STED

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Joost Willemse-2 Joost Willemse-2
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STED

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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
Avi Jacob Avi Jacob
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Re: STED

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*****

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
>
Kyle Douglass Kyle Douglass
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Re: STED

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*****

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
Armstrong, Brian Armstrong, Brian
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Re: STED

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*****

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

*****
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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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Reto Fiolka Reto Fiolka
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Re: STED

In reply to this post by Joost Willemse-2
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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
zdedenn zdedenn
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Re: STED

In reply to this post by Joost Willemse-2
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*****

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:
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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
"
Antonio Jose Pereira Antonio Jose Pereira
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Re: STED

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*****

 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

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*****

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 Aryeh Weiss
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Re: STED

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*****
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*****

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
Sieber, Jochen Sieber, Jochen
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AW: STED

In reply to this post by Joost Willemse-2
*****
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*****

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]
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-----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
-------------------------------------------------------------------------------

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*****

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
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Matthias Reuss Matthias Reuss
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Re: AW: STED

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*****

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


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Avi Jacob Avi Jacob
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Re: AW: STED

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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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