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STED detection

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Alan Smith

STED detection

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Dear list,

Firstly, sorry if this is not confocally enough for this forum, but I hope
someone would know. I was wondering if anyone could help with some a
confusion I have about STED detection.

I understand how the illumination psf is formed in STED using a depletion
beam. However, if the emission is detected is collected in epi-detection,
why is the resolution not determined by the diffraction limit for the objective.

As an example, if a single molecule was producing fluorescence, the image
will be an airy disk determined by the NA of the detecting objective and the
wavelength of light. Despite the fluorescence solely coming from a single
molecule.

I know that for MP excitation the psf is solely determined by the
illumination psf, but again, I am a little unsure as to the reason for this.
Plus STED can obviously be CW also.

Any help or suggestions would be brilliant!
Much appreciated.
Alan Smith

Mark Cannell-2

Re: STED detection

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Hi

The key is that the excitation spot is scanned …

Cheers

On 31/08/2012, at 1:22 PM, Alan Smith <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Firstly, sorry if this is not confocally enough for this forum, but I hope
> someone would know. I was wondering if anyone could help with some a
> confusion I have about STED detection.
>
> I understand how the illumination psf is formed in STED using a depletion
> beam. However, if the emission is detected is collected in epi-detection,
> why is the resolution not determined by the diffraction limit for the objective.
>
> As an example, if a single molecule was producing fluorescence, the image
> will be an airy disk determined by the NA of the detecting objective and the
> wavelength of light. Despite the fluorescence solely coming from a single
> molecule.
>
> I know that for MP excitation the psf is solely determined by the
> illumination psf, but again, I am a little unsure as to the reason for this.
> Plus STED can obviously be CW also.
>
> Any help or suggestions would be brilliant!
> Much appreciated.
> Alan Smith

Mark B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology& Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

Guy Cox-2

Re: STED detection

In reply to this post by Alan Smith

In STED the depletion 'doughnut' (controversial term) limits the fluorescence emission to a spot ~50-80nm radius. So we know that that is where the signal has come from, even though the detection optics cannot actually resolve it.

The parallel with MP is very appropriate - in MP we typically use widefield detection so the detection optics cannot resolve anything at all. But again the scanning beam provides the resolution.

It is just the same in scanning electron microscopy - the resolution all comes from the scanning beam, the detection system has no resolution capability.

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alan Smith
Sent: Friday, 31 August 2012 10:22 PM
To: [hidden email]
Subject: STED detection

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

Dear list,

Firstly, sorry if this is not confocally enough for this forum, but I hope someone would know. I was wondering if anyone could help with some a confusion I have about STED detection.

I understand how the illumination psf is formed in STED using a depletion beam. However, if the emission is detected is collected in epi-detection, why is the resolution not determined by the diffraction limit for the objective.

As an example, if a single molecule was producing fluorescence, the image will be an airy disk determined by the NA of the detecting objective and the wavelength of light. Despite the fluorescence solely coming from a single molecule.

I know that for MP excitation the psf is solely determined by the illumination psf, but again, I am a little unsure as to the reason for this.
Plus STED can obviously be CW also.

Any help or suggestions would be brilliant!
Much appreciated.
Alan Smith

Martin Wessendorf-2

Re: STED detection

In reply to this post by Alan Smith

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Dear Alan--

On 8/31/2012 7:22 AM, Alan Smith wrote:

> I understand how the illumination psf is formed in STED using a depletion
> beam. However, if the emission is detected is collected in epi-detection,
> why is the resolution not determined by the diffraction limit for the objective.
>
> As an example, if a single molecule was producing fluorescence, the image
> will be an airy disk determined by the NA of the detecting objective and the
> wavelength of light. Despite the fluorescence solely coming from a single
> molecule.

You're right: with both a single molecule and with STED, you see a
diffraction-limited Airy disk on the emission side. However, with STED,
you know to a high degree of precision the position of the excitation
beam eliciting that Airy disk. Thus, as you scan that small excitation
beam over a small structure, you can obtain resolution that's many times
better than confocal. In that way, it's similar to near-field
super-resolution methods.

Hope that helps!

Martin Wessendorf
--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

Brian Northan

Re: STED detection

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On a related note can anybody recommend a paper that explains how to
calculate the STED PSF?

Is it worth it to deconvolve this type of data? I've heard conflicting reports.

On Fri, Aug 31, 2012 at 9:56 AM, Martin Wessendorf <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Alan--
>
>
> On 8/31/2012 7:22 AM, Alan Smith wrote:
>
>> I understand how the illumination psf is formed in STED using a depletion
>> beam. However, if the emission is detected is collected in epi-detection,
>> why is the resolution not determined by the diffraction limit for the
>> objective.
>>
>> As an example, if a single molecule was producing fluorescence, the image
>> will be an airy disk determined by the NA of the detecting objective and
>> the
>> wavelength of light. Despite the fluorescence solely coming from a single
>> molecule.
>
>
> You're right: with both a single molecule and with STED, you see a
> diffraction-limited Airy disk on the emission side. However, with STED, you
> know to a high degree of precision the position of the excitation beam
> eliciting that Airy disk. Thus, as you scan that small excitation beam over
> a small structure, you can obtain resolution that's many times better than
> confocal. In that way, it's similar to near-field super-resolution methods.
>
> Hope that helps!
>
> Martin Wessendorf
> --
> Martin Wessendorf, Ph.D. office: (612) 626-0145
> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
> University of Minnesota Preferred FAX: (612) 624-8118
> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
> Minneapolis, MN 55455 e-mail: [hidden email]

James Pawley

Re: STED detection

In reply to this post by Martin Wessendorf-2

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Perhaps Alan is really asking "What happens to the light from all the
molecules excited by the original Airy disk but residing in the
"donut" area of the STED beam?"

These emissions are "swept away" (entrained?) by the STED beam. They
become sort of a mini-laser and the emitted photons leave the area
totally coherent with the STED beam (i.e., in phase, at the same
wavelength as, and going in the same directions as the STED beam).

It is sometimes forgotten that the STED beam is at a wavelength that
is considerably longer than the peak of the emission spectrum. So,
even if you detect your signal fluorescence in the the transition
mode, it is relatively easy to use filters to separate the
fluorescence swept up by the STED beam (not to mention the STED beam
itself) from the fluorescence emitted by molecules near the center of
the donut hole because these latter photons are mostly at wavelenghts
near the emission peak.

Cheers,

Jim Pawley

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Alan--
>
>On 8/31/2012 7:22 AM, Alan Smith wrote:
>
>>I understand how the illumination psf is formed in STED using a depletion
>>beam. However, if the emission is detected is collected in epi-detection,
>>why is the resolution not determined by the diffraction limit for
>>the objective.
>>
>>As an example, if a single molecule was producing fluorescence, the image
>>will be an airy disk determined by the NA of the detecting objective and the
>>wavelength of light. Despite the fluorescence solely coming from a single
>>molecule.
>
>You're right: with both a single molecule and with STED, you see a
>diffraction-limited Airy disk on the emission side. However, with
>STED, you know to a high degree of precision the position of the
>excitation beam eliciting that Airy disk. Thus, as you scan that
>small excitation beam over a small structure, you can obtain
>resolution that's many times better than confocal. In that way,
>it's similar to near-field super-resolution methods.
>
>Hope that helps!
>
>Martin Wessendorf
>--
>Martin Wessendorf, Ph.D. office: (612) 626-0145
>Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>University of Minnesota Preferred FAX: (612) 624-8118
>6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>Minneapolis, MN 55455 e-mail: [hidden email]

--
James and Christine Pawley, PO Box 2348, 5446 Burley Place (PO Box
2348), Sechelt, BC, Canada, V0N3A0, 604-885-0840 NEW! Cell (when I
remember to turn it on!) 1-765-637-1917, <[hidden email]>

Peng Xi-2

Re: STED detection

In reply to this post by Alan Smith

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Dear Alan,
I'd like to quote Paul Silven's solution on single molecule
localization microscopy: "The trick to get around this problem was to
realize that a SINGLE molecule’s position can be located arbitrarily
well. It’s much like a mountain peak, which can be located to within a
few yards, even though the mountain itself may be a a mile wide."
Once you understand that in a wide-field microscopy, you can
determine the position of a single molecule extremely well, you can
bring it to a scanning imaging mode. Take sample scan first because
laser scan is equivalent. All you need to do is to set your scan step
equals to sufficient sampling, so that you can accurately fit the
Gaussian. The Nyquist-Shannon always holds true.
Now come to STED, where you use mini laser and dichroic to have
somehow a multi-molecule with size of 50-80nm. By scanning you get
the high-resolution image.

Sincerely,
Peng Xi
Ph. D. Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Email: [hidden email]
http://xipeng.wordpress.com

On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:

Peng Xi-2

Re: STED detection

In reply to this post by Brian Northan

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Dear Brian,
You can read the following:
1. Volker Westphal and Stefan W. Hell, Nanoscale Resolution in the
Focal Plane of an Optical Microscope, PRL 94, 143903, 2005.
In this paper the excitation and STED PSF were assumed to be sin and
cos, and with Taylor expansion the 1/sqrt(1+I/Is) is obtained.

2. Benjamin Harke et al., Resolution scaling in STED microscopy, OE16
(6) 4154, 2008.
In this paper the excitation is assumed to be Gaussian, and STED is
assumed as x^2. The 1/sqrt(1+a*I/Is) is obtained.

Cheers,
Peng Xi
Ph. D. Associate Professor
Peking University
Email: [hidden email]
http://xipeng.wordpress.com

On Fri, Aug 31, 2012 at 11:22 PM, Brian Northan <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On a related note can anybody recommend a paper that explains how to
> calculate the STED PSF?
>
> Is it worth it to deconvolve this type of data? I've heard conflicting reports.
>
> On Fri, Aug 31, 2012 at 9:56 AM, Martin Wessendorf <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Alan--
>>
>>
>> On 8/31/2012 7:22 AM, Alan Smith wrote:
>>
>>> I understand how the illumination psf is formed in STED using a depletion
>>> beam. However, if the emission is detected is collected in epi-detection,
>>> why is the resolution not determined by the diffraction limit for the
>>> objective.
>>>
>>> As an example, if a single molecule was producing fluorescence, the image
>>> will be an airy disk determined by the NA of the detecting objective and
>>> the
>>> wavelength of light. Despite the fluorescence solely coming from a single
>>> molecule.
>>
>>
>> You're right: with both a single molecule and with STED, you see a
>> diffraction-limited Airy disk on the emission side. However, with STED, you
>> know to a high degree of precision the position of the excitation beam
>> eliciting that Airy disk. Thus, as you scan that small excitation beam over
>> a small structure, you can obtain resolution that's many times better than
>> confocal. In that way, it's similar to near-field super-resolution methods.
>>
>> Hope that helps!
>>
>> Martin Wessendorf
>> --
>> Martin Wessendorf, Ph.D. office: (612) 626-0145
>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>> University of Minnesota Preferred FAX: (612) 624-8118
>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>> Minneapolis, MN 55455 e-mail: [hidden email]

--
席鹏
特聘研究员
北京大学工学院生物医学工程系
地址：中关村北大街北京大学医院A536室
邮编：100084
电话：010-6276 7155
Email: [hidden email]
http://dx.plos.org/10.1371/journal.pone.0040003

Sincerely,
Peng Xi
Ph. D. Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Tel: +86 10-6276 7155
Email: [hidden email]
http://dx.plos.org/10.1371/journal.pone.0040003

Alberto Diaspro

Re: STED detection

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*****

I also would suggest the following paper containing some criteria for optimizing (open access):
Galiani, S., Harke, B., Vicidomini, G., Lignani, G., Benfenati, F., Diaspro, A., & Bianchini, P. (2012). Strategies to maximize the performance of a STED microscope. Optics express, 1–13.
All the best
AD

Il giorno 01/set/2012, alle ore 02:09, Peng Xi <[hidden email]> ha scritto:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Brian,
> You can read the following:
> 1. Volker Westphal and Stefan W. Hell, Nanoscale Resolution in the
> Focal Plane of an Optical Microscope, PRL 94, 143903, 2005.
> In this paper the excitation and STED PSF were assumed to be sin and
> cos, and with Taylor expansion the 1/sqrt(1+I/Is) is obtained.
>
> 2. Benjamin Harke et al., Resolution scaling in STED microscopy, OE16
> (6) 4154, 2008.
> In this paper the excitation is assumed to be Gaussian, and STED is
> assumed as x^2. The 1/sqrt(1+a*I/Is) is obtained.
>
>
> Cheers,
> Peng Xi
> Ph. D. Associate Professor
> Peking University
> Email: [hidden email]
> http://xipeng.wordpress.com
>
>
>
>
>
>
> On Fri, Aug 31, 2012 at 11:22 PM, Brian Northan <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> On a related note can anybody recommend a paper that explains how to
>> calculate the STED PSF?
>>
>> Is it worth it to deconvolve this type of data? I've heard conflicting reports.
>>
>> On Fri, Aug 31, 2012 at 9:56 AM, Martin Wessendorf <[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Alan--
>>>
>>>
>>> On 8/31/2012 7:22 AM, Alan Smith wrote:
>>>
>>>> I understand how the illumination psf is formed in STED using a depletion
>>>> beam. However, if the emission is detected is collected in epi-detection,
>>>> why is the resolution not determined by the diffraction limit for the
>>>> objective.
>>>>
>>>> As an example, if a single molecule was producing fluorescence, the image
>>>> will be an airy disk determined by the NA of the detecting objective and
>>>> the
>>>> wavelength of light. Despite the fluorescence solely coming from a single
>>>> molecule.
>>>
>>>
>>> You're right: with both a single molecule and with STED, you see a
>>> diffraction-limited Airy disk on the emission side. However, with STED, you
>>> know to a high degree of precision the position of the excitation beam
>>> eliciting that Airy disk. Thus, as you scan that small excitation beam over
>>> a small structure, you can obtain resolution that's many times better than
>>> confocal. In that way, it's similar to near-field super-resolution methods.
>>>
>>> Hope that helps!
>>>
>>> Martin Wessendorf
>>> --
>>> Martin Wessendorf, Ph.D. office: (612) 626-0145
>>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>>> University of Minnesota Preferred FAX: (612) 624-8118
>>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>>> Minneapolis, MN 55455 e-mail: [hidden email]
>
>
>
> --
> 席鹏
> 特聘研究员
> 北京大学工学院生物医学工程系
> 地址：中关村北大街北京大学医院A536室
> 邮编：100084
> 电话：010-6276 7155
> Email: [hidden email]
> http://dx.plos.org/10.1371/journal.pone.0040003
>
>
> Sincerely,
> Peng Xi
> Ph. D. Associate Professor
> Dept. of Biomedical Engineering, College of Engineering
> Peking University, Beijing, China
> Tel: +86 10-6276 7155
> Email: [hidden email]
> http://dx.plos.org/10.1371/journal.pone.0040003

Arne Seitz

Re: STED detection

In reply to this post by Peng Xi-2

*****
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*****

What exactly is a "mini laser" and a "multi-molecule"?

Cheers Arne

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
Sent: samedi 1 septembre 2012 01:55
To: [hidden email]
Subject: Re: STED detection

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

Dear Alan,
I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
Now come to STED, where you use mini laser and dichroic to have
somehow a multi-molecule with size of 50-80nm. By scanning you get
the high-resolution image.

Sincerely,
Peng Xi
Ph. D. Associate Professor
Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
Email: [hidden email]
http://xipeng.wordpress.com

On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Firstly, sorry if this is not confocally enough for this forum, but I
> hope someone would know. I was wondering if anyone could help with
> some a confusion I have about STED detection.
>
> I understand how the illumination psf is formed in STED using a
> depletion beam. However, if the emission is detected is collected in
> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>
> As an example, if a single molecule was producing fluorescence, the
> image will be an airy disk determined by the NA of the detecting
> objective and the wavelength of light. Despite the fluorescence solely
> coming from a single molecule.
>
> I know that for MP excitation the psf is solely determined by the
> illumination psf, but again, I am a little unsure as to the reason for this.
> Plus STED can obviously be CW also.
>
> Any help or suggestions would be brilliant!
> Much appreciated.
> Alan Smith

Peng Xi-2

Re: STED detection

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

Dear Arne,
Here "mini laser" referes to the stimulated emission depletion
process happening at the peripherial of the excited fluorescence PSF.
It is indeed light amplification if taking the excitation laser as the
pump source; and sometimes it is called pump-probe process.
"Multi-molecule" is a word I created in contrast to single
molecule, since the ON state, spontaneous emission molecules in STED
is 50-80 diameter, thus certainly contains more than one molecule.

Hope that helps,
Peng Xi
Ph. D. Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Email: [hidden email]
http://xipeng.wordpress.com

On Sun, Sep 2, 2012 at 4:03 AM, Seitz Arne <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> What exactly is a "mini laser" and a "multi-molecule"?
>
> Cheers Arne
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
> Sent: samedi 1 septembre 2012 01:55
> To: [hidden email]
> Subject: Re: STED detection
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Alan,
> I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
> Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
> Now come to STED, where you use mini laser and dichroic to have
> somehow a multi-molecule with size of 50-80nm. By scanning you get
> the high-resolution image.
>
>
> Sincerely,
> Peng Xi
> Ph. D. Associate Professor
> Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
> Email: [hidden email]
> http://xipeng.wordpress.com
>
> On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear list,
>>
>> Firstly, sorry if this is not confocally enough for this forum, but I
>> hope someone would know. I was wondering if anyone could help with
>> some a confusion I have about STED detection.
>>
>> I understand how the illumination psf is formed in STED using a
>> depletion beam. However, if the emission is detected is collected in
>> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>>
>> As an example, if a single molecule was producing fluorescence, the
>> image will be an airy disk determined by the NA of the detecting
>> objective and the wavelength of light. Despite the fluorescence solely
>> coming from a single molecule.
>>
>> I know that for MP excitation the psf is solely determined by the
>> illumination psf, but again, I am a little unsure as to the reason for this.
>> Plus STED can obviously be CW also.
>>
>> Any help or suggestions would be brilliant!
>> Much appreciated.
>> Alan Smith

Guy Cox-2

Re: STED detection

Really it isn't a mini-laser because there is no amplification - it's only when the emitted photon gives rise to further stimulated emission that you get amplification, and laser action.

Fu-Jen Kao, in Taiwan, has done a lot of work on stimulated emisssion microscopy - it's useful because you know in what direction the emitted photon will be travelling. (This is nothing to do with STED, he's detecting the emitted photons, not spontaneous fluorescence).

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
Sent: Sunday, 2 September 2012 9:47 AM
To: [hidden email]
Subject: Re: STED detection

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

Dear Arne,
Here "mini laser" referes to the stimulated emission depletion process happening at the peripherial of the excited fluorescence PSF.
It is indeed light amplification if taking the excitation laser as the pump source; and sometimes it is called pump-probe process.
"Multi-molecule" is a word I created in contrast to single molecule, since the ON state, spontaneous emission molecules in STED is 50-80 diameter, thus certainly contains more than one molecule.

Hope that helps,
Peng Xi
Ph. D. Associate Professor
Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
Email: [hidden email]
http://xipeng.wordpress.com

On Sun, Sep 2, 2012 at 4:03 AM, Seitz Arne <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> What exactly is a "mini laser" and a "multi-molecule"?
>
> Cheers Arne
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Peng Xi
> Sent: samedi 1 septembre 2012 01:55
> To: [hidden email]
> Subject: Re: STED detection
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Alan,
> I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
> Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
> Now come to STED, where you use mini laser and dichroic to have
> somehow a multi-molecule with size of 50-80nm. By scanning you get
> the high-resolution image.
>
>
> Sincerely,
> Peng Xi
> Ph. D. Associate Professor
> Dept. of Biomedical Engineering, College of Engineering Peking
> University, Beijing, China
> Email: [hidden email]
> http://xipeng.wordpress.com
>
> On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear list,
>>
>> Firstly, sorry if this is not confocally enough for this forum, but I
>> hope someone would know. I was wondering if anyone could help with
>> some a confusion I have about STED detection.
>>
>> I understand how the illumination psf is formed in STED using a
>> depletion beam. However, if the emission is detected is collected in
>> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>>
>> As an example, if a single molecule was producing fluorescence, the
>> image will be an airy disk determined by the NA of the detecting
>> objective and the wavelength of light. Despite the fluorescence
>> solely coming from a single molecule.
>>
>> I know that for MP excitation the psf is solely determined by the
>> illumination psf, but again, I am a little unsure as to the reason for this.
>> Plus STED can obviously be CW also.
>>
>> Any help or suggestions would be brilliant!
>> Much appreciated.
>> Alan Smith

Peng Xi-2

Re: STED detection

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

Dear Guy,
If you consider the pump loses one photon, while the probe earns
one photon, you know what I meant by amplification. Of course without
population reversion constantly (the pump is so low), and absence of
the reflective cavity, it is not a normal laser that we are familiar
with. :)
Peng

On Sun, Sep 2, 2012 at 8:49 AM, Guy Cox <[hidden email]> wrote:

> Really it isn't a mini-laser because there is no amplification - it's only when the emitted photon gives rise to further stimulated emission that you get amplification, and laser action.
>
> Fu-Jen Kao, in Taiwan, has done a lot of work on stimulated emisssion microscopy - it's useful because you know in what direction the emitted photon will be travelling. (This is nothing to do with STED, he's detecting the emitted photons, not spontaneous fluorescence).
>
> Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
> Sent: Sunday, 2 September 2012 9:47 AM
> To: [hidden email]
> Subject: Re: STED detection
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Arne,
> Here "mini laser" referes to the stimulated emission depletion process happening at the peripherial of the excited fluorescence PSF.
> It is indeed light amplification if taking the excitation laser as the pump source; and sometimes it is called pump-probe process.
> "Multi-molecule" is a word I created in contrast to single molecule, since the ON state, spontaneous emission molecules in STED is 50-80 diameter, thus certainly contains more than one molecule.
>
> Hope that helps,
> Peng Xi
> Ph. D. Associate Professor
> Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
> Email: [hidden email]
> http://xipeng.wordpress.com
>
> On Sun, Sep 2, 2012 at 4:03 AM, Seitz Arne <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> What exactly is a "mini laser" and a "multi-molecule"?
>>
>> Cheers Arne
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Peng Xi
>> Sent: samedi 1 septembre 2012 01:55
>> To: [hidden email]
>> Subject: Re: STED detection
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Alan,
>> I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
>> Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
>> Now come to STED, where you use mini laser and dichroic to have
>> somehow a multi-molecule with size of 50-80nm. By scanning you get
>> the high-resolution image.
>>
>>
>> Sincerely,
>> Peng Xi
>> Ph. D. Associate Professor
>> Dept. of Biomedical Engineering, College of Engineering Peking
>> University, Beijing, China
>> Email: [hidden email]
>> http://xipeng.wordpress.com
>>
>> On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear list,
>>>
>>> Firstly, sorry if this is not confocally enough for this forum, but I
>>> hope someone would know. I was wondering if anyone could help with
>>> some a confusion I have about STED detection.
>>>
>>> I understand how the illumination psf is formed in STED using a
>>> depletion beam. However, if the emission is detected is collected in
>>> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>>>
>>> As an example, if a single molecule was producing fluorescence, the
>>> image will be an airy disk determined by the NA of the detecting
>>> objective and the wavelength of light. Despite the fluorescence
>>> solely coming from a single molecule.
>>>
>>> I know that for MP excitation the psf is solely determined by the
>>> illumination psf, but again, I am a little unsure as to the reason for this.
>>> Plus STED can obviously be CW also.
>>>
>>> Any help or suggestions would be brilliant!
>>> Much appreciated.
>>> Alan Smith
>
>

Chen, De (NIH/NCI) [C]

Re: STED detection

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

Hi Peng:

For the two energy levels related to STED depletion, there is population inversion exists. So this kind of lasing is best termed the amplified spontaneous emission.

De

________________________________________
From: Peng Xi [[hidden email]]
Sent: Saturday, September 01, 2012 9:31 PM
To: [hidden email]
Subject: Re: STED detection

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

Dear Guy,
If you consider the pump loses one photon, while the probe earns
one photon, you know what I meant by amplification. Of course without
population reversion constantly (the pump is so low), and absence of
the reflective cavity, it is not a normal laser that we are familiar
with. :)
Peng

On Sun, Sep 2, 2012 at 8:49 AM, Guy Cox <[hidden email]> wrote:

Mark Cannell-2

Re: STED detection

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

Why do you think there is a population inversion? In my opinion, this seems rather unlikely given the spill of the depletion beam to the centre and that saturation effects in the excitation beam should reduce resolution -so ground state depletion should be avoided.

I think this introduction of the term 'laser" to the discussion of STED is neither correct nor warranted. Just because there is stimulated emission, this does not imply lasing. I suggest there is no lasing in the excited volume as the fraction of exited molecules is <0.5. -i.e. there is no population inversion. Of course, it would be possible to achieve this but it would not be desirable.

My 2c

Cheers Mark

On 2/09/2012, at 10:13 PM, "Chen, De (NIH/NCI) [C]" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Peng:
>
> For the two energy levels related to STED depletion, there is population inversion exists. So this kind of lasing is best termed the amplified spontaneous emission.
>
> De
>
> ________________________________________
> From: Peng Xi [[hidden email]]
> Sent: Saturday, September 01, 2012 9:31 PM
> To: [hidden email]
> Subject: Re: STED detection
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Guy,
> If you consider the pump loses one photon, while the probe earns
> one photon, you know what I meant by amplification. Of course without
> population reversion constantly (the pump is so low), and absence of
> the reflective cavity, it is not a normal laser that we are familiar
> with. :)
> Peng
>
> On Sun, Sep 2, 2012 at 8:49 AM, Guy Cox <[hidden email]> wrote:
>> Really it isn't a mini-laser because there is no amplification - it's only when the emitted photon gives rise to further stimulated emission that you get amplification, and laser action.
>>
>> Fu-Jen Kao, in Taiwan, has done a lot of work on stimulated emisssion microscopy - it's useful because you know in what direction the emitted photon will be travelling. (This is nothing to do with STED, he's detecting the emitted photons, not spontaneous fluorescence).
>>
>> Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
>> Sent: Sunday, 2 September 2012 9:47 AM
>> To: [hidden email]
>> Subject: Re: STED detection
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Arne,
>> Here "mini laser" referes to the stimulated emission depletion process happening at the peripherial of the excited fluorescence PSF.
>> It is indeed light amplification if taking the excitation laser as the pump source; and sometimes it is called pump-probe process.
>> "Multi-molecule" is a word I created in contrast to single molecule, since the ON state, spontaneous emission molecules in STED is 50-80 diameter, thus certainly contains more than one molecule.
>>
>> Hope that helps,
>> Peng Xi
>> Ph. D. Associate Professor
>> Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
>> Email: [hidden email]
>> http://xipeng.wordpress.com
>>
>> On Sun, Sep 2, 2012 at 4:03 AM, Seitz Arne <[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> What exactly is a "mini laser" and a "multi-molecule"?
>>>
>>> Cheers Arne
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]] On Behalf Of Peng Xi
>>> Sent: samedi 1 septembre 2012 01:55
>>> To: [hidden email]
>>> Subject: Re: STED detection
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Alan,
>>> I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
>>> Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
>>> Now come to STED, where you use mini laser and dichroic to have
>>> somehow a multi-molecule with size of 50-80nm. By scanning you get
>>> the high-resolution image.
>>>
>>>
>>> Sincerely,
>>> Peng Xi
>>> Ph. D. Associate Professor
>>> Dept. of Biomedical Engineering, College of Engineering Peking
>>> University, Beijing, China
>>> Email: [hidden email]
>>> http://xipeng.wordpress.com
>>>
>>> On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear list,
>>>>
>>>> Firstly, sorry if this is not confocally enough for this forum, but I
>>>> hope someone would know. I was wondering if anyone could help with
>>>> some a confusion I have about STED detection.
>>>>
>>>> I understand how the illumination psf is formed in STED using a
>>>> depletion beam. However, if the emission is detected is collected in
>>>> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>>>>
>>>> As an example, if a single molecule was producing fluorescence, the
>>>> image will be an airy disk determined by the NA of the detecting
>>>> objective and the wavelength of light. Despite the fluorescence
>>>> solely coming from a single molecule.
>>>>
>>>> I know that for MP excitation the psf is solely determined by the
>>>> illumination psf, but again, I am a little unsure as to the reason for this.
>>>> Plus STED can obviously be CW also.
>>>>
>>>> Any help or suggestions would be brilliant!
>>>> Much appreciated.
>>>> Alan Smith
>>

Mark B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology& Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

Mark Cannell-2

Re: STED detection

In reply to this post by Chen, De (NIH/NCI) [C]

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

Why do you think there is a population inversion in the excited volume? In my opinion, this seems rather unlikely given the spill of the depletion beam to the centre and that saturation effects in the excitation beam could reduce resolution -so ground state depletion should be avoided. The fact that the centre of the STED PSF is not flat topped implies (to me) no ground state depletion.

I think this introduction of the term 'laser" to the discussion of STED is not warranted (or correct -so I agree with Guy). Just because there is stimulated emission, does not imply lasing. Of course, it would be possible to achieve this but it would not be desirable IMHO.

My 2c

Cheers Mark

On 2/09/2012, at 10:13 PM, "Chen, De (NIH/NCI) [C]" <[hidden email]> wrote:

Mark B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology& Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

Peng Xi-2

Re: STED detection

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

Dear Mark and De,
I am totally lost to Mark's post. I agree with Guy that
mini-laser is not a good term so let's change it to pump-probe. The
population inversion is a statistical process which basically provides
abundant electrons at excite state waiting for stimulated emission.
Apparently this is not the case in a dye labelled biological specimen.
So in STED, it is just stimulted emission dominated, pump-probe
process. With negelegible increase of probe photon.
We have not touch any ground state depletion here...
Peng

On Mon, Sep 3, 2012 at 5:14 PM, Mark Cannell <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Why do you think there is a population inversion in the excited volume? In my opinion, this seems rather unlikely given the spill of the depletion beam to the centre and that saturation effects in the excitation beam could reduce resolution -so ground state depletion should be avoided. The fact that the centre of the STED PSF is not flat topped implies (to me) no ground state depletion.
>
> I think this introduction of the term 'laser" to the discussion of STED is not warranted (or correct -so I agree with Guy). Just because there is stimulated emission, does not imply lasing. Of course, it would be possible to achieve this but it would not be desirable IMHO.
>
> My 2c
>
> Cheers Mark
>
> On 2/09/2012, at 10:13 PM, "Chen, De (NIH/NCI) [C]" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Peng:
>>
>> For the two energy levels related to STED depletion, there is population inversion exists. So this kind of lasing is best termed the amplified spontaneous emission.
>>
>> De
>>
>> ________________________________________
>> From: Peng Xi [[hidden email]]
>> Sent: Saturday, September 01, 2012 9:31 PM
>> To: [hidden email]
>> Subject: Re: STED detection
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Guy,
>> If you consider the pump loses one photon, while the probe earns
>> one photon, you know what I meant by amplification. Of course without
>> population reversion constantly (the pump is so low), and absence of
>> the reflective cavity, it is not a normal laser that we are familiar
>> with. :)
>> Peng
>>
>> On Sun, Sep 2, 2012 at 8:49 AM, Guy Cox <[hidden email]> wrote:
>>> Really it isn't a mini-laser because there is no amplification - it's only when the emitted photon gives rise to further stimulated emission that you get amplification, and laser action.
>>>
>>> Fu-Jen Kao, in Taiwan, has done a lot of work on stimulated emisssion microscopy - it's useful because you know in what direction the emitted photon will be travelling. (This is nothing to do with STED, he's detecting the emitted photons, not spontaneous fluorescence).
>>>
>>> Guy
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
>>> Sent: Sunday, 2 September 2012 9:47 AM
>>> To: [hidden email]
>>> Subject: Re: STED detection
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Arne,
>>> Here "mini laser" referes to the stimulated emission depletion process happening at the peripherial of the excited fluorescence PSF.
>>> It is indeed light amplification if taking the excitation laser as the pump source; and sometimes it is called pump-probe process.
>>> "Multi-molecule" is a word I created in contrast to single molecule, since the ON state, spontaneous emission molecules in STED is 50-80 diameter, thus certainly contains more than one molecule.
>>>
>>> Hope that helps,
>>> Peng Xi
>>> Ph. D. Associate Professor
>>> Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
>>> Email: [hidden email]
>>> http://xipeng.wordpress.com
>>>
>>> On Sun, Sep 2, 2012 at 4:03 AM, Seitz Arne <[hidden email]> wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> What exactly is a "mini laser" and a "multi-molecule"?
>>>>
>>>> Cheers Arne
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]] On Behalf Of Peng Xi
>>>> Sent: samedi 1 septembre 2012 01:55
>>>> To: [hidden email]
>>>> Subject: Re: STED detection
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear Alan,
>>>> I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
>>>> Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
>>>> Now come to STED, where you use mini laser and dichroic to have
>>>> somehow a multi-molecule with size of 50-80nm. By scanning you get
>>>> the high-resolution image.
>>>>
>>>>
>>>> Sincerely,
>>>> Peng Xi
>>>> Ph. D. Associate Professor
>>>> Dept. of Biomedical Engineering, College of Engineering Peking
>>>> University, Beijing, China
>>>> Email: [hidden email]
>>>> http://xipeng.wordpress.com
>>>>
>>>> On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith <[hidden email]> wrote:
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Dear list,
>>>>>
>>>>> Firstly, sorry if this is not confocally enough for this forum, but I
>>>>> hope someone would know. I was wondering if anyone could help with
>>>>> some a confusion I have about STED detection.
>>>>>
>>>>> I understand how the illumination psf is formed in STED using a
>>>>> depletion beam. However, if the emission is detected is collected in
>>>>> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>>>>>
>>>>> As an example, if a single molecule was producing fluorescence, the
>>>>> image will be an airy disk determined by the NA of the detecting
>>>>> objective and the wavelength of light. Despite the fluorescence
>>>>> solely coming from a single molecule.
>>>>>
>>>>> I know that for MP excitation the psf is solely determined by the
>>>>> illumination psf, but again, I am a little unsure as to the reason for this.
>>>>> Plus STED can obviously be CW also.
>>>>>
>>>>> Any help or suggestions would be brilliant!
>>>>> Much appreciated.
>>>>> Alan Smith
>>>
>
> Mark B. Cannell Ph.D. FRSNZ
> Professor of Cardiac Cell Biology
> School of Physiology& Pharmacology
> Medical Sciences Building
> University of Bristol
> Bristol
> BS8 1TD UK
>
> [hidden email]

George McNamara

Re: STED detection

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

Hi Peng,

STED usually operates at high resolution - now a volume around 40x40x200
nm (Z could be less by using more laser power in 3D-STED or 3D-EasySTED)
- so (I suggest) most useful to think about the single molecule case.
That is, one fluorophore is not much of a population, making population
inversion moot. I think it is also useful to think in terms of pulsed
excitation (ex. ~100 fs) and pulsed emission (ex. 300 ps starting a few
ps after the excitation pulse finishes), and a typical fluorophore
excited state lifetimes of ~3 ns (approximately the lifetime of Alexa
Fluor 488, EGFP, Venus).

Also, while STED operates by S.E.R., the same geometry (turn off
fluorophores in the peripheral American style doughnut), but very
different mechanism, applies to rsEGFP and Dreiklang as used by
Grotjohann et al 2011 and Brakemann et al 2011, respectively.

//

Speaking of single molecules, check out Steven Vogel's
VVVVVV </27813/> (Plasmid #27813)
Insert: Venus-5-Venus-5-Venus-5-Venus-5-Venus-5-Venus
http://www.addgene.org/27813/
Also has 5, 4, 3, and 2 repeats versions, plus C5V etc.
Reference: Nguyen TA, Sarkar P, Veetil JV, *Koushik SV*, Vogel SS.
Fluorescence polarization and fluctuation analysis monitors subunit
proximity, stoichiometry, and protein complex hydrodynamics.
</pubmed/22666486> PLoS One. 2012;7(5):e38209. PMID: 22666486.

Question for the listserv: can two or more of these Venus's be excited
at the same time?

Enjoy,

George
p.s. There is also a micro-time component to action in the STED doughnut
volume: an excited fluorophore might emit before a depletion wavelength
photon gets to it - hence the utility of time-gating STED (g-STED) to
clean up the pedastal. Nicely illustrated in Matthias Reuss's PhD
dissertation (near the end of the dissertation mentions that most of the
data used g-STED)
http://archiv.ub.uni-heidelberg.de/volltextserver/volltexte/2011/11539/pdf/Reuss_Dissertation.pdf
see also
http://works.bepress.com/gmcnamara/3/

On 9/3/2012 9:46 AM, Peng Xi wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Mark and De,
> I am totally lost to Mark's post. I agree with Guy that
> mini-laser is not a good term so let's change it to pump-probe. The
> population inversion is a statistical process which basically provides
> abundant electrons at excite state waiting for stimulated emission.
> Apparently this is not the case in a dye labelled biological specimen.
> So in STED, it is just stimulted emission dominated, pump-probe
> process. With negelegible increase of probe photon.
> We have not touch any ground state depletion here...
> Peng
>
> On Mon, Sep 3, 2012 at 5:14 PM, Mark Cannell<[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Why do you think there is a population inversion in the excited volume? In my opinion, this seems rather unlikely given the spill of the depletion beam to the centre and that saturation effects in the excitation beam could reduce resolution -so ground state depletion should be avoided. The fact that the centre of the STED PSF is not flat topped implies (to me) no ground state depletion.
>>
>> I think this introduction of the term 'laser" to the discussion of STED is not warranted (or correct -so I agree with Guy). Just because there is stimulated emission, does not imply lasing. Of course, it would be possible to achieve this but it would not be desirable IMHO.
>>
>> My 2c
>>
>> Cheers Mark
>>
>> On 2/09/2012, at 10:13 PM, "Chen, De (NIH/NCI) [C]"<[hidden email]> wrote:
>>
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Peng:
>>>
>>> For the two energy levels related to STED depletion, there is population inversion exists. So this kind of lasing is best termed the amplified spontaneous emission.
>>>
>>> De
>>>
>>> ________________________________________
>>> From: Peng Xi [[hidden email]]
>>> Sent: Saturday, September 01, 2012 9:31 PM
>>> To: [hidden email]
>>> Subject: Re: STED detection
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear Guy,
>>> If you consider the pump loses one photon, while the probe earns
>>> one photon, you know what I meant by amplification. Of course without
>>> population reversion constantly (the pump is so low), and absence of
>>> the reflective cavity, it is not a normal laser that we are familiar
>>> with. :)
>>> Peng
>>>
>>> On Sun, Sep 2, 2012 at 8:49 AM, Guy Cox<[hidden email]> wrote:
>>>
>>>> Really it isn't a mini-laser because there is no amplification - it's only when the emitted photon gives rise to further stimulated emission that you get amplification, and laser action.
>>>>
>>>> Fu-Jen Kao, in Taiwan, has done a lot of work on stimulated emisssion microscopy - it's useful because you know in what direction the emitted photon will be travelling. (This is nothing to do with STED, he's detecting the emitted photons, not spontaneous fluorescence).
>>>>
>>>> Guy
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peng Xi
>>>> Sent: Sunday, 2 September 2012 9:47 AM
>>>> To: [hidden email]
>>>> Subject: Re: STED detection
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear Arne,
>>>> Here "mini laser" referes to the stimulated emission depletion process happening at the peripherial of the excited fluorescence PSF.
>>>> It is indeed light amplification if taking the excitation laser as the pump source; and sometimes it is called pump-probe process.
>>>> "Multi-molecule" is a word I created in contrast to single molecule, since the ON state, spontaneous emission molecules in STED is 50-80 diameter, thus certainly contains more than one molecule.
>>>>
>>>> Hope that helps,
>>>> Peng Xi
>>>> Ph. D. Associate Professor
>>>> Dept. of Biomedical Engineering, College of Engineering Peking University, Beijing, China
>>>> Email: [hidden email]
>>>> http://xipeng.wordpress.com
>>>>
>>>> On Sun, Sep 2, 2012 at 4:03 AM, Seitz Arne<[hidden email]> wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> What exactly is a "mini laser" and a "multi-molecule"?
>>>>>
>>>>> Cheers Arne
>>>>>
>>>>> -----Original Message-----
>>>>> From: Confocal Microscopy List
>>>>> [mailto:[hidden email]] On Behalf Of Peng Xi
>>>>> Sent: samedi 1 septembre 2012 01:55
>>>>> To: [hidden email]
>>>>> Subject: Re: STED detection
>>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Dear Alan,
>>>>> I'd like to quote Paul Silven's solution on single molecule localization microscopy: "The trick to get around this problem was to realize that a SINGLE molecule's position can be located arbitrarily well. It's much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide."
>>>>> Once you understand that in a wide-field microscopy, you can determine the position of a single molecule extremely well, you can bring it to a scanning imaging mode. Take sample scan first because laser scan is equivalent. All you need to do is to set your scan step equals to sufficient sampling, so that you can accurately fit the Gaussian. The Nyquist-Shannon always holds true.
>>>>> Now come to STED, where you use mini laser and dichroic to have
>>>>> somehow a multi-molecule with size of 50-80nm. By scanning you get
>>>>> the high-resolution image.
>>>>>
>>>>>
>>>>> Sincerely,
>>>>> Peng Xi
>>>>> Ph. D. Associate Professor
>>>>> Dept. of Biomedical Engineering, College of Engineering Peking
>>>>> University, Beijing, China
>>>>> Email: [hidden email]
>>>>> http://xipeng.wordpress.com
>>>>>
>>>>> On Fri, Aug 31, 2012 at 8:22 PM, Alan Smith<[hidden email]> wrote:
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> *****
>>>>>>
>>>>>> Dear list,
>>>>>>
>>>>>> Firstly, sorry if this is not confocally enough for this forum, but I
>>>>>> hope someone would know. I was wondering if anyone could help with
>>>>>> some a confusion I have about STED detection.
>>>>>>
>>>>>> I understand how the illumination psf is formed in STED using a
>>>>>> depletion beam. However, if the emission is detected is collected in
>>>>>> epi-detection, why is the resolution not determined by the diffraction limit for the objective.
>>>>>>
>>>>>> As an example, if a single molecule was producing fluorescence, the
>>>>>> image will be an airy disk determined by the NA of the detecting
>>>>>> objective and the wavelength of light. Despite the fluorescence
>>>>>> solely coming from a single molecule.
>>>>>>
>>>>>> I know that for MP excitation the psf is solely determined by the
>>>>>> illumination psf, but again, I am a little unsure as to the reason for this.
>>>>>> Plus STED can obviously be CW also.
>>>>>>
>>>>>> Any help or suggestions would be brilliant!
>>>>>> Much appreciated.
>>>>>> Alan Smith
>>>>>>
>>>>
>> Mark B. Cannell Ph.D. FRSNZ
>> Professor of Cardiac Cell Biology
>> School of Physiology& Pharmacology
>> Medical Sciences Building
>> University of Bristol
>> Bristol
>> BS8 1TD UK
>>
>> [hidden email]
>>
>

Gitta Hamel

Re: STED detection

In reply to this post by Brian Northan

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

/Commercial response/

Dear Brian,

The Huygens software team has recently developed a STED deconvolution
option that works extremely well.

Have a look at http://www.svi.nl/STED

I'll be happy to give you off-line more background information why
deconvolution of STED data is so successful.

With kind wishes,

Gitta Hamel

/--
Managing Director Huygens SVI tel: +31 (0)35 642 16 26 fax: +31 (0)35
683 79 71 skype: gittahamel cell: +31(0)618 021272 Visiting address
Laapersveld 63, 1213 VB Hilversum, The Netherlands /

On 08/31/2012 05:22 PM, Brian Northan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> On a related note can anybody recommend a paper that explains how to
> calculate the STED PSF?
>
> Is it worth it to deconvolve this type of data? I've heard conflicting reports.
>
> On Fri, Aug 31, 2012 at 9:56 AM, Martin Wessendorf <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Alan--
>>
>>
>> On 8/31/2012 7:22 AM, Alan Smith wrote:
>>
>>> I understand how the illumination psf is formed in STED using a depletion
>>> beam. However, if the emission is detected is collected in epi-detection,
>>> why is the resolution not determined by the diffraction limit for the
>>> objective.
>>>
>>> As an example, if a single molecule was producing fluorescence, the image
>>> will be an airy disk determined by the NA of the detecting objective and
>>> the
>>> wavelength of light. Despite the fluorescence solely coming from a single
>>> molecule.
>>
>> You're right: with both a single molecule and with STED, you see a
>> diffraction-limited Airy disk on the emission side. However, with STED, you
>> know to a high degree of precision the position of the excitation beam
>> eliciting that Airy disk. Thus, as you scan that small excitation beam over
>> a small structure, you can obtain resolution that's many times better than
>> confocal. In that way, it's similar to near-field super-resolution methods.
>>
>> Hope that helps!
>>
>> Martin Wessendorf
>> --
>> Martin Wessendorf, Ph.D. office: (612) 626-0145
>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>> University of Minnesota Preferred FAX: (612) 624-8118
>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>> Minneapolis, MN 55455 e-mail: [hidden email]

--
Managing Director

Huygens SVI

tel: +31 (0)35 642 16 26

fax: +31 (0)35 683 79 71

skype: gittahamel

cell: +31(0)618 021272

Visiting address

Laapersveld 63,
1213 VB Hilversum,
The Netherlands