considerations - Would you use 1p or 2p for FLIM? If
> you
> > will use 2p, you need to make sure that you can excite Cy3 efficiently
> with
> > your 2p laser. In FLIM-FRET analysis, you want your donor-alone (Cy3)
> > decay to be simple - ideally mono-exponential. It is worth searching
> > if anybody has measured the lifetime of Cy3, since you do not have a
> > system
> on
> > site. However, it is always important to know what is the lifetime of
> Cy3
> > in your sample.

> > I hope it helps. And good luck.

> > Sheng
> > Yuansheng Sun, Ph.D.
> > Keck Center for Cellular Imaging
> > University of Virginia

> > We held a workshop on FRET Microscopy
> > http://www.kcci.virginia.edu/workshop/workshop2013/index.php

> >
> > On Mon, Mar 19, 2012 at 8:47 PM, Jean-Pierre CLAMME <
> > [hidden email]> wrote:

> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Hi,
> > >
> > > I was wondering if anyone had done or tried FLIM experiments using
> > > Cy3
> (or
> > > Dy547) as a donor. I have seen a few FLIM publications were Cy3 is
> the
> > > acceptor for GFP but none were Cy3 is the donor.
> > > I don't have a FLIM system handy so I can't just go a try it. So I'm
> > > looking for this information to determine if what I want to do is
> feasible
> > > before I go and find a Lab were I could do the experiment.
> > >
> > > Thank you in advance
> > >
> > > JP
> > >
> > > - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> > > Jean-Pierre CLAMME, PhD Chief Scientist Nitto Denko Technical
> > > 501 Via Del Monte
> > > Oceanside, CA 92058
> > > E-mail: [hidden email]
> > > Phone: +760.435.7065
> > >