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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, I need your expertise to explain something to me. I am teaching 2nd year students a bit about microscopy and we alos have a part about STED. So we show how the donut laser changes the excitation volume of the STED microscope but then a bright student steps up and says: "This is all fine, however based on abbe's diffraction limit the fluorescence coming from that infinitely small exciation volum will diffract again and leave a similar sized fluorescent spot on your detector!" So i go check and find https://www.microscopyu.com/techniques/super-resolution/the-diffraction-barrier-in-optical-microscopy, which says: "A point object in a microscope, such as a fluorescent protein single molecule, generates an image at the intermediate plane that consists of a diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings" So if this is true, why does STED then create super resolution images? He got me confused there and haven't been able to clarify it. I need help! Joost |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Conceptually this is how I understand it: Indeed, even after the depletion laser's donut mask alters the wavelength of the emission from your fluorophore (under the mask), it's still diffracted. Yet the wavelength of the "rings", or outer parts of the diffraction pattern, are then filtered out by the fluorifier disk (filters). So now we don't see it, rather we only see the center part which is smaller than the original diffraction limited airy disk, resulting in improved resolution. If I got this wrong, I am sure someone will correct me :) -- Avi Jacob, Ph.D. Head of Light Microscopy The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 5290002, Israel Cell: 052-5802544 (call here first), Desk: 972-3-5317647 http://tinyurl.com/BIU-Microscopy On Tue, Jan 24, 2017 at 6:20 PM, Joost Willemse <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi all, > > I need your expertise to explain something to me. I am teaching 2nd year > students a bit about microscopy and we alos have a part about STED. > > So we show how the donut laser changes the excitation volume of the STED > microscope but then a bright student steps up and says: > > "This is all fine, however based on abbe's diffraction limit the > fluorescence coming from that infinitely small exciation volum will > diffract again and leave a similar sized fluorescent spot on your > detector!" > > So i go check and find > https://www.microscopyu.com/techniques/super-resolution/ > the-diffraction-barrier-in-optical-microscopy, > which says: > > "A point object in a microscope, such as a fluorescent protein single > molecule, generates an image at the intermediate plane that consists of a > diffraction pattern created by the action of interference. When highly > magnified, the diffraction pattern of the point object is observed to > consist of a central spot (diffraction disk) surrounded by a series of > diffraction rings" > > So if this is true, why does STED then create super resolution images? > He got me confused there and haven't been able to clarify it. > > I need help! > > Joost > |
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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Joost, At the serious risk of being wrong in front of everyone on the confocal list, I would offer the following explanation. I have tried my best to formulate an explanation suitable for your students, but I defer to the expert STED users on the list to correct me if I've made an error :) While it's true that the resulting fluorescence coming from the same region as the intensity null produces a diffraction-limited image in the detector plane, we have additional information we can apply to our measurements. Namely, we know with a precision greater than the diffraction limit where the null is located and that the source of the fluorescence is coming from this region. In many cases (not just microscopy), we can combine measurements with additional assumptions and other pieces of information to arrive at conclusions with a better accuracy or precision than we could if we based our conclusions on the measurements alone. I hope this helps, Kyle Dr. Kyle M. Douglass Post-doctoral Researcher EPFL - The Laboratory of Experimental Biophysics http://leb.epfl.ch/ http://kmdouglass.github.io ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of Joost Willemse [[hidden email]] Sent: Tuesday, January 24, 2017 5:20 PM To: [hidden email] Subject: STED ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, I need your expertise to explain something to me. I am teaching 2nd year students a bit about microscopy and we alos have a part about STED. So we show how the donut laser changes the excitation volume of the STED microscope but then a bright student steps up and says: "This is all fine, however based on abbe's diffraction limit the fluorescence coming from that infinitely small exciation volum will diffract again and leave a similar sized fluorescent spot on your detector!" So i go check and find https://www.microscopyu.com/techniques/super-resolution/the-diffraction-barrier-in-optical-microscopy, which says: "A point object in a microscope, such as a fluorescent protein single molecule, generates an image at the intermediate plane that consists of a diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings" So if this is true, why does STED then create super resolution images? He got me confused there and haven't been able to clarify it. I need help! Joost |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi, I could also be inaccurate here, but I always thought of STED resolution in similar terms as near field super-resolution or NSOM. It is quite simply that if your field of view is 100nm then your effective resolution is 100nm. In the center of the donut in STED is 100nm then your effective resolution is 100nm. So you are limited by your aperture size and not the wavelength; think of Rayleigh criterion rather than the Abbe limit here. Cheers, Brian D Armstrong PhD Light Microscopy Core Facility -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kyle Michael Douglass Sent: Tuesday, January 24, 2017 9:04 AM To: [hidden email] Subject: Re: STED ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Joost, At the serious risk of being wrong in front of everyone on the confocal list, I would offer the following explanation. I have tried my best to formulate an explanation suitable for your students, but I defer to the expert STED users on the list to correct me if I've made an error :) While it's true that the resulting fluorescence coming from the same region as the intensity null produces a diffraction-limited image in the detector plane, we have additional information we can apply to our measurements. Namely, we know with a precision greater than the diffraction limit where the null is located and that the source of the fluorescence is coming from this region. In many cases (not just microscopy), we can combine measurements with additional assumptions and other pieces of information to arrive at conclusions with a better accuracy or precision than we could if we based our conclusions on the measurements alone. I hope this helps, Kyle Dr. Kyle M. Douglass Post-doctoral Researcher EPFL - The Laboratory of Experimental Biophysics http://leb.epfl.ch/ http://kmdouglass.github.io ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of Joost Willemse [[hidden email]] Sent: Tuesday, January 24, 2017 5:20 PM To: [hidden email] Subject: STED ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, I need your expertise to explain something to me. I am teaching 2nd year students a bit about microscopy and we alos have a part about STED. So we show how the donut laser changes the excitation volume of the STED microscope but then a bright student steps up and says: "This is all fine, however based on abbe's diffraction limit the fluorescence coming from that infinitely small exciation volum will diffract again and leave a similar sized fluorescent spot on your detector!" So i go check and find https://www.microscopyu.com/techniques/super-resolution/the-diffraction-barrier-in-optical-microscopy, which says: "A point object in a microscope, such as a fluorescent protein single molecule, generates an image at the intermediate plane that consists of a diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings" So if this is true, why does STED then create super resolution images? He got me confused there and haven't been able to clarify it. I need help! 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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Joost This question can be answered with basic image formation theory: The overall PSF of a confocal microscope is given by the product of the excitation PSF and the detection PSF. In case of a STED microscope, the excitation PSF is very narrow after the stimulated depletion. So when you multiply it with the coarse detection PSF, the finer width of the excitation PSF will dominate the final PSF. Or if you prefer reciprocal space, there you convolve a function with small support (detection OTF) with a function with a very large support. The resulting overall OTF will have a large support, even though the detection one was small. By the way, a scanning microscope does not need to be image forming on the detection side. Think about a two photon microscope in very turbid media (say brain tissue). The emission light can be totally diffuse and may not form any sharp image on the detector. In this case the image is the convolution of the sample’s fluorophore distribution convolved with the excitation PSF. The smaller the excitation PSF is (i.e. the laser focus), the better the resolution. In this regime image forming in the emission light path does not matter. Sincerely, Reto |
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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi, STED is a laser scanning confocal - you're not "imaging the fluorescence spot on your detector". There is just a PMT that measures how much light comes from the spot. The size of the spot is not important (well, still there is the pinhole, but it's role is to suppress significantly-out-of- focus light, just like in normal confocal). Simply the fact that your detected fluorescence comes from very small sub- diffraction volume is what you need to get superresolution image. Maybe another helpful note: in widefield fluorescence the resolution is governed by the emission wavelength, but in confocal it's the exctitation wavelength, that is, the resolution is given by the size of the laser spot you are able to create inside your sample... Best, zdenek -- Zdenek Svindrych, Ph.D. W.M. Keck Center for Cellular Imaging (PLSB 003) University of Virginia, Charlottesville, VA http://www.kcci.virginia.edu/ tel: 434-982-4869 Annual FRET Workshop: http://kcci.virginia.edu/workshop-2017 ---------- Původní zpráva ---------- Od: Joost Willemse <[hidden email]> Komu: [hidden email] Datum: 24. 1. 2017 11:33:06 Předmět: STED "***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, I need your expertise to explain something to me. I am teaching 2nd year students a bit about microscopy and we alos have a part about STED. So we show how the donut laser changes the excitation volume of the STED microscope but then a bright student steps up and says: "This is all fine, however based on abbe's diffraction limit the fluorescence coming from that infinitely small exciation volum will diffract again and leave a similar sized fluorescent spot on your detector!" So i go check and find https://www.microscopyu.com/techniques/super-resolution/the-diffraction- barrier-in-optical-microscopy, which says: "A point object in a microscope, such as a fluorescent protein single molecule, generates an image at the intermediate plane that consists of a diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings" So if this is true, why does STED then create super resolution images? He got me confused there and haven't been able to clarify it. I need help! Joost " |
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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Indeed all things optical are diffraction-limited, even in a STED microscope. The extra amount of information we have is the scanner position, ie, where the doughnut center is. So, you don't really care that the photons coming out of the effective fluorescence source will be blurred when propagating towards the detector. All you need is i)to have sub-diffraction level resolution at the scanner and, for things to be any relevant, ii) to have a non-linear depletion mechanism at the sample so that this effective source is below diffraction level, effectively transferring the limits to the wavelengths at 'the sample' ... de Broglie wavelengths and stuff. I guess we could say that both these two requirements run away from photons, asking help from electrons wavelengths ;-) I hope I'm correct. The principle of STED is really cool. António Pereira, i3S - Universidade do Porto -----Confocal Microscopy List <[hidden email]> escreveu: ----- Para: [hidden email] De: Joost Willemse Enviado por: Confocal Microscopy List Data: 01/24/2017 04:36PM Assunto: STED ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, I need your expertise to explain something to me. I am teaching 2nd year students a bit about microscopy and we alos have a part about STED. So we show how the donut laser changes the excitation volume of the STED microscope but then a bright student steps up and says: "This is all fine, however based on abbe's diffraction limit the fluorescence coming from that infinitely small exciation volum will diffract again and leave a similar sized fluorescent spot on your detector!" So i go check and find https://www.microscopyu.com/techniques/super-resolution/the-diffraction-barrier-in-optical-microscopy, which says: "A point object in a microscope, such as a fluorescent protein single molecule, generates an image at the intermediate plane that consists of a diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings" So if this is true, why does STED then create super resolution images? He got me confused there and haven't been able to clarify it. I need help! Joost |
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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Once you know that the diffraction limited light all comes from a very small spot, it does not matter whether it spreads out on your detector -- you still know it came from that 50nm (for example) spot. Now you must scan this tiny spot in across your surface to build up an image, and you end up with (for example) 1024x1024 spots, each imaged separately. You cannot image them all at the same time because (as your student observed), their images will overlap. However, you can image multiple points if they are separated enough (more than their diffraction), and soe systems do that. Hope that helps. --aryeh On 24/01/2017 18:20, Joost Willemse wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi all, > > I need your expertise to explain something to me. I am teaching 2nd year > students a bit about microscopy and we alos have a part about STED. > > So we show how the donut laser changes the excitation volume of the STED > microscope but then a bright student steps up and says: > > "This is all fine, however based on abbe's diffraction limit the > fluorescence coming from that infinitely small exciation volum will > diffract again and leave a similar sized fluorescent spot on your detector!" > > So i go check and find > https://www.microscopyu.com/techniques/super-resolution/the-diffraction-barrier-in-optical-microscopy, > which says: > > "A point object in a microscope, such as a fluorescent protein single > molecule, generates an image at the intermediate plane that consists of a > diffraction pattern created by the action of interference. When highly > magnified, the diffraction pattern of the point object is observed to > consist of a central spot (diffraction disk) surrounded by a series of > diffraction rings" > > So if this is true, why does STED then create super resolution images? > He got me confused there and haven't been able to clarify it. > > I need help! > > Joost > . > -- Aryeh Weiss Faculty of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384051 |
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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Joost, In STED microscopy - exactly as in confocal microscopy - an excitation volume is scanned over the sample and for each position the according fluorescence intensity is recorded for the focal plane while the pinhole rejects out of focus light. The trick in STED microscopy is shrinking of the effective volume scanned = the area from which fluorescence can derive from. It is reduced to the center of the donut - where no STED light is present. In the area where there is sufficient STED light to suppress the fluorophores ability to emit photons, no signal is obtained. This way a sub-diffraction size volume (= effective excitation PSF) is achieved. An a super-resolved image built up pixel by pixel scanning it over the sample. Tutorials explaining the principle and further background can be found on the Leica Science Lab. E.g. http://www.leica-microsystems.com/science-lab/sted-super-resolution-microscopy-nanoscopy-principles-and-photophysics/ Mit freundlichen Grüßen/Regards Dr. Jochen Sieber Head Product Management Confocal Advanced [hidden email] T +49 621 7028 2709 | F +49 621 7028 1180 Leica Microsystems CMS GmbH Am Friedensplatz 3 | 68165 Mannheim (Germany) Sign-up for the Latest News at Leica-Microsystems.com/Subscribe Leica Microsystems CMS GmbH mit Sitz in Wetzlar | Amtsgericht Wetzlar HRB 2432 Geschäftsführer: Markus Lusser | Melanie Hammerschmidt-Borman | Fedja Bobanovic www.leica-microsystems.com -----Ursprüngliche Nachricht----- Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Joost Willemse Gesendet: Dienstag, 24. Januar 2017 17:21 An: [hidden email] Betreff: STED ---------------------- Information from the mail header ----------------------- Sender: Confocal Microscopy List <[hidden email]> Poster: Joost Willemse <[hidden email]> Subject: STED ------------------------------------------------------------------------------- ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3D3Dconfocalmicroscopy&d=DwIBAg&c=9mghv0deYPYDGP-W745IEdQLV1kHpn4XJRvR6xMRXtA&r=FQa40Y8wSE-XROHPaeLBWL10rvYCjOcVAdNo0UCWEt7KAy97vhNk4joheEtMD3Fk&m=wn4WhQb1jSDx51-l9QPwrqOeg7vyXp9AJrXcWd1uSzE&s=LRIwTzlQtpt4Ov6fW9ljSBk5gveTNS2JZ4l-LKgFpW4&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBAg&c=9mghv0deYPYDGP-W745IEdQLV1kHpn4XJRvR6xMRXtA&r=FQa40Y8wSE-XROHPaeLBWL10rvYCjOcVAdNo0UCWEt7KAy97vhNk4joheEtMD3Fk&m=wn4WhQb1jSDx51-l9QPwrqOeg7vyXp9AJrXcWd1uSzE&s=iJuNyjFwhBqdCgsT2tqmhPJ51tz4nizEhBU0r4ZRmb4&e= and include the link in your posting. ***** Hi all, I need your expertise to explain something to me. I am teaching 2nd year students a bit about microscopy and we alos have a part about STED. So we show how the donut laser changes the excitation volume of the STED microscope but then a bright student steps up and says: "This is all fine, however based on abbe's diffraction limit the fluorescence coming from that infinitely small exciation volum will diffract again and leave a similar sized fluorescent spot on your detecto= r!" So i go check and find https://urldefense.proofpoint.com/v2/url?u=https-3A__www.microscopyu.com_techniques_super-2Dresolution_the-2Ddiffraction-2Db-3D&d=DwIBAg&c=9mghv0deYPYDGP-W745IEdQLV1kHpn4XJRvR6xMRXtA&r=FQa40Y8wSE-XROHPaeLBWL10rvYCjOcVAdNo0UCWEt7KAy97vhNk4joheEtMD3Fk&m=wn4WhQb1jSDx51-l9QPwrqOeg7vyXp9AJrXcWd1uSzE&s=8rxnvF-CYCeWro9Ts2RLWK6P0KarCCyQaqU8sDajZOg&e= arrier-in-optical-microscopy, which says: "A point object in a microscope, such as a fluorescent protein single molecule, generates an image at the intermediate plane that consists of a= diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings" So if this is true, why does STED then create super resolution images? He got me confused there and haven't been able to clarify it. I need help! Joost Please be advised that this email may contain confidential information. If you are not the intended recipient, please notify us by email by replying to the sender and delete this message. The sender disclaims that the content of this email constitutes an offer to enter into, or the acceptance of, any agreement; provided that the foregoing does not invalidate the binding effect of any digital or other electronic reproduction of a manual signature that is included in any attachment. |
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In reply to this post by Joost Willemse-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Joost, This is probably a question everyone stumbles across sooner or later when thinking about the mechanisms behind STED. It is correct that the effective volume of the excitation spot is shrunk by the STED beam. In other words, the region from which spontaneous fluorescence is allowed is much smaller than the original diffraction-limited excitation volume. Also, your student is absolutely correct that the image of this reduced volume on the detector is again diffraction limited. The solution to your question lies in realizing that it doesn’t matter! It’s often helpful to picture an extreme situation, so let’s imagine that we are able to reduce the effective focal volume to a very, very small point. Fluorescence from a molecule located at this point will propagate back through the optical system and end up diffracted on the detector, spread out and with Airy rings and all. However, what we’re interested in is not the image of the molecule per se, but we'd like to know *where* the molecule is. The punchline is that with STED, we are able to get an accurate fix on the molecule, simply because we know the position of the zero point of the STED-PSF. For example, if we do beam scanning, we know where we’re currently pointing the beam, we therefore know where the intensity zero of the STED-donut is, and we therefore know that the molecule can only be at this very place. If it was at another place just a few nanometers away, the molecule would see a non-zero STED intensity and would currently not be able to emit. Without STED, we could only know that it is somewhere within an approx. 200 nm region, the size of the diffraction-limited excitation distribution. All this is 100% independent of the image of the molecule on the detector and whether it is diffraction-limited there or not. Hope this helps. Best, Matthias Matthias Reuss, Dr. Head of Marketing & Sales Abberior Instruments GmbH Hans-Adolf-Krebs-Weg 1 37077 Goettingen Germany phone: +49 (551) 30724 175 fax: +49 (551) 30724 171 http://www.abberior-instruments.com mailto:[hidden email] Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen HRB 201844 | VAT Reg. No.: DE 283588727 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Since this came up some months ago, I was thinking about another way to explain this than my original answer. Indeed some of the smartest STED people on this list also answered, but I think I still have something to add, to conceptually explain scanning confocal resolution and how STED improves it. What I was still ultimately missing here, was how does all the above explain improved resolution between two small points (that are themselves physically under the diffraction limit size), yet are sitting closer to each other than the diffraction limit, and as mentioned, are raster scanned, and whose emissions are not captured "complete" by a camera. If, as mentioned above correctly, the signal is being detected by a PMT from a single point, and I define my pixel as say 150x150nm, which is under the diffraction limit, why do we have a problem to begin with? Well, it's clear - the laser spot itself is also diffracted and is larger than my defined pixel. Thus, if I am exciting one fluorescent point (A), and let's assume that the center of its airy disk is in the center of my defined pixel, and I am detecting the emission from (A) in that pixel, the value I am assigning to the intensity (X) from that pixel, as a result of laser excitation at that one spot, also includes light from the adjacent point (B) that I am trying to resolve from (A). Why? Because (B) is also getting excited (by the diffracted laser spot), is also emitting light, and so (X) is actually the sum of the value of the emission from (A) and the emission from the edges (or closer) of the diffraction pattern (or rings), from the point (B). As the scanning continues, the next pixel will also have light from both (B) and (A). Etc. Thus we have no dip in signal (value assigned to each pixel) and thus cannot resolve (A) from (B). In STED, as mentioned above, we are essentially removing the light from the outskirts of the center point we are detecting, by the mechanism of stimulated depletion, which, as I mentioned in my first post, effectively turns the light under the donut mask into the same wavelength as the depletion laser and is then filtered out ON THE EMISSION SIDE of the light pathway on the way to the detector. Thus, now, the value of (X) only includes light from the center of the airy disk of my currently targeted fluorescent point. Why? Because the light from the edges of (B), having been "STED'd", although is also on the way to the detector, IS FILTERED OUT, and thus does not reach the PMT detector and thus is not being summed to the total value of (X). So to continue this, the next pixel, say in between the two points (A) and (B), can now show a dip in value, because the light from the outer parts of the airy disks of (A) and (B), which would normally reach the detector and be assigned an intensity value that is the reason we *cannot *normally resolve (A) from (B), *is also getting filtered out, *resulting in the dip in intensity values between defined pixels (A) and (B), which gives us superresolution. This is, of course, why you need to have a sufficient number of defined pixels, to be able to SEE the dip in intensity value between (A) and (B). Avi -- Avi Jacob, Ph.D. Head of Light Microscopy The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 5290002, Israel Cell: 052-5802544 (call here first), Desk: 972-3-5317647 http://tinyurl.com/BIU-Microscopy |
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In reply to this post by Matthias Reuss
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear coleagues, This topic has started a nice discussion in our staff Facility. The STED effect, has to be considered in the excitation or the emission volume? Which is the right phrase in STED definition? a) The improving in lateral an axial resolution is provided by reducing the volume of the _emission_ by the fenomenon of stimulated emission. b) The improving in lateral an axial resolution is provided by reducing the volume of the _excitation_ by the fenomenon of stimulated emission. Basically, the term STED is lacking some other explanations behind. Does someone has a better one than "Stimulated Emission Depletion"? Thank you very much in advance. Best regards Juan Luis El 31/01/2017 a las 16:09, Matthias Reuss escribió: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear Joost, > > This is probably a question everyone stumbles across sooner or later when thinking about the mechanisms behind STED. > > It is correct that the effective volume of the excitation spot is shrunk by the STED beam. In other words, the region from which spontaneous fluorescence is allowed is much smaller than the original diffraction-limited excitation volume. Also, your student is absolutely correct that the image of this reduced volume on the detector is again diffraction limited. > > The solution to your question lies in realizing that it doesn’t matter! > > It’s often helpful to picture an extreme situation, so let’s imagine that we are able to reduce the effective focal volume to a very, very small point. Fluorescence from a molecule located at this point will propagate back through the optical system and end up diffracted on the detector, spread out and with Airy rings and all. However, what we’re interested in is not the image of the molecule per se, but we'd like to know *where* the molecule is. > > The punchline is that with STED, we are able to get an accurate fix on the molecule, simply because we know the position of the zero point of the STED-PSF. For example, if we do beam scanning, we know where we’re currently pointing the beam, we therefore know where the intensity zero of the STED-donut is, and we therefore know that the molecule can only be at this very place. If it was at another place just a few nanometers away, the molecule would see a non-zero STED intensity and would currently not be able to emit. Without STED, we could only know that it is somewhere within an approx. 200 nm region, the size of the diffraction-limited excitation distribution. > > All this is 100% independent of the image of the molecule on the detector and whether it is diffraction-limited there or not. > > Hope this helps. > > Best, > Matthias > > > Matthias Reuss, Dr. > Head of Marketing & Sales > Abberior Instruments GmbH > Hans-Adolf-Krebs-Weg 1 > 37077 Goettingen > Germany > > phone: +49 (551) 30724 175 > fax: +49 (551) 30724 171 > http://www.abberior-instruments.com > mailto:[hidden email] > > Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen HRB 201844 | VAT Reg. No.: DE 283588727 > -- Juan Luis Ribas Servicio de Microscopía Centro de Investigación, Tecnología e Innovación Universidad de Sevilla Av. Reina Mercedes 4b 41012 Sevilla Spain |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** You are reducing the emission volume, not preventing excitation. In practice you excite a gaussian-ish spatial volume, then deplete with an annular pattern (+'barbell' for 3D) before the molecules can emit. Craig On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> wrote: > ***** > > This topic has started a nice discussion in our staff Facility. The STED > effect, has to be considered in the excitation or the emission volume? > > Which is the right phrase in STED definition? > > a) The improving in lateral an axial resolution is provided by reducing > the volume of the _emission_ by the fenomenon of stimulated emission. > > b) The improving in lateral an axial resolution is provided by reducing > the volume of the _excitation_ by the fenomenon of stimulated emission. > > Basically, the term STED is lacking some other explanations behind. Does > someone has a better one than "Stimulated Emission Depletion"? > > Thank you very much in advance. > > Best regards > > Juan Luis > > > El 31/01/2017 a las 16:09, Matthias Reuss escribió: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Dear Joost, >> >> This is probably a question everyone stumbles across sooner or later when >> thinking about the mechanisms behind STED. >> >> It is correct that the effective volume of the excitation spot is shrunk >> by the STED beam. In other words, the region from which spontaneous >> fluorescence is allowed is much smaller than the original >> diffraction-limited excitation volume. Also, your student is absolutely >> correct that the image of this reduced volume on the detector is again >> diffraction limited. >> >> The solution to your question lies in realizing that it doesn’t matter! >> >> It’s often helpful to picture an extreme situation, so let’s imagine that >> we are able to reduce the effective focal volume to a very, very small >> point. Fluorescence from a molecule located at this point will propagate >> back through the optical system and end up diffracted on the detector, >> spread out and with Airy rings and all. However, what we’re interested in >> is not the image of the molecule per se, but we'd like to know *where* the >> molecule is. >> >> The punchline is that with STED, we are able to get an accurate fix on >> the molecule, simply because we know the position of the zero point of the >> STED-PSF. For example, if we do beam scanning, we know where we’re >> currently pointing the beam, we therefore know where the intensity zero of >> the STED-donut is, and we therefore know that the molecule can only be at >> this very place. If it was at another place just a few nanometers away, the >> molecule would see a non-zero STED intensity and would currently not be >> able to emit. Without STED, we could only know that it is somewhere within >> an approx. 200 nm region, the size of the diffraction-limited excitation >> distribution. >> >> All this is 100% independent of the image of the molecule on the detector >> and whether it is diffraction-limited there or not. >> >> Hope this helps. >> >> Best, >> Matthias >> >> >> Matthias Reuss, Dr. >> Head of Marketing & Sales >> Abberior Instruments GmbH >> Hans-Adolf-Krebs-Weg 1 >> 37077 Goettingen >> Germany >> >> phone: +49 (551) 30724 175 >> fax: +49 (551) 30724 171 >> http://www.abberior-instruments.com >> mailto:[hidden email] >> >> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen HRB >> 201844 | VAT Reg. No.: DE 283588727 >> >> > -- > Juan Luis Ribas > Servicio de Microscopía > Centro de Investigación, Tecnología e Innovación > Universidad de Sevilla > Av. Reina Mercedes 4b > 41012 Sevilla > Spain > |
 
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George McNamara |
Re: AW: STED ... there are two different emissions in STED experiments
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** neither a nor b, since emission are emissions. There are two different emissions in STED experiments: 1. stimulated emission at exactly the wavelength of the depletion laser wavelength. 2. fluorescence emission, detected at whatever wavelength range the light path to the detector allows. enjoy, George On 11/8/2017 12:44 PM, Craig Brideau wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > You are reducing the emission volume, not preventing excitation. In > practice you excite a gaussian-ish spatial volume, then deplete with an > annular pattern (+'barbell' for 3D) before the molecules can emit. > > Craig > > On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> wrote: > >> ***** >> >> This topic has started a nice discussion in our staff Facility. The STED >> effect, has to be considered in the excitation or the emission volume? >> >> Which is the right phrase in STED definition? >> >> a) The improving in lateral an axial resolution is provided by reducing >> the volume of the _emission_ by the fenomenon of stimulated emission. >> >> b) The improving in lateral an axial resolution is provided by reducing >> the volume of the _excitation_ by the fenomenon of stimulated emission. >> >> Basically, the term STED is lacking some other explanations behind. Does >> someone has a better one than "Stimulated Emission Depletion"? >> >> Thank you very much in advance. >> >> Best regards >> >> Juan Luis >> >> >> El 31/01/2017 a las 16:09, Matthias Reuss escribió: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your posting. >>> ***** >>> >>> Dear Joost, >>> >>> This is probably a question everyone stumbles across sooner or later when >>> thinking about the mechanisms behind STED. >>> >>> It is correct that the effective volume of the excitation spot is shrunk >>> by the STED beam. In other words, the region from which spontaneous >>> fluorescence is allowed is much smaller than the original >>> diffraction-limited excitation volume. Also, your student is absolutely >>> correct that the image of this reduced volume on the detector is again >>> diffraction limited. >>> >>> The solution to your question lies in realizing that it doesn’t matter! >>> >>> It’s often helpful to picture an extreme situation, so let’s imagine that >>> we are able to reduce the effective focal volume to a very, very small >>> point. Fluorescence from a molecule located at this point will propagate >>> back through the optical system and end up diffracted on the detector, >>> spread out and with Airy rings and all. However, what we’re interested in >>> is not the image of the molecule per se, but we'd like to know *where* the >>> molecule is. >>> >>> The punchline is that with STED, we are able to get an accurate fix on >>> the molecule, simply because we know the position of the zero point of the >>> STED-PSF. For example, if we do beam scanning, we know where we’re >>> currently pointing the beam, we therefore know where the intensity zero of >>> the STED-donut is, and we therefore know that the molecule can only be at >>> this very place. If it was at another place just a few nanometers away, the >>> molecule would see a non-zero STED intensity and would currently not be >>> able to emit. Without STED, we could only know that it is somewhere within >>> an approx. 200 nm region, the size of the diffraction-limited excitation >>> distribution. >>> >>> All this is 100% independent of the image of the molecule on the detector >>> and whether it is diffraction-limited there or not. >>> >>> Hope this helps. >>> >>> Best, >>> Matthias >>> >>> >>> Matthias Reuss, Dr. >>> Head of Marketing & Sales >>> Abberior Instruments GmbH >>> Hans-Adolf-Krebs-Weg 1 >>> 37077 Goettingen >>> Germany >>> >>> phone: +49 (551) 30724 175 >>> fax: +49 (551) 30724 171 >>> http://www.abberior-instruments.com >>> mailto:[hidden email] >>> >>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen HRB >>> 201844 | VAT Reg. No.: DE 283588727 >>> >>> >> -- >> Juan Luis Ribas >> Servicio de Microscopía >> Centro de Investigación, Tecnología e Innovación >> Universidad de Sevilla >> Av. Reina Mercedes 4b >> 41012 Sevilla >> Spain >> -- George McNamara, PhD Baltimore, MD 21231 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome) http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 http://confocal.jhu.edu July 2017 Current Protocols article, open access: UNIT 4.4 Microscopy and Image Analysis http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract supporting materials direct link is http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 figures at http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures |
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Craig Brideau |
Re: AW: STED ... there are two different emissions in STED experiments
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Very good point George, although I doubt most people would notice the stimulated emission at the same wavelength as the depletion laser. That said, I'm sure there are applications for pump-probe experiments that would take advantage of the driven emission using lock-in amplification or the like. It's funny to think of the fluorophore as an optical gain medium, but here we are! Craig On Wed, Nov 8, 2017 at 6:53 PM, George McNamara <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > neither a nor b, since emission are emissions. > > There are two different emissions in STED experiments: > > 1. stimulated emission at exactly the wavelength of the depletion laser > wavelength. > > 2. fluorescence emission, detected at whatever wavelength range the light > path to the detector allows. > > enjoy, > > George > > > On 11/8/2017 12:44 PM, Craig Brideau wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> You are reducing the emission volume, not preventing excitation. In >> practice you excite a gaussian-ish spatial volume, then deplete with an >> annular pattern (+'barbell' for 3D) before the molecules can emit. >> >> Craig >> >> On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> wrote: >> >> ***** >>> >>> This topic has started a nice discussion in our staff Facility. The STED >>> effect, has to be considered in the excitation or the emission volume? >>> >>> Which is the right phrase in STED definition? >>> >>> a) The improving in lateral an axial resolution is provided by reducing >>> the volume of the _emission_ by the fenomenon of stimulated emission. >>> >>> b) The improving in lateral an axial resolution is provided by reducing >>> the volume of the _excitation_ by the fenomenon of stimulated emission. >>> >>> Basically, the term STED is lacking some other explanations behind. Does >>> someone has a better one than "Stimulated Emission Depletion"? >>> >>> Thank you very much in advance. >>> >>> Best regards >>> >>> Juan Luis >>> >>> >>> El 31/01/2017 a las 16:09, Matthias Reuss escribió: >>> >>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> Post images on http://www.imgur.com and include the link in your >>>> posting. >>>> ***** >>>> >>>> Dear Joost, >>>> >>>> This is probably a question everyone stumbles across sooner or later >>>> when >>>> thinking about the mechanisms behind STED. >>>> >>>> It is correct that the effective volume of the excitation spot is shrunk >>>> by the STED beam. In other words, the region from which spontaneous >>>> fluorescence is allowed is much smaller than the original >>>> diffraction-limited excitation volume. Also, your student is absolutely >>>> correct that the image of this reduced volume on the detector is again >>>> diffraction limited. >>>> >>>> The solution to your question lies in realizing that it doesn’t matter! >>>> >>>> It’s often helpful to picture an extreme situation, so let’s imagine >>>> that >>>> we are able to reduce the effective focal volume to a very, very small >>>> point. Fluorescence from a molecule located at this point will propagate >>>> back through the optical system and end up diffracted on the detector, >>>> spread out and with Airy rings and all. However, what we’re interested >>>> in >>>> is not the image of the molecule per se, but we'd like to know *where* >>>> the >>>> molecule is. >>>> >>>> The punchline is that with STED, we are able to get an accurate fix on >>>> the molecule, simply because we know the position of the zero point of >>>> the >>>> STED-PSF. For example, if we do beam scanning, we know where we’re >>>> currently pointing the beam, we therefore know where the intensity zero >>>> of >>>> the STED-donut is, and we therefore know that the molecule can only be >>>> at >>>> this very place. If it was at another place just a few nanometers away, >>>> the >>>> molecule would see a non-zero STED intensity and would currently not be >>>> able to emit. Without STED, we could only know that it is somewhere >>>> within >>>> an approx. 200 nm region, the size of the diffraction-limited excitation >>>> distribution. >>>> >>>> All this is 100% independent of the image of the molecule on the >>>> detector >>>> and whether it is diffraction-limited there or not. >>>> >>>> Hope this helps. >>>> >>>> Best, >>>> Matthias >>>> >>>> >>>> Matthias Reuss, Dr. >>>> Head of Marketing & Sales >>>> Abberior Instruments GmbH >>>> Hans-Adolf-Krebs-Weg 1 >>>> 37077 Goettingen >>>> Germany >>>> >>>> phone: +49 (551) 30724 175 >>>> fax: +49 (551) 30724 171 >>>> http://www.abberior-instruments.com >>>> mailto:[hidden email] >>>> >>>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen HRB >>>> 201844 | VAT Reg. No.: DE 283588727 >>>> >>>> >>>> -- >>> Juan Luis Ribas >>> Servicio de Microscopía >>> Centro de Investigación, Tecnología e Innovación >>> Universidad de Sevilla >>> Av. Reina Mercedes 4b >>> 41012 Sevilla >>> Spain >>> >>> > -- > > > George McNamara, PhD > Baltimore, MD 21231 > [hidden email] > https://www.linkedin.com/in/georgemcnamara > https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge > or Firefox, rather than Google Chrome) > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 > http://confocal.jhu.edu > > July 2017 Current Protocols article, open access: > UNIT 4.4 Microscopy and Image Analysis > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract > supporting materials direct link is > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 > figures at > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures > |
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Andrew York |
Re: AW: STED ... there are two different emissions in STED experiments
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Craig's point is a good excuse to bring up one of my favorite physics questions: if you use a plane wave to stimulate a pointlike fluorophore to emit, is the fluorophore's emission a plane wave, or a spherical* outgoing wave? Other ways to phrase the question include: * Does stimulated emission from an isolated pointlike emitter carry information about the emitter location? * Can we use lenses to form images with stimulated emission? We showed some results at the last Focus on Microscopy: https://goo.gl/9Sr812 ...and I think there's lots of fun follow-up questions. *dipole-shaped, of course, not spherically symmetric, but you know what I mean. On Wed, Nov 8, 2017 at 9:10 PM, Craig Brideau <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Very good point George, although I doubt most people would notice the > stimulated emission at the same wavelength as the depletion laser. That > said, I'm sure there are applications for pump-probe experiments that would > take advantage of the driven emission using lock-in amplification or the > like. It's funny to think of the fluorophore as an optical gain medium, but > here we are! > > Craig > > On Wed, Nov 8, 2017 at 6:53 PM, George McNamara <[hidden email] > > > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > neither a nor b, since emission are emissions. > > > > There are two different emissions in STED experiments: > > > > 1. stimulated emission at exactly the wavelength of the depletion laser > > wavelength. > > > > 2. fluorescence emission, detected at whatever wavelength range the light > > path to the detector allows. > > > > enjoy, > > > > George > > > > > > On 11/8/2017 12:44 PM, Craig Brideau wrote: > > > >> ***** > >> To join, leave or search the confocal microscopy listserv, go to: > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >> Post images on http://www.imgur.com and include the link in your > posting. > >> ***** > >> > >> You are reducing the emission volume, not preventing excitation. In > >> practice you excite a gaussian-ish spatial volume, then deplete with an > >> annular pattern (+'barbell' for 3D) before the molecules can emit. > >> > >> Craig > >> > >> On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> wrote: > >> > >> ***** > >>> > >>> This topic has started a nice discussion in our staff Facility. The > STED > >>> effect, has to be considered in the excitation or the emission volume? > >>> > >>> Which is the right phrase in STED definition? > >>> > >>> a) The improving in lateral an axial resolution is provided by reducing > >>> the volume of the _emission_ by the fenomenon of stimulated emission. > >>> > >>> b) The improving in lateral an axial resolution is provided by reducing > >>> the volume of the _excitation_ by the fenomenon of stimulated emission. > >>> > >>> Basically, the term STED is lacking some other explanations behind. > Does > >>> someone has a better one than "Stimulated Emission Depletion"? > >>> > >>> Thank you very much in advance. > >>> > >>> Best regards > >>> > >>> Juan Luis > >>> > >>> > >>> El 31/01/2017 a las 16:09, Matthias Reuss escribió: > >>> > >>> ***** > >>>> To join, leave or search the confocal microscopy listserv, go to: > >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > >>>> Post images on http://www.imgur.com and include the link in your > >>>> posting. > >>>> ***** > >>>> > >>>> Dear Joost, > >>>> > >>>> This is probably a question everyone stumbles across sooner or later > >>>> when > >>>> thinking about the mechanisms behind STED. > >>>> > >>>> It is correct that the effective volume of the excitation spot is > shrunk > >>>> by the STED beam. In other words, the region from which spontaneous > >>>> fluorescence is allowed is much smaller than the original > >>>> diffraction-limited excitation volume. Also, your student is > absolutely > >>>> correct that the image of this reduced volume on the detector is again > >>>> diffraction limited. > >>>> > >>>> The solution to your question lies in realizing that it doesn’t > matter! > >>>> > >>>> It’s often helpful to picture an extreme situation, so let’s imagine > >>>> that > >>>> we are able to reduce the effective focal volume to a very, very small > >>>> point. Fluorescence from a molecule located at this point will > propagate > >>>> back through the optical system and end up diffracted on the detector, > >>>> spread out and with Airy rings and all. However, what we’re interested > >>>> in > >>>> is not the image of the molecule per se, but we'd like to know *where* > >>>> the > >>>> molecule is. > >>>> > >>>> The punchline is that with STED, we are able to get an accurate fix on > >>>> the molecule, simply because we know the position of the zero point of > >>>> the > >>>> STED-PSF. For example, if we do beam scanning, we know where we’re > >>>> currently pointing the beam, we therefore know where the intensity > zero > >>>> of > >>>> the STED-donut is, and we therefore know that the molecule can only be > >>>> at > >>>> this very place. If it was at another place just a few nanometers > away, > >>>> the > >>>> molecule would see a non-zero STED intensity and would currently not > be > >>>> able to emit. Without STED, we could only know that it is somewhere > >>>> within > >>>> an approx. 200 nm region, the size of the diffraction-limited > excitation > >>>> distribution. > >>>> > >>>> All this is 100% independent of the image of the molecule on the > >>>> detector > >>>> and whether it is diffraction-limited there or not. > >>>> > >>>> Hope this helps. > >>>> > >>>> Best, > >>>> Matthias > >>>> > >>>> > >>>> Matthias Reuss, Dr. > >>>> Head of Marketing & Sales > >>>> Abberior Instruments GmbH > >>>> Hans-Adolf-Krebs-Weg 1 > >>>> 37077 Goettingen > >>>> Germany > >>>> > >>>> phone: +49 (551) 30724 175 > >>>> fax: +49 (551) 30724 171 > >>>> http://www.abberior-instruments.com > >>>> mailto:[hidden email] > >>>> > >>>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen HRB > >>>> 201844 | VAT Reg. No.: DE 283588727 > >>>> > >>>> > >>>> -- > >>> Juan Luis Ribas > >>> Servicio de Microscopía > >>> Centro de Investigación, Tecnología e Innovación > >>> Universidad de Sevilla > >>> Av. Reina Mercedes 4b > >>> 41012 Sevilla > >>> Spain > >>> > >>> > > -- > > > > > > George McNamara, PhD > > Baltimore, MD 21231 > > [hidden email] > > https://www.linkedin.com/in/georgemcnamara > > https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge > > or Firefox, rather than Google Chrome) > > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 > > http://confocal.jhu.edu > > > > July 2017 Current Protocols article, open access: > > UNIT 4.4 Microscopy and Image Analysis > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract > > supporting materials direct link is > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 > > figures at > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures > > > |
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Craig Brideau |
Re: AW: STED ... there are two different emissions in STED experiments
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** It's a spherical wavefunction that collapses to a point at the detector. :) Quantum details aside, it should emit in a random direction because fluorescence is a non-coherent process. SHG, CARS, etc on the other hand have particular trajectories governed by the details of the excitation beams. Note that polarization is a different matter for fluorescence: The polarization of the emitted photon will be dependent on tumbling (if molecule is free to tumble, see my work with Ileana Micu on that one...) and the fluorescence lifetime of the molecule. Craig On Thu, Nov 9, 2017 at 1:24 PM, Andrew York < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Craig's point is a good excuse to bring up one of my favorite physics > questions: if you use a plane wave to stimulate a pointlike fluorophore to > emit, is the fluorophore's emission a plane wave, or a spherical* outgoing > wave? > > Other ways to phrase the question include: > * Does stimulated emission from an isolated pointlike emitter carry > information about the emitter location? > * Can we use lenses to form images with stimulated emission? > > We showed some results at the last Focus on Microscopy: > https://goo.gl/9Sr812 > ...and I think there's lots of fun follow-up questions. > > > *dipole-shaped, of course, not spherically symmetric, but you know what I > mean. > > On Wed, Nov 8, 2017 at 9:10 PM, Craig Brideau <[hidden email]> > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Very good point George, although I doubt most people would notice the > > stimulated emission at the same wavelength as the depletion laser. That > > said, I'm sure there are applications for pump-probe experiments that > would > > take advantage of the driven emission using lock-in amplification or the > > like. It's funny to think of the fluorophore as an optical gain medium, > but > > here we are! > > > > Craig > > > > On Wed, Nov 8, 2017 at 6:53 PM, George McNamara < > [hidden email] > > > > > wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > > Post images on http://www.imgur.com and include the link in your > > posting. > > > ***** > > > > > > neither a nor b, since emission are emissions. > > > > > > There are two different emissions in STED experiments: > > > > > > 1. stimulated emission at exactly the wavelength of the depletion laser > > > wavelength. > > > > > > 2. fluorescence emission, detected at whatever wavelength range the > light > > > path to the detector allows. > > > > > > enjoy, > > > > > > George > > > > > > > > > On 11/8/2017 12:44 PM, Craig Brideau wrote: > > > > > >> ***** > > >> To join, leave or search the confocal microscopy listserv, go to: > > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >> Post images on http://www.imgur.com and include the link in your > > posting. > > >> ***** > > >> > > >> You are reducing the emission volume, not preventing excitation. In > > >> practice you excite a gaussian-ish spatial volume, then deplete with > an > > >> annular pattern (+'barbell' for 3D) before the molecules can emit. > > >> > > >> Craig > > >> > > >> On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> > wrote: > > >> > > >> ***** > > >>> > > >>> This topic has started a nice discussion in our staff Facility. The > > STED > > >>> effect, has to be considered in the excitation or the emission > volume? > > >>> > > >>> Which is the right phrase in STED definition? > > >>> > > >>> a) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _emission_ by the fenomenon of stimulated emission. > > >>> > > >>> b) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _excitation_ by the fenomenon of stimulated > emission. > > >>> > > >>> Basically, the term STED is lacking some other explanations behind. > > Does > > >>> someone has a better one than "Stimulated Emission Depletion"? > > >>> > > >>> Thank you very much in advance. > > >>> > > >>> Best regards > > >>> > > >>> Juan Luis > > >>> > > >>> > > >>> El 31/01/2017 a las 16:09, Matthias Reuss escribió: > > >>> > > >>> ***** > > >>>> To join, leave or search the confocal microscopy listserv, go to: > > >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >>>> Post images on http://www.imgur.com and include the link in your > > >>>> posting. > > >>>> ***** > > >>>> > > >>>> Dear Joost, > > >>>> > > >>>> This is probably a question everyone stumbles across sooner or later > > >>>> when > > >>>> thinking about the mechanisms behind STED. > > >>>> > > >>>> It is correct that the effective volume of the excitation spot is > > shrunk > > >>>> by the STED beam. In other words, the region from which spontaneous > > >>>> fluorescence is allowed is much smaller than the original > > >>>> diffraction-limited excitation volume. Also, your student is > > absolutely > > >>>> correct that the image of this reduced volume on the detector is > again > > >>>> diffraction limited. > > >>>> > > >>>> The solution to your question lies in realizing that it doesn’t > > matter! > > >>>> > > >>>> It’s often helpful to picture an extreme situation, so let’s imagine > > >>>> that > > >>>> we are able to reduce the effective focal volume to a very, very > small > > >>>> point. Fluorescence from a molecule located at this point will > > propagate > > >>>> back through the optical system and end up diffracted on the > detector, > > >>>> spread out and with Airy rings and all. However, what we’re > interested > > >>>> in > > >>>> is not the image of the molecule per se, but we'd like to know > *where* > > >>>> the > > >>>> molecule is. > > >>>> > > >>>> The punchline is that with STED, we are able to get an accurate fix > on > > >>>> the molecule, simply because we know the position of the zero point > of > > >>>> the > > >>>> STED-PSF. For example, if we do beam scanning, we know where we’re > > >>>> currently pointing the beam, we therefore know where the intensity > > zero > > >>>> of > > >>>> the STED-donut is, and we therefore know that the molecule can only > be > > >>>> at > > >>>> this very place. If it was at another place just a few nanometers > > away, > > >>>> the > > >>>> molecule would see a non-zero STED intensity and would currently not > > be > > >>>> able to emit. Without STED, we could only know that it is somewhere > > >>>> within > > >>>> an approx. 200 nm region, the size of the diffraction-limited > > excitation > > >>>> distribution. > > >>>> > > >>>> All this is 100% independent of the image of the molecule on the > > >>>> detector > > >>>> and whether it is diffraction-limited there or not. > > >>>> > > >>>> Hope this helps. > > >>>> > > >>>> Best, > > >>>> Matthias > > >>>> > > >>>> > > >>>> Matthias Reuss, Dr. > > >>>> Head of Marketing & Sales > > >>>> Abberior Instruments GmbH > > >>>> Hans-Adolf-Krebs-Weg 1 > > >>>> 37077 Goettingen > > >>>> Germany > > >>>> > > >>>> phone: +49 (551) 30724 175 > > >>>> fax: +49 (551) 30724 171 > > >>>> http://www.abberior-instruments.com > > >>>> mailto:[hidden email] > > >>>> > > >>>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen > HRB > > >>>> 201844 | VAT Reg. No.: DE 283588727 > > >>>> > > >>>> > > >>>> -- > > >>> Juan Luis Ribas > > >>> Servicio de Microscopía > > >>> Centro de Investigación, Tecnología e Innovación > > >>> Universidad de Sevilla > > >>> Av. Reina Mercedes 4b > > >>> 41012 Sevilla > > >>> Spain > > >>> > > >>> > > > -- > > > > > > > > > George McNamara, PhD > > > Baltimore, MD 21231 > > > [hidden email] > > > https://www.linkedin.com/in/georgemcnamara > > > https://works.bepress.com/gmcnamara/75 (may need to use Microsoft > Edge > > > or Firefox, rather than Google Chrome) > > > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 > > > http://confocal.jhu.edu > > > > > > July 2017 Current Protocols article, open access: > > > UNIT 4.4 Microscopy and Image Analysis > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract > > > supporting materials direct link is > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full# > hg0404-sec-0023 > > > figures at > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures > > > > > > |
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Antonio Jose Pereira |
Re: AW: STED ... there are two different emissions in STED experiments
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In reply to this post by George McNamara
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Craig, I guess Andrew was asking about a plane wave 'stimulating emission' on an excited atom/molecule. Contrary to fluorescence, stimulated emission is a coherent process: the stimulated and the stimulating photons are mutually coherent. I may have misunderstood your wording though. António Pereira -----Confocal Microscopy List <[hidden email]> escreveu: ----- Para: [hidden email] De: Craig Brideau Enviado por: Confocal Microscopy List Data: 09-11-2017 21:57 Assunto: Re: AW: STED ... there are two different emissions in STED experiments ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** It's a spherical wavefunction that collapses to a point at the detector. :) Quantum details aside, it should emit in a random direction because fluorescence is a non-coherent process. SHG, CARS, etc on the other hand have particular trajectories governed by the details of the excitation beams. Note that polarization is a different matter for fluorescence: The polarization of the emitted photon will be dependent on tumbling (if molecule is free to tumble, see my work with Ileana Micu on that one...) and the fluorescence lifetime of the molecule. Craig On Thu, Nov 9, 2017 at 1:24 PM, Andrew York < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Craig's point is a good excuse to bring up one of my favorite physics > questions: if you use a plane wave to stimulate a pointlike fluorophore to > emit, is the fluorophore's emission a plane wave, or a spherical* outgoing > wave? > > Other ways to phrase the question include: > * Does stimulated emission from an isolated pointlike emitter carry > information about the emitter location? > * Can we use lenses to form images with stimulated emission? > > We showed some results at the last Focus on Microscopy: > https://goo.gl/9Sr812 > ...and I think there's lots of fun follow-up questions. > > > *dipole-shaped, of course, not spherically symmetric, but you know what I > mean. > > On Wed, Nov 8, 2017 at 9:10 PM, Craig Brideau <[hidden email]> > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Very good point George, although I doubt most people would notice the > > stimulated emission at the same wavelength as the depletion laser. That > > said, I'm sure there are applications for pump-probe experiments that > would > > take advantage of the driven emission using lock-in amplification or the > > like. It's funny to think of the fluorophore as an optical gain medium, > but > > here we are! > > > > Craig > > > > On Wed, Nov 8, 2017 at 6:53 PM, George McNamara < > [hidden email] > > > > > wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > > Post images on http://www.imgur.com and include the link in your > > posting. > > > ***** > > > > > > neither a nor b, since emission are emissions. > > > > > > There are two different emissions in STED experiments: > > > > > > 1. stimulated emission at exactly the wavelength of the depletion laser > > > wavelength. > > > > > > 2. fluorescence emission, detected at whatever wavelength range the > light > > > path to the detector allows. > > > > > > enjoy, > > > > > > George > > > > > > > > > On 11/8/2017 12:44 PM, Craig Brideau wrote: > > > > > >> ***** > > >> To join, leave or search the confocal microscopy listserv, go to: > > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >> Post images on http://www.imgur.com and include the link in your > > posting. > > >> ***** > > >> > > >> You are reducing the emission volume, not preventing excitation. In > > >> practice you excite a gaussian-ish spatial volume, then deplete with > an > > >> annular pattern (+'barbell' for 3D) before the molecules can emit. > > >> > > >> Craig > > >> > > >> On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> > wrote: > > >> > > >> ***** > > >>> > > >>> This topic has started a nice discussion in our staff Facility. The > > STED > > >>> effect, has to be considered in the excitation or the emission > volume? > > >>> > > >>> Which is the right phrase in STED definition? > > >>> > > >>> a) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _emission_ by the fenomenon of stimulated emission. > > >>> > > >>> b) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _excitation_ by the fenomenon of stimulated > emission. > > >>> > > >>> Basically, the term STED is lacking some other explanations behind. > > Does > > >>> someone has a better one than "Stimulated Emission Depletion"? > > >>> > > >>> Thank you very much in advance. > > >>> > > >>> Best regards > > >>> > > >>> Juan Luis > > >>> > > >>> > > >>> El 31/01/2017 a las 16:09, Matthias Reuss escribió: > > >>> > > >>> ***** > > >>>> To join, leave or search the confocal microscopy listserv, go to: > > >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >>>> Post images on http://www.imgur.com and include the link in your > > >>>> posting. > > >>>> ***** > > >>>> > > >>>> Dear Joost, > > >>>> > > >>>> This is probably a question everyone stumbles across sooner or later > > >>>> when > > >>>> thinking about the mechanisms behind STED. > > >>>> > > >>>> It is correct that the effective volume of the excitation spot is > > shrunk > > >>>> by the STED beam. In other words, the region from which spontaneous > > >>>> fluorescence is allowed is much smaller than the original > > >>>> diffraction-limited excitation volume. Also, your student is > > absolutely > > >>>> correct that the image of this reduced volume on the detector is > again > > >>>> diffraction limited. > > >>>> > > >>>> The solution to your question lies in realizing that it doesn’t > > matter! > > >>>> > > >>>> It’s often helpful to picture an extreme situation, so let’s imagine > > >>>> that > > >>>> we are able to reduce the effective focal volume to a very, very > small > > >>>> point. Fluorescence from a molecule located at this point will > > propagate > > >>>> back through the optical system and end up diffracted on the > detector, > > >>>> spread out and with Airy rings and all. However, what we’re > interested > > >>>> in > > >>>> is not the image of the molecule per se, but we'd like to know > *where* > > >>>> the > > >>>> molecule is. > > >>>> > > >>>> The punchline is that with STED, we are able to get an accurate fix > on > > >>>> the molecule, simply because we know the position of the zero point > of > > >>>> the > > >>>> STED-PSF. For example, if we do beam scanning, we know where we’re > > >>>> currently pointing the beam, we therefore know where the intensity > > zero > > >>>> of > > >>>> the STED-donut is, and we therefore know that the molecule can only > be > > >>>> at > > >>>> this very place. If it was at another place just a few nanometers > > away, > > >>>> the > > >>>> molecule would see a non-zero STED intensity and would currently not > > be > > >>>> able to emit. Without STED, we could only know that it is somewhere > > >>>> within > > >>>> an approx. 200 nm region, the size of the diffraction-limited > > excitation > > >>>> distribution. > > >>>> > > >>>> All this is 100% independent of the image of the molecule on the > > >>>> detector > > >>>> and whether it is diffraction-limited there or not. > > >>>> > > >>>> Hope this helps. > > >>>> > > >>>> Best, > > >>>> Matthias > > >>>> > > >>>> > > >>>> Matthias Reuss, Dr. > > >>>> Head of Marketing & Sales > > >>>> Abberior Instruments GmbH > > >>>> Hans-Adolf-Krebs-Weg 1 > > >>>> 37077 Goettingen > > >>>> Germany > > >>>> > > >>>> phone: +49 (551) 30724 175 > > >>>> fax: +49 (551) 30724 171 > > >>>> http://www.abberior-instruments.com > > >>>> mailto:[hidden email] > > >>>> > > >>>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen > HRB > > >>>> 201844 | VAT Reg. No.: DE 283588727 > > >>>> > > >>>> > > >>>> -- > > >>> Juan Luis Ribas > > >>> Servicio de Microscopía > > >>> Centro de Investigación, Tecnología e Innovación > > >>> Universidad de Sevilla > > >>> Av. Reina Mercedes 4b > > >>> 41012 Sevilla > > >>> Spain > > >>> > > >>> > > > -- > > > > > > > > > George McNamara, PhD > > > Baltimore, MD 21231 > > > [hidden email] > > > https://www.linkedin.com/in/georgemcnamara > > > https://works.bepress.com/gmcnamara/75 (may need to use Microsoft > Edge > > > or Firefox, rather than Google Chrome) > > > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 > > > http://confocal.jhu.edu > > > > > > July 2017 Current Protocols article, open access: > > > UNIT 4.4 Microscopy and Image Analysis > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract > > > supporting materials direct link is > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full# > hg0404-sec-0023 > > > figures at > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures > > > > > > |
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Zdenek Svindrych-2 |
Re: AW: STED ... there are two different emissions in STED experiments
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Yes, it's getting interesting :-). There is one problem with the well-known cartoon where the emitted photon travels just next to the stimulating one, coherently and in the same direction. It's called the uncertainty principle. If we know precisely where the photon comes from (from the molecule), the momentum (and hence the direction) is essentially random! The situation is the same if you're looking at SHG or CARS from a single molecule. The trick is, nobody is really looking at a single molecule. When you have an ensemble of molecules experiencing stimulated emission, their wave functions (read "spherical waves" for simplicity) are in precise phase relation to each other, resulting in constructive interference in the direction of propagation of the stimulating light ("plane wave"). You can think of it as Huygen's principle turned upside down (or inside out :-). As to the original question, I think it's impossible to observe the stimulated emission in a typical STED scenario, the additional signal is too weak (like one photon in a billion) and I can't imagine a way to separate it from the depletion beam. But lasing inside individual cells has been demonstrated: doi:10.1038/nphoton.2011.99 Best, zdenek -- Zdenek Svindrych, Ph.D. Research Associate - Imaging Specialist Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth ---------- Původní e-mail ---------- Od: Antonio Jose Pereira <[hidden email]> Komu: [hidden email] Datum: 9. 11. 2017 19:05:01 Předmět: Re: AW: STED ... there are two different emissions in STED experiments "***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Craig, I guess Andrew was asking about a plane wave 'stimulating emission' on an excited atom/molecule. Contrary to fluorescence, stimulated emission is a coherent process: the stimulated and the stimulating photons are mutually coherent. I may have misunderstood your wording though. António Pereira -----Confocal Microscopy List <[hidden email]> escreveu: - ---- Para: [hidden email] De: Craig Brideau Enviado por: Confocal Microscopy List Data: 09-11-2017 21:57 Assunto: Re: AW: STED ... there are two different emissions in STED experiments ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** It's a spherical wavefunction that collapses to a point at the detector. :) Quantum details aside, it should emit in a random direction because fluorescence is a non-coherent process. SHG, CARS, etc on the other hand have particular trajectories governed by the details of the excitation beams. Note that polarization is a different matter for fluorescence: The polarization of the emitted photon will be dependent on tumbling (if molecule is free to tumble, see my work with Ileana Micu on that one...) and the fluorescence lifetime of the molecule. Craig On Thu, Nov 9, 2017 at 1:24 PM, Andrew York < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Craig's point is a good excuse to bring up one of my favorite physics > questions: if you use a plane wave to stimulate a pointlike fluorophore to > emit, is the fluorophore's emission a plane wave, or a spherical* outgoing > wave? > > Other ways to phrase the question include: > * Does stimulated emission from an isolated pointlike emitter carry > information about the emitter location? > * Can we use lenses to form images with stimulated emission? > > We showed some results at the last Focus on Microscopy: > https://goo.gl/9Sr812 > ...and I think there's lots of fun follow-up questions. > > > *dipole-shaped, of course, not spherically symmetric, but you know what I > mean. > > On Wed, Nov 8, 2017 at 9:10 PM, Craig Brideau <[hidden email]> > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Very good point George, although I doubt most people would notice the > > stimulated emission at the same wavelength as the depletion laser. That > > said, I'm sure there are applications for pump-probe experiments that > would > > take advantage of the driven emission using lock-in amplification or the > > like. It's funny to think of the fluorophore as an optical gain medium, > but > > here we are! > > > > Craig > > > > On Wed, Nov 8, 2017 at 6:53 PM, George McNamara < > [hidden email] > > > > > wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > > Post images on http://www.imgur.com and include the link in your > > posting. > > > ***** > > > > > > neither a nor b, since emission are emissions. > > > > > > There are two different emissions in STED experiments: > > > > > > 1. stimulated emission at exactly the wavelength of the depletion > > > wavelength. > > > > > > 2. fluorescence emission, detected at whatever wavelength range the > light > > > path to the detector allows. > > > > > > enjoy, > > > > > > George > > > > > > > > > On 11/8/2017 12:44 PM, Craig Brideau wrote: > > > > > >> ***** > > >> To join, leave or search the confocal microscopy listserv, go to: > > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >> Post images on http://www.imgur.com and include the link in your > > posting. > > >> ***** > > >> > > >> You are reducing the emission volume, not preventing excitation. In > > >> practice you excite a gaussian-ish spatial volume, then deplete with > an > > >> annular pattern (+'barbell' for 3D) before the molecules can emit. > > >> > > >> Craig > > >> > > >> On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> > wrote: > > >> > > >> ***** > > >>> > > >>> This topic has started a nice discussion in our staff Facility. The > > STED > > >>> effect, has to be considered in the excitation or the emission > volume? > > >>> > > >>> Which is the right phrase in STED definition? > > >>> > > >>> a) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _emission_ by the fenomenon of stimulated > > >>> > > >>> b) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _excitation_ by the fenomenon of stimulated > emission. > > >>> > > >>> Basically, the term STED is lacking some other explanations behind. > > Does > > >>> someone has a better one than "Stimulated Emission Depletion"? > > >>> > > >>> Thank you very much in advance. > > >>> > > >>> Best regards > > >>> > > >>> Juan Luis > > >>> > > >>> > > >>> El 31/01/2017 a las 16:09, Matthias Reuss escribió: > > >>> > > >>> ***** > > >>>> To join, leave or search the confocal microscopy listserv, go to: > > >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >>>> Post images on http://www.imgur.com and include the link in your > > >>>> posting. > > >>>> ***** > > >>>> > > >>>> Dear Joost, > > >>>> > > >>>> This is probably a question everyone stumbles across sooner or > > >>>> when > > >>>> thinking about the mechanisms behind STED. > > >>>> > > >>>> It is correct that the effective volume of the excitation spot is > > shrunk > > >>>> by the STED beam. In other words, the region from which spontaneous > > >>>> fluorescence is allowed is much smaller than the original > > >>>> diffraction-limited excitation volume. Also, your student is > > absolutely > > >>>> correct that the image of this reduced volume on the detector is > again > > >>>> diffraction limited. > > >>>> > > >>>> The solution to your question lies in realizing that it doesn’t > > matter! > > >>>> > > >>>> It’s often helpful to picture an extreme situation, so let’s > > >>>> that > > >>>> we are able to reduce the effective focal volume to a very, very > small > > >>>> point. Fluorescence from a molecule located at this point will > > propagate > > >>>> back through the optical system and end up diffracted on the > detector, > > >>>> spread out and with Airy rings and all. However, what we’re > interested > > >>>> in > > >>>> is not the image of the molecule per se, but we'd like to know > *where* > > >>>> the > > >>>> molecule is. > > >>>> > > >>>> The punchline is that with STED, we are able to get an accurate fix > on > > >>>> the molecule, simply because we know the position of the zero point > of > > >>>> the > > >>>> STED-PSF. For example, if we do beam scanning, we know where we’re > > >>>> currently pointing the beam, we therefore know where the intensity > > zero > > >>>> of > > >>>> the STED-donut is, and we therefore know that the molecule can only > be > > >>>> at > > >>>> this very place. If it was at another place just a few nanometers > > away, > > >>>> the > > >>>> molecule would see a non-zero STED intensity and would currently not > > be > > >>>> able to emit. Without STED, we could only know that it is somewhere > > >>>> within > > >>>> an approx. 200 nm region, the size of the diffraction-limited > > excitation > > >>>> distribution. > > >>>> > > >>>> All this is 100% independent of the image of the molecule on the > > >>>> detector > > >>>> and whether it is diffraction-limited there or not. > > >>>> > > >>>> Hope this helps. > > >>>> > > >>>> Best, > > >>>> Matthias > > >>>> > > >>>> > > >>>> Matthias Reuss, Dr. > > >>>> Head of Marketing & Sales > > >>>> Abberior Instruments GmbH > > >>>> Hans-Adolf-Krebs-Weg 1 > > >>>> 37077 Goettingen > > >>>> Germany > > >>>> > > >>>> phone: +49 (551) 30724 175 > > >>>> fax: +49 (551) 30724 171 > > >>>> http://www.abberior-instruments.com > > >>>> mailto:[hidden email] > > >>>> > > >>>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen > HRB > > >>>> 201844 | VAT Reg. No.: DE 283588727 > > >>>> > > >>>> > > >>>> -- > > >>> Juan Luis Ribas > > >>> Servicio de Microscopía > > >>> Centro de Investigación, Tecnología e Innovación > > >>> Universidad de Sevilla > > >>> Av. Reina Mercedes 4b > > >>> 41012 Sevilla > > >>> Spain > > >>> > > >>> > > > -- > > > > > > > > > George McNamara, PhD > > > Baltimore, MD 21231 > > > [hidden email] > > > https://www.linkedin.com/in/georgemcnamara > > > https://works.bepress.com/gmcnamara/75 (may need to use Microsoft > Edge > > > or Firefox, rather than Google Chrome) > > > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 > > > http://confocal.jhu.edu > > > > > > July 2017 Current Protocols article, open access: > > > UNIT 4.4 Microscopy and Image Analysis > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract > > > supporting materials direct link is > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full# > hg0404-sec-0023 > > > figures at > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures > > > > > > |
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Antonio Jose Pereira |
Re: AW: STED ... there are two different emissions in STED experiments
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Zdenek, Yes. Andrew's question is really ... stimulating. I used the expression 'mutually coherent' meaning exactly that, not that photons have the same direction, or even a direction. From what I understand of it, mutual coherence means that the two photons (bosons) will be indistinguishable, as they share the same mode of propagation after the stimulated emission event. This mode is certainly a wide cone due to diffraction, i.e., due to the uncertainty principle. Now I'm stuck in thinking of the following embodiment of the experiment: We have a 4pi detector around the excited atom. If the atom is allowed time to fluoresce, I think the photon will be detected anywhere over 4pi. On the other hand, if it is stimulated to emit, will it (or them) be detected anywhere in ... 2pi stereo-rad? A half-space? Antonio Pereira i3S, Porto -----Confocal Microscopy List <[hidden email]> escreveu: ----- Para: [hidden email] De: [hidden email] Enviado por: Confocal Microscopy List Data: 10-11-2017 00:48 Assunto: Re: AW: STED ... there are two different emissions in STED experiments ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Yes, it's getting interesting :-). There is one problem with the well-known cartoon where the emitted photon travels just next to the stimulating one, coherently and in the same direction. It's called the uncertainty principle. If we know precisely where the photon comes from (from the molecule), the momentum (and hence the direction) is essentially random! The situation is the same if you're looking at SHG or CARS from a single molecule. The trick is, nobody is really looking at a single molecule. When you have an ensemble of molecules experiencing stimulated emission, their wave functions (read "spherical waves" for simplicity) are in precise phase relation to each other, resulting in constructive interference in the direction of propagation of the stimulating light ("plane wave"). You can think of it as Huygen's principle turned upside down (or inside out :-). As to the original question, I think it's impossible to observe the stimulated emission in a typical STED scenario, the additional signal is too weak (like one photon in a billion) and I can't imagine a way to separate it from the depletion beam. But lasing inside individual cells has been demonstrated: doi:10.1038/nphoton.2011.99 Best, zdenek -- Zdenek Svindrych, Ph.D. Research Associate - Imaging Specialist Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth ---------- Původní e-mail ---------- Od: Antonio Jose Pereira <[hidden email]> Komu: [hidden email] Datum: 9. 11. 2017 19:05:01 Předmět: Re: AW: STED ... there are two different emissions in STED experiments "***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Craig, I guess Andrew was asking about a plane wave 'stimulating emission' on an excited atom/molecule. Contrary to fluorescence, stimulated emission is a coherent process: the stimulated and the stimulating photons are mutually coherent. I may have misunderstood your wording though. António Pereira -----Confocal Microscopy List <[hidden email]> escreveu: - ---- Para: [hidden email] De: Craig Brideau Enviado por: Confocal Microscopy List Data: 09-11-2017 21:57 Assunto: Re: AW: STED ... there are two different emissions in STED experiments ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** It's a spherical wavefunction that collapses to a point at the detector. :) Quantum details aside, it should emit in a random direction because fluorescence is a non-coherent process. SHG, CARS, etc on the other hand have particular trajectories governed by the details of the excitation beams. Note that polarization is a different matter for fluorescence: The polarization of the emitted photon will be dependent on tumbling (if molecule is free to tumble, see my work with Ileana Micu on that one...) and the fluorescence lifetime of the molecule. Craig On Thu, Nov 9, 2017 at 1:24 PM, Andrew York < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Craig's point is a good excuse to bring up one of my favorite physics > questions: if you use a plane wave to stimulate a pointlike fluorophore to > emit, is the fluorophore's emission a plane wave, or a spherical* outgoing > wave? > > Other ways to phrase the question include: > * Does stimulated emission from an isolated pointlike emitter carry > information about the emitter location? > * Can we use lenses to form images with stimulated emission? > > We showed some results at the last Focus on Microscopy: > https://goo.gl/9Sr812 > ...and I think there's lots of fun follow-up questions. > > > *dipole-shaped, of course, not spherically symmetric, but you know what I > mean. > > On Wed, Nov 8, 2017 at 9:10 PM, Craig Brideau <[hidden email]> > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Very good point George, although I doubt most people would notice the > > stimulated emission at the same wavelength as the depletion laser. That > > said, I'm sure there are applications for pump-probe experiments that > would > > take advantage of the driven emission using lock-in amplification or the > > like. It's funny to think of the fluorophore as an optical gain medium, > but > > here we are! > > > > Craig > > > > On Wed, Nov 8, 2017 at 6:53 PM, George McNamara < > [hidden email] > > > > > wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > > Post images on http://www.imgur.com and include the link in your > > posting. > > > ***** > > > > > > neither a nor b, since emission are emissions. > > > > > > There are two different emissions in STED experiments: > > > > > > 1. stimulated emission at exactly the wavelength of the depletion > > > wavelength. > > > > > > 2. fluorescence emission, detected at whatever wavelength range the > light > > > path to the detector allows. > > > > > > enjoy, > > > > > > George > > > > > > > > > On 11/8/2017 12:44 PM, Craig Brideau wrote: > > > > > >> ***** > > >> To join, leave or search the confocal microscopy listserv, go to: > > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >> Post images on http://www.imgur.com and include the link in your > > posting. > > >> ***** > > >> > > >> You are reducing the emission volume, not preventing excitation. In > > >> practice you excite a gaussian-ish spatial volume, then deplete with > an > > >> annular pattern (+'barbell' for 3D) before the molecules can emit. > > >> > > >> Craig > > >> > > >> On Wed, Nov 8, 2017 at 9:53 AM, Juan Luis Ribas <[hidden email]> > wrote: > > >> > > >> ***** > > >>> > > >>> This topic has started a nice discussion in our staff Facility. The > > STED > > >>> effect, has to be considered in the excitation or the emission > volume? > > >>> > > >>> Which is the right phrase in STED definition? > > >>> > > >>> a) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _emission_ by the fenomenon of stimulated > > >>> > > >>> b) The improving in lateral an axial resolution is provided by > reducing > > >>> the volume of the _excitation_ by the fenomenon of stimulated > emission. > > >>> > > >>> Basically, the term STED is lacking some other explanations behind. > > Does > > >>> someone has a better one than "Stimulated Emission Depletion"? > > >>> > > >>> Thank you very much in advance. > > >>> > > >>> Best regards > > >>> > > >>> Juan Luis > > >>> > > >>> > > >>> El 31/01/2017 a las 16:09, Matthias Reuss escribió: > > >>> > > >>> ***** > > >>>> To join, leave or search the confocal microscopy listserv, go to: > > >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > >>>> Post images on http://www.imgur.com and include the link in your > > >>>> posting. > > >>>> ***** > > >>>> > > >>>> Dear Joost, > > >>>> > > >>>> This is probably a question everyone stumbles across sooner or > > >>>> when > > >>>> thinking about the mechanisms behind STED. > > >>>> > > >>>> It is correct that the effective volume of the excitation spot is > > shrunk > > >>>> by the STED beam. In other words, the region from which spontaneous > > >>>> fluorescence is allowed is much smaller than the original > > >>>> diffraction-limited excitation volume. Also, your student is > > absolutely > > >>>> correct that the image of this reduced volume on the detector is > again > > >>>> diffraction limited. > > >>>> > > >>>> The solution to your question lies in realizing that it doesn’t > > matter! > > >>>> > > >>>> It’s often helpful to picture an extreme situation, so let’s > > >>>> that > > >>>> we are able to reduce the effective focal volume to a very, very > small > > >>>> point. Fluorescence from a molecule located at this point will > > propagate > > >>>> back through the optical system and end up diffracted on the > detector, > > >>>> spread out and with Airy rings and all. However, what we’re > interested > > >>>> in > > >>>> is not the image of the molecule per se, but we'd like to know > *where* > > >>>> the > > >>>> molecule is. > > >>>> > > >>>> The punchline is that with STED, we are able to get an accurate fix > on > > >>>> the molecule, simply because we know the position of the zero point > of > > >>>> the > > >>>> STED-PSF. For example, if we do beam scanning, we know where we’re > > >>>> currently pointing the beam, we therefore know where the intensity > > zero > > >>>> of > > >>>> the STED-donut is, and we therefore know that the molecule can only > be > > >>>> at > > >>>> this very place. If it was at another place just a few nanometers > > away, > > >>>> the > > >>>> molecule would see a non-zero STED intensity and would currently not > > be > > >>>> able to emit. Without STED, we could only know that it is somewhere > > >>>> within > > >>>> an approx. 200 nm region, the size of the diffraction-limited > > excitation > > >>>> distribution. > > >>>> > > >>>> All this is 100% independent of the image of the molecule on the > > >>>> detector > > >>>> and whether it is diffraction-limited there or not. > > >>>> > > >>>> Hope this helps. > > >>>> > > >>>> Best, > > >>>> Matthias > > >>>> > > >>>> > > >>>> Matthias Reuss, Dr. > > >>>> Head of Marketing & Sales > > >>>> Abberior Instruments GmbH > > >>>> Hans-Adolf-Krebs-Weg 1 > > >>>> 37077 Goettingen > > >>>> Germany > > >>>> > > >>>> phone: +49 (551) 30724 175 > > >>>> fax: +49 (551) 30724 171 > > >>>> http://www.abberior-instruments.com > > >>>> mailto:[hidden email] > > >>>> > > >>>> Managing Director: Dr. Gerald Donnert | Trade Register: Göttingen > HRB > > >>>> 201844 | VAT Reg. No.: DE 283588727 > > >>>> > > >>>> > > >>>> -- > > >>> Juan Luis Ribas > > >>> Servicio de Microscopía > > >>> Centro de Investigación, Tecnología e Innovación > > >>> Universidad de Sevilla > > >>> Av. Reina Mercedes 4b > > >>> 41012 Sevilla > > >>> Spain > > >>> > > >>> > > > -- > > > > > > > > > George McNamara, PhD > > > Baltimore, MD 21231 > > > [hidden email] > > > https://www.linkedin.com/in/georgemcnamara > > > https://works.bepress.com/gmcnamara/75 (may need to use Microsoft > Edge > > > or Firefox, rather than Google Chrome) > > > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 > > > http://confocal.jhu.edu > > > > > > July 2017 Current Protocols article, open access: > > > UNIT 4.4 Microscopy and Image Analysis > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract > > > supporting materials direct link is > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full# > hg0404-sec-0023 > > > figures at > > > http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures > > > > > > |
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