Leica STED machine

[Martin Wessendorf]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey folks!

I just got back from the Society for Neuroscience meeting in San Diego,
where Leica was giving a demo of it's Stimulated Emission Depletion
(STED) instrument.

Seeing it was an interesting experience.  The microscope appeared to
improve resolution of some specimens (specifically, histones) by a
factor of probably 5-fold--there was a very pronounced improvement and
unless their scale bar was lying, they seemed to be down into the 50 nm
range.  It did not seem to offer as much improvement on their muscle
specimen, and photobleaching was a serious problem on that one as well
when they went up to high zooms.

The STED module works only for one color (far red) and does not work
well with all fluorophores (--specifically, Cy5 apparently bleaches too
fast to be useful).  The fluorophores they recommended are the ATTO 647
and 655 dyes.  Although STED provides an improvement in x-y resolution,
there's little or no improvement in the z-axis resolution.

The instrument is essentially a Leica multiphoton microscope with the
STED unit as an attachment.  It can be used in single-photon,
multiphoton or STED modes.  Price is $1.3 million USD.

I'd be interested in hearing from other folks who talked to Leica about
this machine and who saw one of the demos.  If I were buying one of
these items, it'd be worried about it suddenly becoming obsolete (as
happened with some of the early 2-photon instruments) due to some new
development in the pipeline.  Does anyone have any sense of how likely
that is?  I'd also be concerned about how suited it is to all
preparations--will it work only with the strongest labeling?  Do the
ATTO dyes require particular mounting media?

Thanks in advance!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

[Armstrong, Brian]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you for sharing your experience.
I talked with Leica at SFN, but did not arrange a specific demo time
because I was previously aware of the $1.3 mil price tag. I attended a
"Super-Resolution Microscopy" meeting held by Leica at UCLA where the
experts from Germany on STED and 4Pi spoke. However, I was not aware
that the instrument was so particular about the fluorophores.
My take is that Zeiss (Big-Blue) does not like to be outdone and
certainly the alliance of Stefan Hell with Leica could cause them
concern. I have also heard a rumor that Zeiss is coming out with a
STED-like instrument, or at least a sub-diffration limit microscopy
system.
Moreover, it seems that Leica or Zeiss could make a system that utilizes
sub-diffraction techniques such as FPALM or STORM that could be
performed via software after imaging (similar to having a dedicated
deconvolution microscopy system).
As for my prediction for the "cutting edge time" for the STED
microscope???
I have no idea.

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Martin Wessendorf
Sent: Thursday, November 08, 2007 11:23 AM
To: [hidden email]
Subject: Leica STED machine

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey folks!

I just got back from the Society for Neuroscience meeting in San Diego,
where Leica was giving a demo of it's Stimulated Emission Depletion
(STED) instrument.

Seeing it was an interesting experience.  The microscope appeared to
improve resolution of some specimens (specifically, histones) by a
factor of probably 5-fold--there was a very pronounced improvement and
unless their scale bar was lying, they seemed to be down into the 50 nm
range.  It did not seem to offer as much improvement on their muscle
specimen, and photobleaching was a serious problem on that one as well
when they went up to high zooms.

The STED module works only for one color (far red) and does not work
well with all fluorophores (--specifically, Cy5 apparently bleaches too
fast to be useful).  The fluorophores they recommended are the ATTO 647
and 655 dyes.  Although STED provides an improvement in x-y resolution,
there's little or no improvement in the z-axis resolution.

The instrument is essentially a Leica multiphoton microscope with the
STED unit as an attachment.  It can be used in single-photon,
multiphoton or STED modes.  Price is $1.3 million USD.

I'd be interested in hearing from other folks who talked to Leica about
this machine and who saw one of the demos.  If I were buying one of
these items, it'd be worried about it suddenly becoming obsolete (as
happened with some of the early 2-photon instruments) due to some new
development in the pipeline.  Does anyone have any sense of how likely
that is?  I'd also be concerned about how suited it is to all
preparations--will it work only with the strongest labeling?  Do the
ATTO dyes require particular mounting media?

Thanks in advance!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

mms1.coh.org made the following annotations
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[kspencer007]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello all;
        I was also impressed with the STED microscope at SFN. Great
improvement in resolution. However, as Martin said, it only works with
two dyes now (although someone brought a sample with them to the demo,
and found that their dye also worked...now there are three), and only
one dye at a time. The system is only good for fixed samples...it didn't
appear to even be useful for cell surface labeling of live cells.
Because of the requirement of having two lasers at different wavelengths
perfectly aligned, penetration depth was limited to 15-20 microns. I'm
still sending them some samples from our labs, but I too am waiting for
the "second-generation" STED before I send my PO.

        Kathy Spencer, PhD
        Microscopy Core Manager
        ICND
        The Scripps Research Institute
        La Jolla, CA 92037


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Armstrong, Brian
Sent: Thursday, November 08, 2007 12:38 PM
To: [hidden email]
Subject: Re: Leica STED machine

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you for sharing your experience.
I talked with Leica at SFN, but did not arrange a specific demo time
because I was previously aware of the $1.3 mil price tag. I attended a
"Super-Resolution Microscopy" meeting held by Leica at UCLA where the
experts from Germany on STED and 4Pi spoke. However, I was not aware
that the instrument was so particular about the fluorophores.
My take is that Zeiss (Big-Blue) does not like to be outdone and
certainly the alliance of Stefan Hell with Leica could cause them
concern. I have also heard a rumor that Zeiss is coming out with a
STED-like instrument, or at least a sub-diffration limit microscopy
system.
Moreover, it seems that Leica or Zeiss could make a system that utilizes
sub-diffraction techniques such as FPALM or STORM that could be
performed via software after imaging (similar to having a dedicated
deconvolution microscopy system).
As for my prediction for the "cutting edge time" for the STED
microscope???
I have no idea.

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Martin Wessendorf
Sent: Thursday, November 08, 2007 11:23 AM
To: [hidden email]
Subject: Leica STED machine

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey folks!

I just got back from the Society for Neuroscience meeting in San Diego,
where Leica was giving a demo of it's Stimulated Emission Depletion
(STED) instrument.

Seeing it was an interesting experience.  The microscope appeared to
improve resolution of some specimens (specifically, histones) by a
factor of probably 5-fold--there was a very pronounced improvement and
unless their scale bar was lying, they seemed to be down into the 50 nm
range.  It did not seem to offer as much improvement on their muscle
specimen, and photobleaching was a serious problem on that one as well
when they went up to high zooms.

The STED module works only for one color (far red) and does not work
well with all fluorophores (--specifically, Cy5 apparently bleaches too
fast to be useful).  The fluorophores they recommended are the ATTO 647
and 655 dyes.  Although STED provides an improvement in x-y resolution,
there's little or no improvement in the z-axis resolution.

The instrument is essentially a Leica multiphoton microscope with the
STED unit as an attachment.  It can be used in single-photon,
multiphoton or STED modes.  Price is $1.3 million USD.

I'd be interested in hearing from other folks who talked to Leica about
this machine and who saw one of the demos.  If I were buying one of
these items, it'd be worried about it suddenly becoming obsolete (as
happened with some of the early 2-photon instruments) due to some new
development in the pipeline.  Does anyone have any sense of how likely
that is?  I'd also be concerned about how suited it is to all
preparations--will it work only with the strongest labeling?  Do the
ATTO dyes require particular mounting media?

Thanks in advance!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

mms1.coh.org made the following annotations
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financial information). Because this e-mail has been sent without
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information without the knowledge or consent of the sender. If you are
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you received the communication in error, please notify the sender
immediately by replying to this message and deleting the message and any
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further e-mail from the sender.  

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[Ella Tour]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Martin,

I saw the STED demo yesterday in San Diego as well. I was also very
impressed by the gain in resolution of the histone staining sample
detected with STED comparing to the normal confocal mode of this
Leica SP5 machine. As you said, photobleaching might be a problem,
and the STED image looked great only when detected with Avalanche
Photodiode Detector, and very dim with poor signal/noise ratio when
detected with PMT. We've tried to image our sample stained with Alexa
647 dye, but it did not work with STED.

For STED imaging, the Leica people recommended using
2,2'-Thiodiethanol (Fluka, 88559), the new mounting medium described
in the following paper from Stefan Hell lab:

Staudt, Lang, Medda, Engelhardt and Hell (2007) Microscopy Research
Technique 70:1-9.

Best wishes,

>Ella

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hey folks!
>
>I just got back from the Society for Neuroscience meeting in San
>Diego, where Leica was giving a demo of it's Stimulated Emission
>Depletion (STED) instrument.
>
>Seeing it was an interesting experience.  The microscope appeared to
>improve resolution of some specimens (specifically, histones) by a
>factor of probably 5-fold--there was a very pronounced improvement
>and unless their scale bar was lying, they seemed to be down into
>the 50 nm range.  It did not seem to offer as much improvement on
>their muscle specimen, and photobleaching was a serious problem on
>that one as well when they went up to high zooms.
>
>The STED module works only for one color (far red) and does not work
>well with all fluorophores (--specifically, Cy5 apparently bleaches
>too fast to be useful).  The fluorophores they recommended are the
>ATTO 647 and 655 dyes.  Although STED provides an improvement in x-y
>resolution, there's little or no improvement in the z-axis
>resolution.
>
>The instrument is essentially a Leica multiphoton microscope with
>the STED unit as an attachment.  It can be used in single-photon,
>multiphoton or STED modes.  Price is $1.3 million USD.
>
>I'd be interested in hearing from other folks who talked to Leica
>about this machine and who saw one of the demos.  If I were buying
>one of these items, it'd be worried about it suddenly becoming
>obsolete (as happened with some of the early 2-photon instruments)
>due to some new development in the pipeline.  Does anyone have any
>sense of how likely that is?  I'd also be concerned about how suited
>it is to all preparations--will it work only with the strongest
>labeling?  Do the ATTO dyes require particular mounting media?
>
>Thanks in advance!
>
>Martin
>--
>Martin Wessendorf, Ph.D.                   office: [phone]
>Assoc Prof, Dept Neuroscience                 lab: [phone]
>University of Minnesota             Preferred FAX: [phone]
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

--




Ella Tour
Department of Cell and Developmental Biology, 0349
University of California, San Diego
9500 Gilman Drive, 4305 Bonner Hall
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

[Martin Wessendorf]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Kathryn Spencer wrote:
> I was also impressed with the STED microscope at SFN. Great
> improvement in resolution. However, as Martin said, it only works with
> two dyes now (although someone brought a sample with them to the demo,
> and found that their dye also worked...now there are three), and only
> one dye at a time.

...Just to make sure that this point is clear--it can deal with more
than one dye, but the dyes that can be used are all far-red (i.e.
emission between about 650 and 700 nm).

> The system is only good for fixed samples...it didn't
> appear to even be useful for cell surface labeling of live cells.
> Because of the requirement of having two lasers at different wavelengths
> perfectly aligned, penetration depth was limited to 15-20 microns. I'm
> still sending them some samples from our labs, but I too am waiting for
> the "second-generation" STED before I send my PO.

When I spoke to the Leica people, they said (as best I recollect) that
development of multi-color STED depended on development of the
appropriate fluorophores and that there were no hardware improvements
that were in the immediate pipeline.  That's a bit surprising since (as
was pointed out to me off-line) Gerald Donnert in Stefan Hell's group
has already published on multicolor STED (Biophys. J. 2007 92: L67-69L)
and hints that improvements in lasers and related hardware should make
implementation easier in the near future.  --In that paper they also
mention the depletion laser for the far-red fluorphore as not being
optimal...which again seems odd since far-red STED is what Leica is
selling.

My aged memory has probably turned the story around 180 degrees, though,
and I should probably invite the reps from Leica chime in and set the
story straight!  Maybe they could also comment on how easy (or
difficult) it is to keep the microscope aligned and the related issue of
improving depth penetration.

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

[Lenaldo Rocha]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In a recent event in my institution, Leica representative mentioned  
that visible (70 nm resolution) and TREX STED (25 nm resolution) are  
feasible. I as far as I can remenber the problem with these techniques  
is the power of the laser required to deplete the fluorophore  
emission. It requires very powerful pulsed lasers and there is no  
commercially available visible laser with enough power to do this. And  
the prototypes are very expensive.

I searched the web and found papers describing TREX-STED. I won't  
comment on the numbers of laser power they give because that is beyond  
my competence, but perhaps someone here with more expertise could look  
at this numbers and enlighten rest of us.

Lenaldo Rocha

[John Oreopoulos]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

For anyone wanting to learn more about the various "super-resolution" optical techniques being developed, there is a very nice review article that came out on the topic:

Title:

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Author Full Names:

Rice, James H.

Source:

MOLECULAR BIOSYSTEMS 3 (11): 781-793 2007

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

On 8-Nov-07, at 7:14 PM, Kathryn Spencer wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello all;

I was also impressed with the STED microscope at SFN. Great

improvement in resolution. However, as Martin said, it only works with

two dyes now (although someone brought a sample with them to the demo,

and found that their dye also worked...now there are three), and only

one dye at a time. The system is only good for fixed samples...it didn't

appear to even be useful for cell surface labeling of live cells.

Because of the requirement of having two lasers at different wavelengths

perfectly aligned, penetration depth was limited to 15-20 microns. I'm

still sending them some samples from our labs, but I too am waiting for

the "second-generation" STED before I send my PO.

Kathy Spencer, PhD

Microscopy Core Manager

ICND

The Scripps Research Institute

La Jolla, CA 92037

-----Original Message-----

From: Confocal Microscopy List [[hidden email]] On

Behalf Of Armstrong, Brian

Sent: Thursday, November 08, 2007 12:38 PM

To: [hidden email]

Subject: Re: Leica STED machine

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you for sharing your experience. 

I talked with Leica at SFN, but did not arrange a specific demo time

because I was previously aware of the $1.3 mil price tag. I attended a

"Super-Resolution Microscopy" meeting held by Leica at UCLA where the

experts from Germany on STED and 4Pi spoke. However, I was not aware

that the instrument was so particular about the fluorophores.

My take is that Zeiss (Big-Blue) does not like to be outdone and

certainly the alliance of Stefan Hell with Leica could cause them

concern. I have also heard a rumor that Zeiss is coming out with a

STED-like instrument, or at least a sub-diffration limit microscopy

system.

Moreover, it seems that Leica or Zeiss could make a system that utilizes

sub-diffraction techniques such as FPALM or STORM that could be

performed via software after imaging (similar to having a dedicated

deconvolution microscopy system). 

As for my prediction for the "cutting edge time" for the STED

microscope???

I have no idea. 

Brian D Armstrong PhD

Light Microscopy Core Manager

Beckman Research Institute

City of Hope

1450 E Duarte Rd

Duarte, CA 91010

626-359-8111 x62872

http://www.cityofhope.org/SharedResources/LightMicroscopy

-----Original Message-----

From: Confocal Microscopy List [[hidden email]] On

Behalf Of Martin Wessendorf

Sent: Thursday, November 08, 2007 11:23 AM

To: [hidden email]

Subject: Leica STED machine

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey folks!

I just got back from the Society for Neuroscience meeting in San Diego,

where Leica was giving a demo of it's Stimulated Emission Depletion

(STED) instrument.

Seeing it was an interesting experience.  The microscope appeared to

improve resolution of some specimens (specifically, histones) by a

factor of probably 5-fold--there was a very pronounced improvement and

unless their scale bar was lying, they seemed to be down into the 50 nm

range.  It did not seem to offer as much improvement on their muscle

specimen, and photobleaching was a serious problem on that one as well

when they went up to high zooms.

The STED module works only for one color (far red) and does not work

well with all fluorophores (--specifically, Cy5 apparently bleaches too

fast to be useful).  The fluorophores they recommended are the ATTO 647

and 655 dyes.  Although STED provides an improvement in x-y resolution,

there's little or no improvement in the z-axis resolution.

The instrument is essentially a Leica multiphoton microscope with the

STED unit as an attachment.  It can be used in single-photon,

multiphoton or STED modes.  Price is $1.3 million USD.

I'd be interested in hearing from other folks who talked to Leica about

this machine and who saw one of the demos.  If I were buying one of

these items, it'd be worried about it suddenly becoming obsolete (as

happened with some of the early 2-photon instruments) due to some new

development in the pipeline.  Does anyone have any sense of how likely

that is?  I'd also be concerned about how suited it is to all

preparations--will it work only with the strongest labeling?  Do the

ATTO dyes require particular mounting media?

Thanks in advance!

Martin

-- 

Martin Wessendorf, Ph.D.                   office: [phone]

Assoc Prof, Dept Neuroscience                 lab: [phone]

University of Minnesota             Preferred FAX: [phone]

6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]

Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

mms1.coh.org made the following annotations

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financial information). Because this e-mail has been sent without

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[Martin Hoppe-2]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Commercial Vendor Response

 

All,

 

details about technology and benefits of the Leica TCS STED system can be found on our website http://www.confocal-microscopy.com

Go to the STED section. You can also download the full product brochure from there, which highlights STED technology and applications more extensively.

 

Best regards

Martin

 

--------------------------------------------------------
Martin Hoppe, Ph.D.
Head Market Management Life Science Research Division

 

Leica Microsystems CMS GmbH
Am Friedensplatz 3 | 68165 Mannheim (Germany)
Phone : [phone] | Fax : [phone]
Cell: [phone]

 

Leica Microsystems CMS GmbH  | GmbH mit Sitz in Wetzlar | Amtsgericht Wetzlar  HRB 2432
Geschäftsführer : Dr. David Martyr | Colin Davis | Dr. Wolf-Otto Reuter

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[George McNamara]

Search the CONFOCAL archive at
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Stefan Hell recently published STED with continuous wave beams
(Willig et al Nat Methods. 2007 Nov;4(11):915-8), so the beam shaping
and co-alignment of the depletion beam may end up being more
important than having a pulsed laser, and thus price might go down.

I am disappointed to hear that Z-resolution is not significantly
improved. Did Leica neglect to put depletion "bars" in the system?
Sounds like they were effective only in the XY "doughnut".

A lot of interesting papers have been published in 2007 on various
methods for fluorescence nanoscopy. STED is by no means the only way
to get to 100x smaller voxels than conventional confocal, multiphoton and TIRF.

STED Question: what happens to the fluorophore's excited energy when
depletion occurs? Does the molecule return to the ground state (a)
without emitting a photon or (b) emits a photon of the same
wavelength (and direction? as in a laser) as the depletion photon?



At 02:22 PM 11/8/2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hey folks!
>
>I just got back from the Society for Neuroscience meeting in San
>Diego, where Leica was giving a demo of it's Stimulated Emission
>Depletion (STED) instrument.
>
>Seeing it was an interesting experience.  The microscope appeared to
>improve resolution of some specimens (specifically, histones) by a
>factor of probably 5-fold--there was a very pronounced improvement
>and unless their scale bar was lying, they seemed to be down into
>the 50 nm range.  It did not seem to offer as much improvement on
>their muscle specimen, and photobleaching was a serious problem on
>that one as well when they went up to high zooms.
>
>The STED module works only for one color (far red) and does not work
>well with all fluorophores (--specifically, Cy5 apparently bleaches
>too fast to be useful).  The fluorophores they recommended are the
>ATTO 647 and 655 dyes.  Although STED provides an improvement in x-y
>resolution, there's little or no improvement in the z-axis resolution.
>
>The instrument is essentially a Leica multiphoton microscope with
>the STED unit as an attachment.  It can be used in single-photon,
>multiphoton or STED modes.  Price is $1.3 million USD.
>
>I'd be interested in hearing from other folks who talked to Leica
>about this machine and who saw one of the demos.  If I were buying
>one of these items, it'd be worried about it suddenly becoming
>obsolete (as happened with some of the early 2-photon instruments)
>due to some new development in the pipeline.  Does anyone have any
>sense of how likely that is?  I'd also be concerned about how suited
>it is to all preparations--will it work only with the strongest
>labeling?  Do the ATTO dyes require particular mounting media?
>
>Thanks in advance!
>
>Martin
>--
>Martin Wessendorf, Ph.D.                   office: [phone]
>Assoc Prof, Dept Neuroscience                 lab: [phone]
>University of Minnesota             Preferred FAX: [phone]
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see
Analytical Imaging Core Facility)

[Mark Cannell]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

George McNamara wrote:
>
>
> STED Question: what happens to the fluorophore's excited energy when
> depletion occurs? Does the molecule return to the ground state (a)
> without emitting a photon or (b) emits a photon of the same wavelength
> (and direction? as in a laser) as the depletion photon?
Hi George

As I understand it, in Stimulated Emission, the molecule emits a photon
similar just like as occurs in a laser. This is possible because the
emission spectrum is broad (lots of vibrational levels possible).

Cheers Mark Cannell

[Martin Hoppe-2]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

George,

 

if you shape phase filtering in STED to increase z resolution, you will automatically lose xy resolution. Based on multiple customer requests, we have focused on optimal xy resolution. z resolution stays confocal.

Best regards
Martin

--------------------------------------------------------

Martin Hoppe, Ph.D.

Head Market Management Life Science Research Division

Leica Microsystems CMS GmbH

Am Friedensplatz 3 | 68165 Mannheim (Germany)

Phone : [phone] | Fax : [phone]

Cell: [phone]

Leica Microsystems CMS GmbH | GmbH mit Sitz in Wetzlar | Amtsgericht Wetzlar HRB 2432

Geschäftsführer : Dr. David Martyr | Colin Davis | Dr. Wolf-Otto Reuter

-----Ursprüngliche Mitteilung-----
Von: George McNamara <[hidden email]>
An: [hidden email]
Verschickt: So., 11. Nov. 2007, 15:29
Thema: Re: Leica STED machine

Search the CONFOCAL archive at 
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
 
Stefan Hell recently published STED with continuous wave beams (Willig et al Nat Methods. 2007 Nov;4(11):915-8), so the beam shaping and co-alignment of the depletion beam may end up being more important than having a pulsed laser, and thus price might go down. 
 
I am disappointed to hear that Z-resolution is not significantly improved. Did Leica neglect to put depletion "bars" in the system? Sounds like they were effective only in the XY "doughnut". 
 
A lot of interesting papers have been published in 2007 on various methods for fluorescence nanoscopy. STED is by no means the only way to get to 100x smaller voxels than conventional confocal, multiphoton and TIRF. 
 
STED Question: what happens to the fluorophore's excited energy when depletion occurs? Does the molecule return to the ground state (a) without emitting a photon or (b) emits a photon of the same wavelength (and direction? as in a laser) as the depletion photon? 
 
 
At 02:22 PM 11/8/2007, you wrote: 
>Search the CONFOCAL archive at 
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
> 
>Hey folks! 
> 
>I just got back from the Society for Neuroscience meeting in San >Diego, where Leica was giving a demo of it's Stimulated Emission >Depletion (STED) instrument. 
> 
>Seeing it was an interesting experience. The microscope appeared to >improve resolution of some specimens (specifically, histones) by a >factor of probably 5-fold--there was a very pronounced improvement >and unless their scale bar was lying, they seemed to be down into >the 50 nm range. It did not seem to offer as much improvement on >their muscle specimen, and photobleaching was a serious problem on >that one as well when they went up to high zooms. 
> 
>The STED module works only for one color (far red) and does not work >well with all fluorophores (--specifically, Cy5 apparently bleaches >too fast to be useful). The fluorophores they recommended are the >ATTO 647 and 655 dyes. Although STED provides an improvement in x-y >resolution, there's little or no improvement in the z-axis resolution. 
> 
>The instrument is essentially a Leica multiphoton microscope with >the STED unit as an attachment. It can be used in single-photon, >multiphoton or STED modes. Price is $1.3 million USD. 
> 
>I'd be interested in hearing from other folks who talked to Leica >about this machine and who saw one of the demos. If I were buying >one of these items, it'd be worried about it suddenly becoming >obsolete (as happened with some of the early 2-photon instruments) >due to some new development in the pipeline. Does anyone have any >sense of how likely that is? I'd also be concerned about how suited >it is to all preparations--will it work only with the strongest >labeling? Do the ATTO dyes require particular mounting media? 
> 
>Thanks in advance! 
> 
>Martin 
>-- 
>Martin Wessendorf, Ph.D. office: [phone] 
>Assoc Prof, Dept Neuroscience lab: [phone] 
>University of Minnesota Preferred FAX: [phone] 
>6-145 Jackson Hall, 321 Church St. SE Dept Fax: [phone] 
>Minneapolis, MN 55455 E-mail: martinw[at]med.umn.edu 
 
 
 
George McNamara, Ph.D. 
University of Miami, Miller School of Medicine 
Image Core 
Miami, FL 33010 
[hidden email] 
[hidden email] 
305-243-8436 office 
http://home.earthlink.net/~pubspectra/ 
http://home.earthlink.net/~geomcnamara/ 
http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) 

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[Gert van Cappellen]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In STED, like in some other high resolution imaging techniques, were a
smaller excitation spot is used to get a higher resolution, the result
will be less photons coming out of the sample. So you either increase
your laser power resulting in more bleaching or you increase your
detection sensitivity by using the most sensitive detectors. With such
low amount of photons you off course never get very high signal to noise
ratio's. Sadly enough you can't get it all.

Gert

on 9-11-2007 4:11 Ella Tour said the following:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Martin,
>
> I saw the STED demo yesterday in San Diego as well. I was also very
> impressed by the gain in resolution of the histone staining sample
> detected with STED comparing to the normal confocal mode of this Leica
> SP5 machine. As you said, photobleaching might be a problem, and the
> STED image looked great only when detected with Avalanche Photodiode
> Detector, and very dim with poor signal/noise ratio when detected with
> PMT. We've tried to image our sample stained with Alexa 647 dye, but
> it did not work with STED.
>
> For STED imaging, the Leica people recommended using
> 2,2'-Thiodiethanol (Fluka, 88559), the new mounting medium described
> in the following paper from Stefan Hell lab:
>
> Staudt, Lang, Medda, Engelhardt and Hell (2007) Microscopy Research
> Technique 70:1-9.
>
> Best wishes,
>
>> Ella
>
>
>
>
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hey folks!
>>
>> I just got back from the Society for Neuroscience meeting in San
>> Diego, where Leica was giving a demo of it's Stimulated Emission
>> Depletion (STED) instrument.
>>
>> Seeing it was an interesting experience.  The microscope appeared to
>> improve resolution of some specimens (specifically, histones) by a
>> factor of probably 5-fold--there was a very pronounced improvement
>> and unless their scale bar was lying, they seemed to be down into the
>> 50 nm range.  It did not seem to offer as much improvement on their
>> muscle specimen, and photobleaching was a serious problem on that one
>> as well when they went up to high zooms.
>>
>> The STED module works only for one color (far red) and does not work
>> well with all fluorophores (--specifically, Cy5 apparently bleaches
>> too fast to be useful).  The fluorophores they recommended are the
>> ATTO 647 and 655 dyes.  Although STED provides an improvement in x-y
>> resolution, there's little or no improvement in the z-axis resolution.
>>
>> The instrument is essentially a Leica multiphoton microscope with the
>> STED unit as an attachment.  It can be used in single-photon,
>> multiphoton or STED modes.  Price is $1.3 million USD.
>>
>> I'd be interested in hearing from other folks who talked to Leica
>> about this machine and who saw one of the demos.  If I were buying
>> one of these items, it'd be worried about it suddenly becoming
>> obsolete (as happened with some of the early 2-photon instruments)
>> due to some new development in the pipeline.  Does anyone have any
>> sense of how likely that is?  I'd also be concerned about how suited
>> it is to all preparations--will it work only with the strongest
>> labeling?  Do the ATTO dyes require particular mounting media?
>>
>> Thanks in advance!
>>
>> Martin
>> --
>> Martin Wessendorf, Ph.D.                   office: [phone]
>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>> University of Minnesota             Preferred FAX: [phone]
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu
>
>

--
wigGert van Cappellen, [hidden email]
TEL [phone]; FAX [phone]
Dept. of Reproduction and Development; http://www.erasmusmc.nl/rede
Chairman Optical Imaging Centre; http://www.erasmusmc.nl/oic/
Erasmus MC, room Ee914, Dr. Molenwaterplein 50, 3015 GE ROTTERDAM, The Netherlands
Erasmus MC, P.O. box 2040, 3000 CA Rotterdam, The Netherlands
Delivery adres:
Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands

[Brian Bennett-6]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear martin W

with regards to the comments about mounting medium, I was the person
responsible for making the Histone samples that Leica used at the
meeting.  I followed the protocol my co-author and I used in our PNAS, 4Pi
paper, November 2006.  In fact the entire protocol was the same, with the
exception being the ATTO dyes which I conjugated to a Maximally
crossabsorbed IgG here in My lab.  the Primary was Histone H3 K27
The mounting medium I used was simply 87%GLY with 13%1MTris pH7.4 I
believe Leica uses a different medium which I have not had a chance to
test yet. Additionally the block was a big part of this as well, the
sample specifity that is.  
Incidentally I am quite impressed with STED, having looked at and
continuing to research histone mods by 4Pi, it is interesting that the
continued increase in resolution motivates new thinking as to biological
function, each new picture seems to tell us something more, putting the
pieces together - well that is the hard part.  
What scares with regard to STED is its ease of use, I fear Dogma in
various fields established by interpreting new images too rapidly.  This
is the Hubble telescope for the cellular environment, taking time to
pondor what we see will be important

Brian

 
> Dr. Brian T. Bennett >
> Chief Scientist - Microscopy Research and Development>
> Lake Placid Biologicals >
> 1915 Saranac Ave, STE 2 >
> Lake Placid, NY 12946 >
> 518-523-7025 X202
>
> [hidden email]
>

[Chris Wood-5]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

With regard to Leica claiming no equipment advances are in the pipeline...
well they would, wouldn't they!  I have no idea if there are or not, but
either way Leica *have* to claim not, otherwise they won't sell any of the
model they have on sale now.

Taking a rational guess, if advances are already published in the
literature, then they will (or at least should) be made available as as
soon as they can reasonably be implemented. Leica could deliberately 'sit
on' these improvements to extend the life-cycles of the current products,
but remember, even if you do have an impervious patent, there are plenty
of up-and-coming alternative techniques for improving resolution beyond
the diffraction limit. Competition tends to speed the rate of upgrades and
improvements, and deliberately retarding them can be a risky gamble.

Saludos

Chris

Dr Chris Wood
Instituto de Biotecnología
Universidad Nacional Autónoma de México
Av. Universidad 2001
Col. Chamilpa
Cuernavaca 62150
Morelos
México

On Fri, 9 Nov 2007 09:17:58 -0600, Martin Wessendorf <[hidden email]>
wrote:

>When I spoke to the Leica people, they said (as best I recollect) that
>development of multi-color STED depended on development of the
>appropriate fluorophores and that there were no hardware improvements
>that were in the immediate pipeline.  That's a bit surprising since (as
>was pointed out to me off-line) Gerald Donnert in Stefan Hell's group
>has already published on multicolor STED (Biophys. J. 2007 92: L67-69L)
>and hints that improvements in lasers and related hardware should make
>implementation easier in the near future.  --In that paper they also
>mention the depletion laser for the far-red fluorphore as not being
>optimal...which again seems odd since far-red STED is what Leica is
>selling.
>
>My aged memory has probably turned the story around 180 degrees, though,
>and I should probably invite the reps from Leica chime in and set the
>story straight!  Maybe they could also comment on how easy (or
>difficult) it is to keep the microscope aligned and the related issue of
>improving depth penetration.
>
>Martin Wessendorf
>
>--
>Martin Wessendorf, Ph.D.                   office: [phone]
>Assoc Prof, Dept Neuroscience                 lab: [phone]
>University of Minnesota             Preferred FAX: [phone]
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu
>=========================================================================

[Martin Hoppe-2]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

     Commercial Vendor

 

All,

 

As there have been several questions posted related to the Leica TCS STED, we have briefly summarized the relevance of STED for applications and have included some comments to sample preparation and technology:

 

Microscopic fluorescence imaging resolution in the range of a few tens of nanometers will have a dramatic impact on a large variety of key questions in life science research. STED provides this resolution and at the same time is non-invasive, 3D-capable and fully compatible with confocal techniques.

The Leica TCS STED actually contains a full spectral confocal and multiphoton system.

 

In general, STED microscopy is suited for every question related to structural information that cannot be obtained by conventional microscopy due to diffraction limited resolution. STED microscopy can separate up to 9 individual structures in a spot of approx. 250 nm while a standard confocal can resolve only 1 structure. Thus STED imaging not only gives access to previously unreachable detail inside a morphologically intact cell, but can also be used to quantitatively analyze dimensions of cell compartments.

 

A very important aspect of STED microscopy is the fact that STED is an enhancement to Confocal Laser Scanning technology and can fully exploit confocal benefits. This includes 3D sectioning capability – a diffraction unlimited image can be easily recorded from a defined 3D confocal volume inside the sample. STED is also compatible with most advanced confocal microscopy techniques, e.g. multi-color recording with additional confocal channels. Furthermore, STED allows recording of real 3D stacks.

Big range of biological applications

STED will allow scientists to study structures previously inaccessible to light microscopy in the fields of Neurobiology and Neuroscience, Membrane biology and Membrane rafts/Intracellular transport

 

This new and astonishing improvement to lateral resolution within a confocal volume has already produced excellent experimental results in imaging Drosophila larvae, Mammalian cell cultures, Plant cells, Brain slices and Membrane sheets.

 

And a lot more will to come as the instruments are spread around the globe and scientists use them for every day research.

STED: Physical resolution, no mathematics

A very important aspect is the purely physical character of STED microscopy. STED microscopy provides real, optical resolution. No mathematics at all (e.g. deconvolution, image restoration, statistical analysis, etc…) is needed. You get superresolution directly in the raw data.

 

However, STED images can benefit from further deconvolution and image restoration techniques, as well. That means: researchers can enhance STED image quality further by mathematics, optionally.

Sample preparation/labeling/dyes

The labeling procedures used to prepare a STED sample are not at all different from the ones used for confocal microscopy. The only thing one has to keep in mind is that STED needs optimized dyes and that therefore these dyes rather than others need to be used. The standard application is labeling the protein of interest by immunofluorescence. Nice results were also obtained with other labeling techniques as FISH (fluorescence in situ hybridization) and staining the actin cytoskeleton with by phalloidin-Atto647N.

 

Leica recommends the use of Atto 647N and Atto 655 (from AttoTec GmbH) for STED imaging and instrument specifications are given for this dyes. Atto655 and Atto647N both are very resistant to photobleaching and show high quantum yields, making them also ideal probes for confocal microscopy. Sample preparation is in no way different from sample preparation for confocal microscopy. For example: in an immunostaining you just use secondary antibodies conjugated to Atto655 or Atto647N instead of antibodies conjugated e.g. to Cy5.

 

However, also other dyes may function, in future. For performance checks, e.g. Crimson(tm) beads from Molecular Probes can also be used.

 

As embedding medium, standards like Mowiol, Glycerol, Vector shield work nicely and good results were obtained at penetration depths of more than 15 µm. All the samples at the launch in San Diego were embedded in one of these standard media.

Penetration depth depends on appropriate refractive index matching of immersion and embedding medium, therefore to penetrate even deeper it might be interesting to have a look at the new adjustable mounting medium TDE. See the following publication for details: Staudt, Lang, Medda, Engelhardt, Hell; “2,2'-Thiodiethanol: A New Water Soluble Mounting Medium for High Resolution Optical Microscopy”, Microscopy Research and Technique, 2007. TDE allows perfect refractive index matching and therefore should open the possibility to obtain good STED results far more than 15 µm deep in the sample. Atto647N and Atto655 perform nicely in this new embedding medium.

STED is single molecule sensitive

Breaking the diffraction limit by shrinking the focal spot results in a smaller volume from which fluorescence can be emitted and makes the use of smaller pixels mandatory. Therefore there is less signal emitted per pixel. Nevertheless, with very sensitive and low noise APDs (Avalanche Photo Diodes) even very weakly fluorescing samples can be imaged in the STED channel. With APD detectors the STED techniques is single molecule sensitive. For bright samples the internal PMT´s and Spectral Detectors can be used. So there is flexibility on the detector side and the user is able to select the detector that fits best to the sample.

 

Marcus Dyba       
Jochen Sieber      
Tanjef Szellas       
Martin Hoppe       

--------------------------------------------------------

Leica Microsystems CMS GmbH

Am Friedensplatz 3 | 68165 Mannheim (Germany)
Phone : [phone] | Fax : [phone]

Cell: [phone]

Email: [hidden email]

 

Leica Microsystems CMS GmbH  | GmbH mit Sitz in Wetzlar | Amtsgericht Wetzlar  HRB 2432 I Geschäftsführer : Dr. David Martyr | Colin Davis | Dr. Wolf-Otto Reuter

 

 

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[vb-2]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 

Dear Marcus,       
Dear Jochen,   
Dear Tanjef,      
Dear Martin,

      

>Breaking the diffraction limit by shrinking the focal spot results in a smaller volume from which fluorescence can be emitted and makes the use of smaller pixels mandatory. Therefore there is >less signal emitted per pixel. Nevertheless, with very sensitive and low noise APDs (Avalanche Photo Diodes) even very weakly fluorescing samples can be imaged in the STED channel. With >APD detectors the STED techniques is single molecule sensitive. For bright samples the internal PMT´s and Spectral Detectors can be used. So there is flexibility on the detector side and the >user is able to select the detector that fits best to the sample.

 

 

Could you, please provide an example of a single fluorescent molecule detection experiment, with the complete description of an image acquisition parameters?

Also, could you please describe the APDs (e.g. technical specifications in great detail)!!!???

 

Thank you in advance,

 

Vitaly

NCI-Frederick

 

   

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Thursday, November 15, 2007 11:26 AM

Subject: Re: Leica STED machine

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

     Commercial Vendor

 

All,

 

As there have been several questions posted related to the Leica TCS STED, we have briefly summarized the relevance of STED for applications and have included some comments to sample preparation and technology:

 

Microscopic fluorescence imaging resolution in the range of a few tens of nanometers will have a dramatic impact on a large variety of key questions in life science research. STED provides this resolution and at the same time is non-invasive, 3D-capable and fully compatible with confocal techniques.

The Leica TCS STED actually contains a full spectral confocal and multiphoton system.

 

In general, STED microscopy is suited for every question related to structural information that cannot be obtained by conventional microscopy due to diffraction limited resolution. STED microscopy can separate up to 9 individual structures in a spot of approx. 250 nm while a standard confocal can resolve only 1 structure. Thus STED imaging not only gives access to previously unreachable detail inside a morphologically intact cell, but can also be used to quantitatively analyze dimensions of cell compartments.

 

A very important aspect of STED microscopy is the fact that STED is an enhancement to Confocal Laser Scanning technology and can fully exploit confocal benefits. This includes 3D sectioning capability – a diffraction unlimited image can be easily recorded from a defined 3D confocal volume inside the sample. STED is also compatible with most advanced confocal microscopy techniques, e.g. multi-color recording with additional confocal channels. Furthermore, STED allows recording of real 3D stacks.

Big range of biological applications

STED will allow scientists to study structures previously inaccessible to light microscopy in the fields of Neurobiology and Neuroscience, Membrane biology and Membrane rafts/Intracellular transport

 

This new and astonishing improvement to lateral resolution within a confocal volume has already produced excellent experimental results in imaging Drosophila larvae, Mammalian cell cultures, Plant cells, Brain slices and Membrane sheets.

 

And a lot more will to come as the instruments are spread around the globe and scientists use them for every day research.

STED: Physical resolution, no mathematics

A very important aspect is the purely physical character of STED microscopy. STED microscopy provides real, optical resolution. No mathematics at all (e.g. deconvolution, image restoration, statistical analysis, etc…) is needed. You get superresolution directly in the raw data.

 

However, STED images can benefit from further deconvolution and image restoration techniques, as well. That means: researchers can enhance STED image quality further by mathematics, optionally.

Sample preparation/labeling/dyes

The labeling procedures used to prepare a STED sample are not at all different from the ones used for confocal microscopy. The only thing one has to keep in mind is that STED needs optimized dyes and that therefore these dyes rather than others need to be used. The standard application is labeling the protein of interest by immunofluorescence. Nice results were also obtained with other labeling techniques as FISH (fluorescence in situ hybridization) and staining the actin cytoskeleton with by phalloidin-Atto647N.

 

Leica recommends the use of Atto 647N and Atto 655 (from AttoTec GmbH) for STED imaging and instrument specifications are given for this dyes. Atto655 and Atto647N both are very resistant to photobleaching and show high quantum yields, making them also ideal probes for confocal microscopy. Sample preparation is in no way different from sample preparation for confocal microscopy. For example: in an immunostaining you just use secondary antibodies conjugated to Atto655 or Atto647N instead of antibodies conjugated e.g. to Cy5.

 

However, also other dyes may function, in future. For performance checks, e.g. Crimson(tm) beads from Molecular Probes can also be used.

 

As embedding medium, standards like Mowiol, Glycerol, Vector shield work nicely and good results were obtained at penetration depths of more than 15 µm. All the samples at the launch in San Diego were embedded in one of these standard media.

Penetration depth depends on appropriate refractive index matching of immersion and embedding medium, therefore to penetrate even deeper it might be interesting to have a look at the new adjustable mounting medium TDE. See the following publication for details: Staudt, Lang, Medda, Engelhardt, Hell; “2,2'-Thiodiethanol: A New Water Soluble Mounting Medium for High Resolution Optical Microscopy”, Microscopy Research and Technique, 2007. TDE allows perfect refractive index matching and therefore should open the possibility to obtain good STED results far more than 15 µm deep in the sample. Atto647N and Atto655 perform nicely in this new embedding medium.

STED is single molecule sensitive

Breaking the diffraction limit by shrinking the focal spot results in a smaller volume from which fluorescence can be emitted and makes the use of smaller pixels mandatory. Therefore there is less signal emitted per pixel. Nevertheless, with very sensitive and low noise APDs (Avalanche Photo Diodes) even very weakly fluorescing samples can be imaged in the STED channel. With APD detectors the STED techniques is single molecule sensitive. For bright samples the internal PMT´s and Spectral Detectors can be used. So there is flexibility on the detector side and the user is able to select the detector that fits best to the sample.

 

Marcus Dyba       
Jochen Sieber      
Tanjef Szellas       
Martin Hoppe       

--------------------------------------------------------

Leica Microsystems CMS GmbH

Am Friedensplatz 3 | 68165 Mannheim (Germany)
Phone : [phone] | Fax : [phone]

Cell: [phone]

Email: [hidden email]

 

Leica Microsystems CMS GmbH  | GmbH mit Sitz in Wetzlar | Amtsgericht Wetzlar  HRB 2432 I Geschäftsführer : Dr. David Martyr | Colin Davis | Dr. Wolf-Otto Reuter

 

 

---

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[John Oreopoulos]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all,

I've been asked to do some single molecule fluorescence imaging for a student from another lab using our microscope which is equipped with a Roper Cascade 512B EMMCD camera and total internal reflection fluorescence (TIRF) illumination optics. I know that this should be possible with this type of equipment, but I haven't had much experience with it before. I was wondering if anyone on the listserver could provide me with some practical tips and pointers for this type of imaging.

Unfortunately, the student is using FITC as a label, and I know that FITC is not very photostable and it bleaches quickly. Is it possible to do single molecule imaging with FITC?

Is there a special type of immersion oil I should be using that has minimal autofluorescence?

Should I be using the electron multiplying amplifier or the "traditional"/normal camera amplifier? The noise is a lot less with the normal amplifier, but I find the laser power or exposure time needs to  be increased significantly to get a reasonable image (when used with experiments where lots of dye is present).

Should I be trying to fill up the dynamic range in the image? Ie: should I set laser levels / exposure time / EM gain setting so that the single molecules come close to hitting the 16 bit maximum count level? What are the typical exposure times, laser powers, EM gain settings to use for single-molecule imaging? Should I be binning?

What is an acceptable SNR for single molecule imaging and how do I determine that on the fly during the experiment? Is there a better way to visualize the molecules on the preview screen (something like a live histogram stretch / auto contrast, a HI/LO look up table, etc.) I tried single-molecule imaging once before, and I found the molecules would bleach by the time I had found a good ROI to image in the preview screen and pushed the snap button in the software. 

How do I know if I'm looking at one molecule vs two or more within a diffraction-limited spot? Do you only know after the fact when they've bleached (looking for step heights in the pixel intensity vs. time trace)?. 

Is there a "legal" way to clean up the image noise after the experiment using post-processing software like ImageJ? What's the best way to present single-molecule data/images?

Thank you very much in advance if you can help me answer any of these questions or provide any additional tips.

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

[Kurt Thorn]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi John -

Single molecule imaging shouldn't be too hard with this setup.  The
Cascade 512B is kind of noisy (the Cascade II is much better) but you
should still be able to see single molecules.  We routinely image single
molecules (usually GFP) on a Nikon TIRF setup with a Cascade II camera
without doing anything very special.

Unfortunately, using FITC for single molecule imaging is somewhat
problematic.  My experience with it years ago was that it would survive
for only 2-3 seconds before bleaching - just long enough to see that
something was there but not long enough to do anything with it.  
Alexa488 is much better for single molecule imaging.

I think you should be able to use either amplifier on the camera, though
I always use the EM amplifier as it allows much shorter exposures.  With
our Cascade II or QuantEM cameras, we can image at 10 Hz easily with the
EM amplifier and can do 30 Hz on bright specimens.  I think you will
have trouble filling the dynamic range of the camera unless you do very
long exposure times.  We typically use the EM gain at max (for the
Cascade 512B you'd probably want to be a little lower, around 3600-3800,
as the high gain settings are pretty noisy).

To determine whether you're seeing one molecule or two, the best way is
to look at the bleaching trace and see whether you see single step or
multi-step bleaching.  You can certainly clean up your data after the
fact with median filtering or a similar process.

Hope that helps,
Kurt

John Oreopoulos wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all,
>
> I've been asked to do some single molecule fluorescence imaging for a
> student from another lab using our microscope which is equipped with a
> Roper Cascade 512B EMMCD camera and total internal reflection
> fluorescence (TIRF) illumination optics. I know that this should be
> possible with this type of equipment, but I haven't had much
> experience with it before. I was wondering if anyone on the listserver
> could provide me with some practical tips and pointers for this type
> of imaging.
>
> Unfortunately, the student is using FITC as a label, and I know that
> FITC is not very photostable and it bleaches quickly. Is it possible
> to do single molecule imaging with FITC?
>
> Is there a special type of immersion oil I should be using that has
> minimal autofluorescence?
>
> Should I be using the electron multiplying amplifier or the
> "traditional"/normal camera amplifier? The noise is a lot less with
> the normal amplifier, but I find the laser power or exposure time
> needs to  be increased significantly to get a reasonable image (when
> used with experiments where lots of dye is present).
>
> Should I be trying to fill up the dynamic range in the image? Ie:
> should I set laser levels / exposure time / EM gain setting so that
> the single molecules come close to hitting the 16 bit maximum count
> level? What are the typical exposure times, laser powers, EM gain
> settings to use for single-molecule imaging? Should I be binning?
>
> What is an acceptable SNR for single molecule imaging and how do I
> determine that on the fly during the experiment? Is there a better way
> to visualize the molecules on the preview screen (something like a
> live histogram stretch / auto contrast, a HI/LO look up table, etc.) I
> tried single-molecule imaging once before, and I found the molecules
> would bleach by the time I had found a good ROI to image in the
> preview screen and pushed the snap button in the software.
>
> How do I know if I'm looking at one molecule vs two or more within a
> diffraction-limited spot? Do you only know after the fact when they've
> bleached (looking for step heights in the pixel intensity vs. time
> trace)?.
>
> Is there a "legal" way to clean up the image noise after the
> experiment using post-processing software like ImageJ? What's the best
> way to present single-molecule data/images?
>
> Thank you very much in advance if you can help me answer any of these
> questions or provide any additional tips.
>
>
> John Oreopoulos, BSc,
>
> PhD Candidate
>
> University of Toronto
>
> Institute For Biomaterials and Biomedical Engineering
>
> Centre For Studies in Molecular Imaging
>
>
> Tel: W:416-946-5022
>
>
>

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone [phone]
fax   [phone]

[George McNamara]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,

Q. Should I be trying to fill up the dynamic range in the image?

Maybe - but what really matters is the photon flux per pixel. the goal of EM mode is to get above the read noise limit.

The November 2007 issue of Photonics Spectra has an article on p66 by Mayank Tripathi of Andor on the cross-over point on SMR curves between EM and conventional mode:

" ... the SNR curves for the 2.5 MHz readout rate in the EM and conventional modes intersect at B2; that is, at 125 photons per pixel the SNRs of both curves is 7.5. For photon fluxes below this strength, the EM mode will have better SNRs than the conventional mode at 2.5 MHz readout."

The reason for "maybe" is that I don't know (and don't need to know) your camera details. However, your specifications sheet should give you the answer. If your EMCCD has a gain of 1000x, 125 photons per pixel crossover point (as in  the camera above) and (say) a maximum 100,000 electrons at the last element of the readout register, you'd saturate that last element at 100 photons per pixel, well before the crossover point. My understanding is that you should just increase the gain to get above the read noise. This has been covered by others in previous EMCCD threads.


I've not done single molecule imaging (well, maybe at a UConn/Zeiss multiphoton course during a non-descanned detector demonstration), seems to me that you should use parameters that let you tell 0 from 1 from 2 molecules in the focal volume. This is typically done by watching for 2 (or more) steps in photobleaching. Otherwise, diluting your dye to lower area coverage should give you a corresponding decrease in single molecule spots.



George


At 12:44 PM 11/15/2007, you wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all,

I've been asked to do some single molecule fluorescence imaging for a student from another lab using our microscope which is equipped with a Roper Cascade 512B EMMCD camera and total internal reflection fluorescence (TIRF) illumination optics. I know that this should be possible with this type of equipment, but I haven't had much experience with it before. I was wondering if anyone on the listserver could provide me with some practical tips and pointers for this type of imaging.

Unfortunately, the student is using FITC as a label, and I know that FITC is not very photostable and it bleaches quickly. Is it possible to do single molecule imaging with FITC?

Is there a special type of immersion oil I should be using that has minimal autofluorescence?

Should I be using the electron multiplying amplifier or the "traditional"/normal camera amplifier? The noise is a lot less with the normal amplifier, but I find the laser power or exposure time needs to  be increased significantly to get a reasonable image (when used with experiments where lots of dye is present).

Should I be trying to fill up the dynamic range in the image? Ie: should I set laser levels / exposure time / EM gain setting so that the single molecules come close to hitting the 16 bit maximum count level? What are the typical exposure times, laser powers, EM gain settings to use for single-molecule imaging? Should I be binning?

What is an acceptable SNR for single molecule imaging and how do I determine that on the fly during the experiment? Is there a better way to visualize the molecules on the preview screen (something like a live histogram stretch / auto contrast, a HI/LO look up table, etc.) I tried single-molecule imaging once before, and I found the molecules would bleach by the time I had found a good ROI to image in the preview screen and pushed the snap button in the software.

How do I know if I'm looking at one molecule vs two or more within a diffraction-limited spot? Do you only know after the fact when they've bleached (looking for step heights in the pixel intensity vs. time trace)?.

Is there a "legal" way to clean up the image noise after the experiment using post-processing software like ImageJ? What's the best way to present single-molecule data/images?

Thank you very much in advance if you can help me answer any of these questions or provide any additional tips.


John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging


Tel: W:416-946-5022

 

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility)