Confocal Microscopy List

DsRed vs 2P

Classic

List

Threaded

13 messages Options

Staffan Nyström

DsRed vs 2P

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

Dear list members,

Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?
I have problems getting any decent fluorescence signal through between 700 and 1000nm excitation and there is significant bleedthrough
with EGFP. In the end I gave up since the "regular" 1 photon excitation way gave a much better S/N ratio with minimal bleed through.

Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA water dipping objectives.

Best

Staffan Nystrom

PhD student

MBB / Oncology & Pathology

Karolinska Institutet
Stockholm, Sweden

Craig Brideau

Re: DsRed vs 2P

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

What wavelength did you have your Ti:Saph tuned to? The 2p wavelength which
a dye prefers is often different from what you would expect. Try tuning the
laser around its full range and see if you get better signal anywhere else.
Do you have any filters in your microscope that could be blocking the laser
or causing other problems?

Craig

2010/12/13 Staffan Nyström <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list members,
>
> Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?
> I have problems getting any decent fluorescence signal through between 700
> and 1000nm excitation and there is significant bleedthrough
> with EGFP. In the end I gave up since the "regular" 1 photon excitation way
> gave a much better S/N ratio with minimal bleed through.
>
> Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
> water dipping objectives.
>
> Best
>
> Staffan Nystrom
>
> PhD student
>
> MBB / Oncology & Pathology
>
> Karolinska Institutet
> Stockholm, Sweden
>

Haberman, Ann

Re: DsRed vs 2P

In reply to this post by Staffan Nyström

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

Hi Staffan,

Which variant of DsRed are you using? Some of them are easy to excite between 850 - 950 nm. The immature state of DsRed has a pronounced green emission, but a variant created by Ben Glick is rapidly maturing and has very little green emission.

It can be purchased through Clontech. http://www.clontech.com/products/detail.asp?product_id=157258

Ann Haberman

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear list members,
>
>Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?
>I have problems getting any decent fluorescence signal through between 700 and 1000nm excitation and there is significant bleedthrough
>with EGFP. In the end I gave up since the "regular" 1 photon excitation way gave a much better S/N ratio with minimal bleed through.
>
>Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA water dipping objectives.
>
>Best
>
>Staffan Nystrom
>
>PhD student
>
>MBB / Oncology & Pathology
>
>Karolinska Institutet
>Stockholm, Sweden

--

Ann Haberman, PhD
Assistant Professor
Department of Laboratory Medicine
Yale University School of Medicine

1 Gilbert St.
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]

Staffan Nyström

Re: DsRed vs 2P

In reply to this post by Craig Brideau

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

@Craig

I have tried 10 nm steps from around 710 up to 1000 nm but to no great awail.
I do get some signal out, but nowhere near the output I get with the 561 laser.
Since the emission of for example EGFP (at ~910nm) or DAPI (at ~750nm) is detected without problems (using NDDs) there shouldn't be any problems with blocking from filters (- or?)..

Best Staffan

--

What wavelength did you have your Ti:Saph tuned to? The 2p wavelength which
a dye prefers is often different from what you would expect. Try tuning the
laser around its full range and see if you get better signal anywhere else.
Do you have any filters in your microscope that could be blocking the laser
or causing other problems?

Craig

2010/12/13 Staffan Nyström <[hidden email]>

Shivaprasad Bhuvanendran-2

Re: DsRed vs 2P

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

Hi Staffan,

On the Zeiss LSM system that you are using, you can try an 'Excitation
Fingerprinting' macro to generate the excitation spectra of different
fluorochromes. The macro, available from Zeiss, automates the process of
recording the emission signal at different IR wavelengths at a pre-defined
step size while keeping the laser power constant. When manually changing the
wavelengths, you may not be able to accurately adjust the AOM settings to
compensate for change in laser power. Using this technique, you should be
able to determine the optimum excitation wavelength for the combination of
fluorochromes you have.

We have successfully excited DsRed (in combination with other
fluorochromes) at 910-940nm, without bleedthrough in the green
channel(bandpass 490-540), on a Olympus FV-1000MPE microscope using a
Coherent's Chameleon Vision laser .

In your case, as Ann has suggested, the variant of the DsRed that you are
using may also be a problem.

Best,
Shiva

Shivaprasad Bhuvanendran
Research Support Specialist - Bio-Imaging
The Rockefeller University
1230 York Avenue
Box 209 (DWB 201)
New York NY 10065
tel +1 212 327 7487
fax +1 212 327 7489

2010/12/14 Staffan Nyström <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> @Craig
>
> I have tried 10 nm steps from around 710 up to 1000 nm but to no great
> awail.
> I do get some signal out, but nowhere near the output I get with the 561
> laser.
> Since the emission of for example EGFP (at ~910nm) or DAPI (at ~750nm) is
> detected without problems (using NDDs) there shouldn't be any problems with
> blocking from filters (- or?)..
>
> Best Staffan
>
> --
>
> What wavelength did you have your Ti:Saph tuned to? The 2p wavelength
> which
> a dye prefers is often different from what you would expect. Try tuning
> the
> laser around its full range and see if you get better signal anywhere else.
> Do you have any filters in your microscope that could be blocking the
> laser
> or causing other problems?
>
> Craig
>
>
> 2010/12/13 Staffan Nyström <[hidden email]>
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear list members,
> >
> > Has anyone any experience with visualizing DsRed using Ti:Saph (2
> photon)?
> > I have problems getting any decent fluorescence signal through between
> 700
> > and 1000nm excitation and there is significant bleedthrough
> > with EGFP. In the end I gave up since the "regular" 1 photon excitation
> way
> > gave a much better S/N ratio with minimal bleed through.
> >
> > Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
> > water dipping objectives.
> >
> > Best
> >
> > Staffan Nystrom
> >
> > PhD student
> >
> > MBB / Oncology & Pathology
> >
> > Karolinska Institutet
> > Stockholm, Sweden
> >
>

Craig Brideau

Re: DsRed vs 2P

In reply to this post by Staffan Nyström

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

Hmm... doesn't sound like a filter problem if the unit is designed for
non-descanned detection: It should expect a Ti:Saph laser input so the
filters should be set accordingly. As the others have said, it may be your
dye. You might want to retry it at 910 to 940 as Shiva suggested, but
otherwise maybe obtain a different sample of dye?

Craig

2010/12/14 Staffan Nyström <[hidden email]>

Paul Herzmark

Re: DsRed vs 2P

In reply to this post by Staffan Nyström

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

Using my Tai-Saphire laser, I measured the optimal 2P excitation wavelength
for TD Tomato (a different red fluorescent protein).

Since the wattage coming out of the laser varies with wavelength you need to
correct for that. I have a pickoff after my laser power attenuator that goes
to a power meter so I can keep the power delivered to the cells constant.
Scan through the wavelengths changing the attenuator to keep the power
constant and look for the brightest emission. I found 1040 nm (the end of
what my laser will deliver) to be about 5X more efficient per watt than 920
nm.

It was still going up when I got to 1040 so I am going to try an OPO
(Optical Parametric Oscillator) to look at even longer wavelengths.

Keeping the delivered power constant and scanning the spectrum is also a
good way to find the optimum compromise power for simultaneously exciting
several fluorophores.

Paul

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax

2010/12/13 Staffan Nyström <[hidden email]>

Jennifer Clarke

Re: DsRed vs 2P

In reply to this post by Craig Brideau

Dear list

(Not strictly a confocal question:)

I heard a while ago that the use of mercury lamps on fluorescence microscopes would be discontinued in the United States, and possibly other countries, in the next couple of years,due to the safer replacement illumination systems now available.

Can anyone verify this rumour?

Kind regards
Jen
--
Jennifer Clarke BSc (Hons) PhD
Research Associate, Anatomy and Histology
Centre for Neuroscience, School of Medicine
&
Facility Manager, Optical Microscopy Suite, Flinders Microscopy

Flinders University
GPO Box 2100, Adelaide 5001
Phone: 61 8 8204 6454/ 61 8 8204 6637
Email: [hidden email]

Kate Luby-Phelps

Re: DsRed vs 2P

In reply to this post by Staffan Nyström

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

The power of the laser drops off as you go out in wavelength and in any case the
one photon excitation maximum for DsRed is approx. 550 nm so even 1000 is
not optimal. At 500 nm the excitation of DsRed is less than half of maximum.
The problem will be even worse for mCherry. I gather there is some hope for
using an OPO to excite the red fluorescent proteins. The two photon cross-
section for DsRed is here:

http://www.biophysics.leidenuniv.nl/schmidt/Paper/CPL01_2pXFPs.pdf

They were able to get the best fluorescence at 980 but if you look at the
intensity relative to the other FPs it is weak.

Also, apparently there is some three-photon absorption by DsRed at 760 nm that
changes it from red to green, which might account for your "bleedthrough".
The immature form of DsRed is green.

Stick with the 561.

Andreas Bruckbauer

Re: DsRed vs 2P

In reply to this post by Paul Herzmark

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

The pick up from the laser is a good idea and i think microscope manufacturers should implement this and get rid of the percentage numbers for laser powers which are widely used. However this will not account for the differences in transmission of the different optical elements in the microscope. So the Zeiss macro is a good idea if you are interested in the fluorophore characteristics. The only thing to keep in mind is that, if your sample allows you to use more power and you are operating at 100%, you still might gain from using the lower wavelength. The fluorescence goes with the square of the laser power, so 2.5x more power translates to 6x more fluorescence, if the fluorescent protein is 3x more efficient at the longer wavelength (at same power level), you would still get 2x more fluorescence using the lower wavelength at higher power. But these are just theoretical values.

best wishes

Andreas

-----Original Message-----
From: Paul Herzmark <[hidden email]>
To: [hidden email]
Sent: Tue, 14 Dec 2010 22:20
Subject: Re: DsRed vs 2P

*****

To join, leave or search the confocal microscopy listserv, go to:

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

*****

Using my Tai-Saphire laser, I measured the optimal 2P excitation wavelength

for TD Tomato (a different red fluorescent protein).

Since the wattage coming out of the laser varies with wavelength you need to

correct for that. I have a pickoff after my laser power attenuator that goes

to a power meter so I can keep the power delivered to the cells constant.

Scan through the wavelengths changing the attenuator to keep the power

constant and look for the brightest emission. I found 1040 nm (the end of

what my laser will deliver) to be about 5X more efficient per watt than 920

nm.

It was still going up when I got to 1040 so I am going to try an OPO

(Optical Parametric Oscillator) to look at even longer wavelengths.

Keeping the delivered power constant and scanning the spectrum is also a

good way to find the optimum compromise power for simultaneously exciting

several fluorophores.

Paul

Paul Herzmark

Specialist

[hidden email]

Department of Molecular and Cell Biology

479 Life Science Addition

University of California, Berkeley

Berkeley, CA 94720-3200

(510) 643-9603

(510) 643-9500 fax

2010/12/13 Staffan Nyström <[hidden email]>

> *****

> To join, leave or search the confocal microscopy listserv, go to:

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

> *****

>

> Dear list members,

>

> Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?

> I have problems getting any decent fluorescence signal through between 700

> and 1000nm excitation and there is significant bleedthrough

> with EGFP. In the end I gave up since the "regular" 1 photon excitation way

> gave a much better S/N ratio with minimal bleed through.

>

> Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA

> water dipping objectives.

>

> Best

>

> Staffan Nystrom

>

> PhD student

>

> MBB / Oncology & Pathology

>

> Karolinska Institutet

> Stockholm, Sweden

>

=

Sudipta Maiti

Re: DsRed vs 2P

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

Remember that the pulsewidth will also be changing as you tune, so if you have
a pulse compressor, you may want to tweak that too to keep it constant. Though
the dependence for 2P is linear, pulsewidths can change quite drastically
towards the ends of the tuning range of a Ti:sapph.
Sudipta
On Wed, 15 Dec 2010 06:38:48 -0500, Andreas Bruckbauer wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The pick up from the laser is a good idea and i think microscope
> manufacturers should implement this and get rid of the percentage
> numbers for laser powers which are widely used. However this will
> not account for the differences in transmission of the different
> optical elements in the microscope. So the Zeiss macro is a good
> idea if you are interested in the fluorophore characteristics. The
> only thing to keep in mind is that, if your sample allows you to use
> more power and you are operating at 100%, you still might gain from
> using the lower wavelength. The fluorescence goes with the square of
> the laser power, so 2.5x more power translates to 6x more
> fluorescence, if the fluorescent protein is 3x more efficient at the
> longer wavelength (at same power level), you would still get 2x more
> fluorescence using the lower wavelength at higher power. But these
> are just theoretical values.
>
> best wishes
>
> Andreas
>
> -----Original Message-----
> From: Paul Herzmark <[hidden email]>
> To: [hidden email]
> Sent: Tue, 14 Dec 2010 22:20
> Subject: Re: DsRed vs 2P
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
> Using my Tai-Saphire laser, I measured the optimal 2P excitation wavelength
>
> for TD Tomato (a different red fluorescent protein).
>
> Since the wattage coming out of the laser varies with wavelength you
> need to
>
> correct for that. I have a pickoff after my laser power attenuator
> that goes
>
> to a power meter so I can keep the power delivered to the cells constant.
>
> Scan through the wavelengths changing the attenuator to keep the power
>
> constant and look for the brightest emission. I found 1040 nm (the
> end of
>
> what my laser will deliver) to be about 5X more efficient per watt
> than 920
>
> nm.
>
> It was still going up when I got to 1040 so I am going to try an OPO
>
> (Optical Parametric Oscillator) to look at even longer wavelengths.
>
> Keeping the delivered power constant and scanning the spectrum is
> also a
>
> good way to find the optimum compromise power for simultaneously exciting
>
> several fluorophores.
>
> Paul
>
> Paul Herzmark
>
> Specialist
>
> [hidden email]
>
> Department of Molecular and Cell Biology
>
> 479 Life Science Addition
>
> University of California, Berkeley
>
> Berkeley, CA 94720-3200
>
> (510) 643-9603
>
> (510) 643-9500 fax
>
> 2010/12/13 Staffan Nyström <[hidden email]>
>
> > *****
>
> > To join, leave or search the confocal microscopy listserv, go to:
>
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> > *****
>
> >
>
> > Dear list members,
>
> >
>
> > Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?
>
> > I have problems getting any decent fluorescence signal through between 700
>
> > and 1000nm excitation and there is significant bleedthrough
>
> > with EGFP. In the end I gave up since the "regular" 1 photon excitation

way

>
> > gave a much better S/N ratio with minimal bleed through.
>
> >
>
> > Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
>
> > water dipping objectives.
>
> >
>
> > Best
>
> >
>
> > Staffan Nystrom
>
> >
>
> > PhD student
>
> >
>
> > MBB / Oncology & Pathology
>
> >
>
> > Karolinska Institutet
>
> > Stockholm, Sweden
>
> >
>
> =

Dr. Sudipta Maiti
Associate Professor
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: 91-22-2280-4610
alternate e-mail: [hidden email]
url: biophotonics.wetpaint.com

Craig Brideau

Re: DsRed vs 2P

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

That's a very good point! I found from my own compressor that the longer
you tune the less sensitive the pulse is to dispersion. This may account
for some of the brightening seen as the laser was tuned more to the
infrared! Thanks for bringing that up and jogging my memory.
There are so many factors to consider when characterizing these things!

Craig

On Wed, Dec 15, 2010 at 5:30 AM, Sudipta Maiti
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Remember that the pulsewidth will also be changing as you tune, so if you
> have
> a pulse compressor, you may want to tweak that too to keep it constant.
> Though
> the dependence for 2P is linear, pulsewidths can change quite drastically
> towards the ends of the tuning range of a Ti:sapph.
> Sudipta
> On Wed, 15 Dec 2010 06:38:48 -0500, Andreas Bruckbauer wrote
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > The pick up from the laser is a good idea and i think microscope
> > manufacturers should implement this and get rid of the percentage
> > numbers for laser powers which are widely used. However this will
> > not account for the differences in transmission of the different
> > optical elements in the microscope. So the Zeiss macro is a good
> > idea if you are interested in the fluorophore characteristics. The
> > only thing to keep in mind is that, if your sample allows you to use
> > more power and you are operating at 100%, you still might gain from
> > using the lower wavelength. The fluorescence goes with the square of
> > the laser power, so 2.5x more power translates to 6x more
> > fluorescence, if the fluorescent protein is 3x more efficient at the
> > longer wavelength (at same power level), you would still get 2x more
> > fluorescence using the lower wavelength at higher power. But these
> > are just theoretical values.
> >
> > best wishes
> >
> > Andreas
> >
> > -----Original Message-----
> > From: Paul Herzmark <[hidden email]>
> > To: [hidden email]
> > Sent: Tue, 14 Dec 2010 22:20
> > Subject: Re: DsRed vs 2P
> >
> > *****
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> > *****
> >
> > Using my Tai-Saphire laser, I measured the optimal 2P excitation
> wavelength
> >
> > for TD Tomato (a different red fluorescent protein).
> >
> > Since the wattage coming out of the laser varies with wavelength you
> > need to
> >
> > correct for that. I have a pickoff after my laser power attenuator
> > that goes
> >
> > to a power meter so I can keep the power delivered to the cells constant.
> >
> > Scan through the wavelengths changing the attenuator to keep the power
> >
> > constant and look for the brightest emission. I found 1040 nm (the
> > end of
> >
> > what my laser will deliver) to be about 5X more efficient per watt
> > than 920
> >
> > nm.
> >
> > It was still going up when I got to 1040 so I am going to try an OPO
> >
> > (Optical Parametric Oscillator) to look at even longer wavelengths.
> >
> > Keeping the delivered power constant and scanning the spectrum is
> > also a
> >
> > good way to find the optimum compromise power for simultaneously exciting
> >
> > several fluorophores.
> >
> > Paul
> >
> > Paul Herzmark
> >
> > Specialist
> >
> > [hidden email]
> >
> > Department of Molecular and Cell Biology
> >
> > 479 Life Science Addition
> >
> > University of California, Berkeley
> >
> > Berkeley, CA 94720-3200
> >
> > (510) 643-9603
> >
> > (510) 643-9500 fax
> >
> > 2010/12/13 Staffan Nyström <[hidden email]>
> >
> > > *****
> >
> > > To join, leave or search the confocal microscopy listserv, go to:
> >
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> > > *****
> >
> > >
> >
> > > Dear list members,
> >
> > >
> >
> > > Has anyone any experience with visualizing DsRed using Ti:Saph (2
> photon)?
> >
> > > I have problems getting any decent fluorescence signal through between
> 700
> >
> > > and 1000nm excitation and there is significant bleedthrough
> >
> > > with EGFP. In the end I gave up since the "regular" 1 photon excitation
> way
> >
> > > gave a much better S/N ratio with minimal bleed through.
> >
> > >
> >
> > > Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
> >
> > > water dipping objectives.
> >
> > >
> >
> > > Best
> >
> > >
> >
> > > Staffan Nystrom
> >
> > >
> >
> > > PhD student
> >
> > >
> >
> > > MBB / Oncology & Pathology
> >
> > >
> >
> > > Karolinska Institutet
> >
> > > Stockholm, Sweden
> >
> > >
> >
> > =
>
>
> Dr. Sudipta Maiti
> Associate Professor
> Dept. of Chemical Sciences
> Tata Institute of Fundamental Research
> Homi Bhabha Raod, Colaba, Mumbai 400005
> Ph. 91-22-2278-2716 / 2539
> Fax: 91-22-2280-4610
> alternate e-mail: [hidden email]
> url: biophotonics.wetpaint.com
>

Gavin Rumbaugh

Re: DsRed vs 2P

In reply to this post by Staffan Nyström

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

There is a very nice paper showing that the DsRED and variants are excited by two-photon energy from 660-780nm (depending on the variant; http://pubs.acs.org/doi/abs/10.1021/jp8087379 ). IN fact, some of these variants are reported to be excited more efficiently in this range versus the >1000nm peak (http://f1000.com/1147971). The paper was highlighted in Faculty of 1000, and people have have commented that this excitation peak exists when DsRed variants are expressed in neurons in vivo. Personally, i have excited mCHerry in brain slices at 760 and signal to noise was as good as single photon excitation at 560. We are presently experimenting with TdTomato in neurons from brain slice. Will report our observations in the future if people are interested.

Best, Gavin

Gavin Rumbaugh, PhD
Assistant Professor
Department of Neuroscience, #3C2
The Scripps Research Institute
Jupiter, FL 33458
205-306-7096
[hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Staffan Nyström [[hidden email]]
Sent: Monday, December 13, 2010 9:04 AM
To: [hidden email]
Subject: DsRed vs 2P

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

Dear list members,

Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?
I have problems getting any decent fluorescence signal through between 700 and 1000nm excitation and there is significant bleedthrough
with EGFP. In the end I gave up since the "regular" 1 photon excitation way gave a much better S/N ratio with minimal bleed through.

Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA water dipping objectives.

Best

Staffan Nystrom

PhD student

MBB / Oncology & Pathology

Karolinska Institutet
Stockholm, Sweden