Fluorescence illumination NA vs. Z Resolution.

[Daniel White-2]

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Hi all

Geeky question :

What is the physical process by which illumination NA in a confocal or WF
fluorescence microscope affects the z resolution, the axial response (OTF)?

Since fluorescence lifetime is nanoseconds, emission is incoherent wrt
excitation. Assuming free dye rotation during fluorescence lifetime, how
can angle range of incident photon affect angle range of emitted photon?

What am I missing?

What's the quantum electrodynamics explanation here? What would Feynman
have said?

Cheers

[0000001ed7f52e4a-dmarc-request]

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Hi Daniel,
I would say simple wave optics, think Huygens principle.  Only in the focus you have constructive interference, if you move away from it you get destructive interference. Where depends on the angle of the incoming light. The emitted light is isotrop. But you only get fluorescence where you excite it.

Best wishes

Andreas

-----Original Message-----
From: "Daniel White" <[hidden email]>
Sent: ‎21/‎07/‎2017 18:52
To: "[hidden email]" <[hidden email]>
Subject: Fluorescence illumination NA vs. Z Resolution.

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Hi all

Geeky question :

What is the physical process by which illumination NA in a confocal or WF
fluorescence microscope affects the z resolution, the axial response (OTF)?

Since fluorescence lifetime is nanoseconds, emission is incoherent wrt
excitation. Assuming free dye rotation during fluorescence lifetime, how
can angle range of incident photon affect angle range of emitted photon?

What am I missing?

What's the quantum electrodynamics explanation here? What would Feynman
have said?

Cheers

[Craig Brideau]

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If you look at the point-spread function of a focused laser in 3D (or an XZ
or YZ projection), you will see the interference pattern (as Andreas says)
in all directions (including Z). The interference is what helps confine the
energy to the focal point. Using geometry you can realize that by having a
larger variety of angles you give more interferometric possibilities, and
thus a tighter focus. The same general concept applies to widefield, except
instead of a laser point source the field of view is considered an infinite
collection of point sources. If you have lots of time on your hands,
consider the propagation of a semicircle of point emitters, each point
emitting a spherical wave, and see how the waves arrive at the 'center' of
the semicircle in terms of phase.

Craig

On Fri, Jul 21, 2017 at 12:12 PM, Andreas Bruckbauer <
[hidden email]> wrote:

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> *****
>
> Hi Daniel,
> I would say simple wave optics, think Huygens principle.  Only in the
> focus you have constructive interference, if you move away from it you get
> destructive interference. Where depends on the angle of the incoming light.
> The emitted light is isotrop. But you only get fluorescence where you
> excite it.
>
> Best wishes
>
> Andreas
>
> -----Original Message-----
> From: "Daniel White" <[hidden email]>
> Sent: ‎21/‎07/‎2017 18:52
> To: "[hidden email]" <[hidden email]>
> Subject: Fluorescence illumination NA vs. Z Resolution.
>
> *****
> 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
>
> Geeky question :
>
> What is the physical process by which illumination NA in a confocal or WF
> fluorescence microscope affects the z resolution, the axial response (OTF)?
>
> Since fluorescence lifetime is nanoseconds, emission is incoherent wrt
> excitation. Assuming free dye rotation during fluorescence lifetime, how
> can angle range of incident photon affect angle range of emitted photon?
>
> What am I missing?
>
> What's the quantum electrodynamics explanation here? What would Feynman
> have said?
>
> Cheers
>

[mmodel]

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I think NA of illumination does not affect resolution in wide field fluorescence, only in confocal

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel White
Sent: Friday, July 21, 2017 1:52 PM
To: [hidden email]
Subject: Fluorescence illumination NA vs. Z Resolution.

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Hi all

Geeky question :

What is the physical process by which illumination NA in a confocal or WF fluorescence microscope affects the z resolution, the axial response (OTF)?

Since fluorescence lifetime is nanoseconds, emission is incoherent wrt excitation. Assuming free dye rotation during fluorescence lifetime, how can angle range of incident photon affect angle range of emitted photon?

What am I missing?

What's the quantum electrodynamics explanation here? What would Feynman have said?

Cheers

[Craig Brideau]

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It still influences the depth of field in widefield, which is analogous to
the 'optical slice' in confocal. The thing is that widefield's confinement
is so poor you don't necessarily see much difference unless working with
two very different NAs. See depth of field in photography for the same idea.

Craig

On Fri, Jul 21, 2017 at 2:34 PM, MODEL, MICHAEL <[hidden email]> wrote:

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> *****
>
> I think NA of illumination does not affect resolution in wide field
> fluorescence, only in confocal
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Daniel White
> Sent: Friday, July 21, 2017 1:52 PM
> To: [hidden email]
> Subject: Fluorescence illumination NA vs. Z Resolution.
>
> *****
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> and include the link in your posting.
> *****
>
> Hi all
>
> Geeky question :
>
> What is the physical process by which illumination NA in a confocal or WF
> fluorescence microscope affects the z resolution, the axial response (OTF)?
>
> Since fluorescence lifetime is nanoseconds, emission is incoherent wrt
> excitation. Assuming free dye rotation during fluorescence lifetime, how
> can angle range of incident photon affect angle range of emitted photon?
>
> What am I missing?
>
> What's the quantum electrodynamics explanation here? What would Feynman
> have said?
>
> Cheers
>

[James Pawley]

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Hi Daniel,

The simple answer is that the illumination angle affects the rate at which the intensity of the excitation decreases as one leaves the focus plane.  And because, in fluorescence microscopy, emission is proportional to excitation and z-resolution is defined in terms of the reduction in collected signal as the fluorescent object moves away from the focus plane.

In confocal, this is fairly straight forward as we have a cone of light that shrinks almost to zero area at the focus plane and then expands again (minus absorption and reflection in the specimen). As the area of any circular cross-section of this cone varies with the square of the distance from the focus plane, the intensity of the excitation also varies inversely with the square of this distance.

Now imagine WF but with a very small setting of the field diaphragm, say 5µm in the image plane. In this case also, the excitation will decrease with the distance from the focus plane, just not quite as fast as in confocal.

In an early paper on WF 3D deconvolution, Agard et all, called this a pseudo-cofocal effect and it was pronounced enough to give a different 3D PSF when they were looking at small isolated nuclei.

In WF, this effect becomes less important as one goes to more common settings of the field diaphragm (When 512x512 sensors were used with 0.1 µm pixels, the diagonal field of view was about 70µm), you would only notice much change once you were tens of µm out of focus.

While it is common to state that in WF the illumination doesn’t vary with Z, this is only an approximation and only really true if axial illumination fills only the central point in the BFP of the illuminating optic.

JP


James and Christine Pawley, 5446 Burley Place, Box 2348, Sechelt BC, Canada, V0N3A0 [hidden email]<mailto:[hidden email]>, Phone [phone], cell 1-604-989-6146



On Jul 21, 2017, at 10:52 AM, Daniel White <[hidden email]<mailto:[hidden email]>> wrote:

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Hi all

Geeky question :

What is the physical process by which illumination NA in a confocal or WF
fluorescence microscope affects the z resolution, the axial response (OTF)?

Since fluorescence lifetime is nanoseconds, emission is incoherent wrt
excitation. Assuming free dye rotation during fluorescence lifetime, how
can angle range of incident photon affect angle range of emitted photon?

What am I missing?

What's the quantum electrodynamics explanation here? What would Feynman
have said?

Cheers

[Benjamin Smith]

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The key is that you also have to keep in mind that the NA objective impacts
your ability to detect said light, and diffraction limited resolution then
applies where:

lateral resolution ∝ 1/NA
Z-resolution ∝ 1/NA^2

This means as you go up in NA, lateral resolution gradually increases,
meaning you won't see much difference, however z-resolution increases
exponentially.  So going from 0.7NA to 1.4NA will result in a 2-fold
increase in XY resolution and a 4-fold increase in Z-resolution.
This is why low NA confocal PSFs look like severely elongated ellipsoids,
while high NA confocal PSFs look nearly spherical.

For a compound microscope, this means you change the depth of focus, as
Craig has already explained.  For thin sections under 1 um thick, the
section thickness is less than the Z-resolution, so there is no chance in
Z-res with NA as the sample is limiting.

However, if you do a focus series and then do a deconvolution or high-pass
filter, you will notice a much sharper image in the XZ and YZ axes with
high NA, which reflects the improved Z resolution.

One other thing to note when it comes to fluorescence, not only does higher
NA improve resolution, but it also improves your ability to detect
fluorescence per unit area.  As you pointed out, biological fluorochromes
behave as spherical emitters, so the higher the NA, the more of the emitted
light you collect.

Your initial observation does lead to one neat feature, which is that
because changing the NA of the excitation does not change the resolution of
the objective (unlike with transmitted light images), you can use the
aperture stop on the reflected light path to attenuate the intensity of the
arc lamp, without any considerable effect on the image in most cases.  If
your sample is thick, a higher excitation NA would still help, though, as
the excitation energy is then predominantly concentrated at the focal
plane, and rapidly attenuates outside the focal plane.  This means that
there is less out-of-focus light to remove at a pinhole aperture or
post-processing and therefore you will get better contrast.

Hope this helps,
   Ben Smith

On Fri, Jul 21, 2017 at 1:52 PM, Craig Brideau <[hidden email]>
wrote:

> *****
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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.
> *****
>
> It still influences the depth of field in widefield, which is analogous to
> the 'optical slice' in confocal. The thing is that widefield's confinement
> is so poor you don't necessarily see much difference unless working with
> two very different NAs. See depth of field in photography for the same
> idea.
>
> Craig
>
> On Fri, Jul 21, 2017 at 2:34 PM, MODEL, MICHAEL <[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.
> > *****
> >
> > I think NA of illumination does not affect resolution in wide field
> > fluorescence, only in confocal
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Daniel White
> > Sent: Friday, July 21, 2017 1:52 PM
> > To: [hidden email]
> > Subject: Fluorescence illumination NA vs. Z Resolution.
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://na01.safelinks.protection.outlook.com/?url=
> > http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%
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> > 7C36adaa158de34113205508d4d06140d1%7Ce5a06f4a1ec44d018f73e7dd15f2
> > 6134%7C1&sdata=IQXntzXiBHDoSi%2Fi0aMKiC0U1Qw%
> > 2BqYQasI74eB5Bxc4%3D&reserved=0
> > Post images on https://na01.safelinks.protection.outlook.com/?url=
> > http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%
> > 7C36adaa158de34113205508d4d06140d1%7Ce5a06f4a1ec44d018f73e7dd15f2
> > 6134%7C1&sdata=mVJGCykA4XhVh8DnNCu2V2uNIfQfOk1CTu0Zn7wJNgE%3D&reserved=0
> > and include the link in your posting.
> > *****
> >
> > Hi all
> >
> > Geeky question :
> >
> > What is the physical process by which illumination NA in a confocal or WF
> > fluorescence microscope affects the z resolution, the axial response
> (OTF)?
> >
> > Since fluorescence lifetime is nanoseconds, emission is incoherent wrt
> > excitation. Assuming free dye rotation during fluorescence lifetime, how
> > can angle range of incident photon affect angle range of emitted photon?
> >
> > What am I missing?
> >
> > What's the quantum electrodynamics explanation here? What would Feynman
> > have said?
> >
> > Cheers
> >
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
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e-mail: [hidden email]
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