Illumination source coherence requirement for SIM

[Lu Yan]

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Hi all,


I have no SIM experience so far. But I had always thought that in order to generate the grating/grid/structured pattern on the sample plane by interfering two beams would require coherent light sources like a laser with reasonable coherent length (or temporal coherence).


But then I realized there are also SIM using low-coherence light source like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes used. So I got a little confused. What is the fundamental difference bewteen these implementation? Is it just structured pattern contrast difference? What is the coherence requirement for SIM or similar tech. or is there any?


Thanks much in advance,

Lu

[Marc Reinig]

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Coherent light will produce speckled images.  You need the coherence
length to be considerably less than the exposure time.  We decorrelate
our laser with a spinning disk.  That results in both spatial and
temporal decorrelation.

Marco

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order to generate the grating/grid/structured pattern on the sample plane by interfering two beams would require coherent light sources like a laser with reasonable coherent length (or temporal coherence).
>
>
> But then I realized there are also SIM using low-coherence light source like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes used. So I got a little confused. What is the fundamental difference bewteen these implementation? Is it just structured pattern contrast difference? What is the coherence requirement for SIM or similar tech. or is there any?
>
>
> Thanks much in advance,
>
> Lu

[Zdenek Svindrych-2]

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*****

Hi Lu,
the quick answer is: yes, you can do SIM with LEDs (we used to do that in
Guy Hagen's lab, there are other papers out there as well), but the contrast
is lower and you have to use lower spatial frequencies of the patterns...
There are a number of ways to create the pattern in the sample space with
lasers. But with LEDs the (probably) only way is to image an amplitude
grating (or a pattern on an amplitude spatial light modulator) into the
sample space. Then the contrast of the pattern is given by the Opical
Transfer Function (OTF) or better the Modulation Transfer Function (MTF) of
the objective lens. That means the contrast of the high-frequency patterns
(at the resolution limit of the objective) is essentially zero. At lower
frequencies you can get some useful contrast, but the superresolution
improvement will be less than factor of 2. Also, this incoherent pattern has
different z-profile than that of either 2D or 3D laser-based SIM.

You can increase the spatial coherence of the LED source with iris stops
cleverly position in the optical setup, but that will cut your illumination
intensity dramatically (and I'm not sure how much contrast improvement you
get).
So, you can use LEDs for SIM, but to get the most of it,  the image
processing involved will be different than what has been done traditionally.
You can think of it as improved deconvolution, where the missing cone
problem is eliminated thanks to the patterned illumination.

Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
tel: [phone]

---------- Původní e-mail ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 27. 7. 2017 13:32:37
Předmět: Illumination source coherence requirement for SIM
"*****
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*****

Hi all,


I have no SIM experience so far. But I had always thought that in order to
generate the grating/grid/structured pattern on the sample plane by
interfering two beams would require coherent light sources like a laser with
reasonable coherent length (or temporal coherence).


But then I realized there are also SIM using low-coherence light source like
diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes
used. So I got a little confused. What is the fundamental difference bewteen
these implementation? Is it just structured pattern contrast difference?
What is the coherence requirement for SIM or similar tech. or is there any?


Thanks much in advance,

Lu
"

[Marc Reinig]

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

One further thought on the SIM illumination.  You should have the
illumination polarization lined up with your pattern for optimum
contrast.  We use a laser and a polarization rotator for that reason.
Plus we often image at 488 nm and LED's are hard to come by that are
bright enough once you've filtered out the unwanted polarizations.

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 11:54 AM,  <[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 Lu,
> the quick answer is: yes, you can do SIM with LEDs (we used to do that in
> Guy Hagen's lab, there are other papers out there as well), but the contrast
> is lower and you have to use lower spatial frequencies of the patterns...
> There are a number of ways to create the pattern in the sample space with
> lasers. But with LEDs the (probably) only way is to image an amplitude
> grating (or a pattern on an amplitude spatial light modulator) into the
> sample space. Then the contrast of the pattern is given by the Opical
> Transfer Function (OTF) or better the Modulation Transfer Function (MTF) of
> the objective lens. That means the contrast of the high-frequency patterns
> (at the resolution limit of the objective) is essentially zero. At lower
> frequencies you can get some useful contrast, but the superresolution
> improvement will be less than factor of 2. Also, this incoherent pattern has
> different z-profile than that of either 2D or 3D laser-based SIM.
>
> You can increase the spatial coherence of the LED source with iris stops
> cleverly position in the optical setup, but that will cut your illumination
> intensity dramatically (and I'm not sure how much contrast improvement you
> get).
> So, you can use LEDs for SIM, but to get the most of it,  the image
> processing involved will be different than what has been done traditionally.
> You can think of it as improved deconvolution, where the missing cone
> problem is eliminated thanks to the patterned illumination.
>
> Best, zdenek
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> Department of Biology,University of Virginia
> 409 McCormick Rd, Charlottesville, VA-22904
> http://www.kcci.virginia.edu/
> tel: [phone]
>
> ---------- Původní e-mail ----------
> Od: Yan, Lu <[hidden email]>
> Komu: [hidden email]
> Datum: 27. 7. 2017 13:32:37
> Předmět: Illumination source coherence requirement for SIM
> "*****
> 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 have no SIM experience so far. But I had always thought that in order to
> generate the grating/grid/structured pattern on the sample plane by
> interfering two beams would require coherent light sources like a laser with
> reasonable coherent length (or temporal coherence).
>
>
> But then I realized there are also SIM using low-coherence light source like
> diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes
> used. So I got a little confused. What is the fundamental difference bewteen
> these implementation? Is it just structured pattern contrast difference?
> What is the coherence requirement for SIM or similar tech. or is there any?
>
>
> Thanks much in advance,
>
> Lu
> "

[Lu Yan]

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

Hi zdenek,


Thanks for your reply.


Now I understand that using the LED sources the grid pattern has to be re-imaged onto the sample, and the contrast depends on the MTF of the lens. But for interference-based pattern generation, i.e. using a grating to essentially split a beam (into +1 and -1 orders) and then bring them together at the sample plane to get the interference pattern, this configuration will need coherent source, i.e. coherent length larger than the beam path difference, right?


The resultant fringe/pattern spacing depends on the angle between two beams and of course the wavelength. However, in this configuration the sample plane is also the image plane of the grating, so how is this interference not same as re-imaging the grating pattern? This is what I am confused about.


Thanks,

Lu

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Thursday, July 27, 2017 2:54:55 PM
To: [hidden email]
Subject: Re: Illumination source coherence requirement for SIM

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

Hi Lu,
the quick answer is: yes, you can do SIM with LEDs (we used to do that in
Guy Hagen's lab, there are other papers out there as well), but the contrast
is lower and you have to use lower spatial frequencies of the patterns...
There are a number of ways to create the pattern in the sample space with
lasers. But with LEDs the (probably) only way is to image an amplitude
grating (or a pattern on an amplitude spatial light modulator) into the
sample space. Then the contrast of the pattern is given by the Opical
Transfer Function (OTF) or better the Modulation Transfer Function (MTF) of
the objective lens. That means the contrast of the high-frequency patterns
(at the resolution limit of the objective) is essentially zero. At lower
frequencies you can get some useful contrast, but the superresolution
improvement will be less than factor of 2. Also, this incoherent pattern has
different z-profile than that of either 2D or 3D laser-based SIM.

You can increase the spatial coherence of the LED source with iris stops
cleverly position in the optical setup, but that will cut your illumination
intensity dramatically (and I'm not sure how much contrast improvement you
get).
So, you can use LEDs for SIM, but to get the most of it,  the image
processing involved will be different than what has been done traditionally.
You can think of it as improved deconvolution, where the missing cone
problem is eliminated thanks to the patterned illumination.

Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
tel: [phone]

---------- Původní e-mail ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 27. 7. 2017 13:32:37
Předmět: Illumination source coherence requirement for SIM
"*****
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 have no SIM experience so far. But I had always thought that in order to
generate the grating/grid/structured pattern on the sample plane by
interfering two beams would require coherent light sources like a laser with
reasonable coherent length (or temporal coherence).


But then I realized there are also SIM using low-coherence light source like
diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes
used. So I got a little confused. What is the fundamental difference bewteen
these implementation? Is it just structured pattern contrast difference?
What is the coherence requirement for SIM or similar tech. or is there any?


Thanks much in advance,

Lu
"

[Lu Yan]

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

Hi Marco,


Agreed. But isn't the two beam interference depending on the temporal coherence instead of spatial? Please correct me if I am wrong.


Thanks,

Lu

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Marc Reinig <[hidden email]>
Sent: Thursday, July 27, 2017 1:59:34 PM
To: [hidden email]
Subject: Re: Illumination source coherence requirement for SIM

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

Coherent light will produce speckled images.  You need the coherence
length to be considerably less than the exposure time.  We decorrelate
our laser with a spinning disk.  That results in both spatial and
temporal decorrelation.

Marco

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order to generate the grating/grid/structured pattern on the sample plane by interfering two beams would require coherent light sources like a laser with reasonable coherent length (or temporal coherence).
>
>
> But then I realized there are also SIM using low-coherence light source like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes used. So I got a little confused. What is the fundamental difference bewteen these implementation? Is it just structured pattern contrast difference? What is the coherence requirement for SIM or similar tech. or is there any?
>
>
> Thanks much in advance,
>
> Lu

[Zdenek Svindrych-2]

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

Exactly!
The two optical arrangements are the same, the only difference is the
illumination source and possibly the grating period.

Imagine this: you start with laser-based 2- or 3- beam SIM, that is, you
have two or three small spot in the BFP of the objective lens. Now, if you
increase the spatial (or angular) extent of the light source, that is,
increase etendue and decrease spatial coherence, the spots in the BFP start
to grow. E.g. 50 um / 0.22 NA fiber is still a good source for SIM, the
'spots' in the BFP are bigger, but still distinctly separated (depends on
the size of the illuminated field of view, too), pattern contrast is high
and you can reduce the annoying speckle...

But if you increase the source etendue further, the 'spots' start to overlap
and the contrast suffers. With a decent power LED (1 by 1 mm chip,
Lambertian emission) you always overfill the BFP of a high magnification
objective, so you are working in the incoherent regime, governed by the MTF.

The temporal (in-)coherence and spectral bandwidth play minor role, imho.

If you like fourier optics, the pattern intensity in focus should be the
modulus of the FFT of the electric field distribution in the BFP (in the
coherent case, ignoring vectorial and polarization effects). From that
calculation it appears that the contrast decreases as the spots in the BFP
grow, even if the field in the BFP is otherwise perfectly temporally
coherent...
best, zdenek


---------- Původní e-mail ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 27. 7. 2017 20:26:46
Předmět: Re: Illumination source coherence requirement for SIM
"*****
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*****

Hi zdenek,

Thanks for your reply.

Now I understand that using the LED sources the grid pattern has to be re-
imaged onto the sample, and the contrast depends on the MTF of the lens. But
for interference-based pattern generation, i.e. using a grating to
essentially split a beam (into +1 and -1 orders) and then bring them
together at the sample plane to get the interference pattern, this
configuration will need coherent source, i.e. coherent length larger than
the beam path difference, right?

The resultant fringe/pattern spacing depends on the angle between two beams
and of course the wavelength. However, in this configuration the sample
plane is also the image plane of the grating, so how is this interference
not same as re-imaging the grating pattern? This is what I am confused
about.

Thanks,
Lu
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf
of [hidden email] <[hidden email]>
Sent: Thursday, July 27, 2017 2:54:55 PM
To: [hidden email]
Subject: Re: Illumination source coherence requirement for SIM

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

Hi Lu,
the quick answer is: yes, you can do SIM with LEDs (we used to do that in
Guy Hagen's lab, there are other papers out there as well), but the contrast

is lower and you have to use lower spatial frequencies of the patterns...
There are a number of ways to create the pattern in the sample space with
lasers. But with LEDs the (probably) only way is to image an amplitude
grating (or a pattern on an amplitude spatial light modulator) into the
sample space. Then the contrast of the pattern is given by the Opical
Transfer Function (OTF) or better the Modulation Transfer Function (MTF) of
the objective lens. That means the contrast of the high-frequency patterns
(at the resolution limit of the objective) is essentially zero. At lower
frequencies you can get some useful contrast, but the superresolution
improvement will be less than factor of 2. Also, this incoherent pattern has

different z-profile than that of either 2D or 3D laser-based SIM.

You can increase the spatial coherence of the LED source with iris stops
cleverly position in the optical setup, but that will cut your illumination
intensity dramatically (and I'm not sure how much contrast improvement you
get).
So, you can use LEDs for SIM, but to get the most of it, the image
processing involved will be different than what has been done traditionally.

You can think of it as improved deconvolution, where the missing cone
problem is eliminated thanks to the patterned illumination.

Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
tel: [phone]

---------- Původní e-mail ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 27. 7. 2017 13:32:37
Předmět: Illumination source coherence requirement for SIM
"*****
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*****

Hi all,

I have no SIM experience so far. But I had always thought that in order to
generate the grating/grid/structured pattern on the sample plane by
interfering two beams would require coherent light sources like a laser with

reasonable coherent length (or temporal coherence).

But then I realized there are also SIM using low-coherence light source like

diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes
used. So I got a little confused. What is the fundamental difference bewteen

these implementation? Is it just structured pattern contrast difference?
What is the coherence requirement for SIM or similar tech. or is there any?

Thanks much in advance,
Lu
"
"

[Marc Reinig]

*****
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 Lu,

If you were able to only temporally decorrelate the light over time,
the relative phases of the light interacting at different places would
stay the same.  The speckles, the places where they constructively and
destructively interfere, would stay the same because the path lengths
would stay constant.

When you spatially decorrelate over time, the light at different
places is changing phase relative to each other over time and you do
not get a stable speckle field because the places of constructive and
destructive interference are changing.

You need them both.

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 5:27 PM, Yan, Lu <[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 Marco,
>
>
> Agreed. But isn't the two beam interference depending on the temporal coherence instead of spatial? Please correct me if I am wrong.
>
>
> Thanks,
>
> Lu
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Marc Reinig <[hidden email]>
> Sent: Thursday, July 27, 2017 1:59:34 PM
> To: [hidden email]
> Subject: Re: Illumination source coherence requirement for SIM
>
> *****
> 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.
> *****
>
> Coherent light will produce speckled images.  You need the coherence
> length to be considerably less than the exposure time.  We decorrelate
> our laser with a spinning disk.  That results in both spatial and
> temporal decorrelation.
>
> Marco
>
> Marco
>
> Marc R. Reinig
> W. M. Keck Center for Adaptive Optical Microscopy
> University of California Santa Cruz
>
>
>
> On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order to generate the grating/grid/structured pattern on the sample plane by interfering two beams would require coherent light sources like a laser with reasonable coherent length (or temporal coherence).
>>
>>
>> But then I realized there are also SIM using low-coherence light source like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also sometimes used. So I got a little confused. What is the fundamental difference bewteen these implementation? Is it just structured pattern contrast difference? What is the coherence requirement for SIM or similar tech. or is there any?
>>
>>
>> Thanks much in advance,
>>
>> Lu

[Zdenek Svindrych-2]

*****
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*****

Let me just add, that it depends a lot on the way you create your pattern.
If you use fancy beamsplitters to get your two or three spots in the
objective BFP (the "GE OMX SIM" arrangement, for example), then the temporal
coherence (and, conversely, the spectral width) is of utmost importance. In
this case the spatial frequency of the pattern depends linearly on
wavelength, so if you want a good pattern contrast across, lets say, 1000
lines in your field of view, you need linewidth much smaller than 1/1000 of
the central wavelength. Easy with lasers, impossible with LEDs.

On the other hand, in the more traditional setup (e.g. Nikon N-SIM), where
you are imaging the pattern into the sample (as Lu and Marco mentioned) the
frequency of the pattern is given by the grating (or SLM patter) period, so
the contrast is high even with broadband source (note that for the shorter-
wavelength components the pattern frequency is less than the maximum
possible, and the long-wavelength components may miss the BFP aperture
completely, leading to zero contrast). Or, from another point of view: the
path difference between the two (or three) beams in the BFP is always zero
in this case. Also, the +/-1 "spots" in the BFP are no longer spots, but
little patches of rainbow...

Hope it's not too confusing.

Disclaimer: no commercial interest.

Disclaimer: I have not confirmed this experimentally. I still may be wrong.
Has anyone tried "broadband" SIM, e.g. with superluminescent diodes (not
really useful for fluorescence excitation due to red-NIR wavelengths), or
supercontinuum lasers?
Best, zdenek




---------- Původní e-mail ----------
Od: Marc Reinig <[hidden email]>
Komu: [hidden email]
Datum: 28. 7. 2017 14:28:27
Předmět: Re: Illumination source coherence requirement for SIM
"*****
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*****

Hi Lu,

If you were able to only temporally decorrelate the light over time,
the relative phases of the light interacting at different places would
stay the same. The speckles, the places where they constructively and
destructively interfere, would stay the same because the path lengths
would stay constant.

When you spatially decorrelate over time, the light at different
places is changing phase relative to each other over time and you do
not get a stable speckle field because the places of constructive and
destructive interference are changing.

You need them both.

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 5:27 PM, Yan, Lu <[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 Marco,
>
>
> Agreed. But isn't the two beam interference depending on the temporal

coherence instead of spatial? Please correct me if I am wrong.
>
>
> Thanks,
>
> Lu
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
behalf of Marc Reinig <[hidden email]>

> Sent: Thursday, July 27, 2017 1:59:34 PM
> To: [hidden email]
> Subject: Re: Illumination source coherence requirement for SIM
>
> *****
> 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.
> *****
>
> Coherent light will produce speckled images. You need the coherence
> length to be considerably less than the exposure time. We decorrelate
> our laser with a spinning disk. That results in both spatial and
> temporal decorrelation.
>
> Marco
>
> Marco
>
> Marc R. Reinig
> W. M. Keck Center for Adaptive Optical Microscopy
> University of California Santa Cruz
>
>
>
> On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order
to generate the grating/grid/structured pattern on the sample plane by
interfering two beams would require coherent light sources like a laser with
reasonable coherent length (or temporal coherence).
>>
>>
>> But then I realized there are also SIM using low-coherence light source
like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also
sometimes used. So I got a little confused. What is the fundamental
difference bewteen these implementation? Is it just structured pattern
contrast difference? What is the coherence requirement for SIM or similar
tech. or is there any?
>>
>>
>> Thanks much in advance,
>>
>> Lu
"

[Lu Yan]

*****
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.
*****

zdenek, Marco,

Thanks for your input. I found a pretty neat picture of explaining spatial and temporal coherence here https://www.rp-photonics.com/coherence.html (no commercial interest 😊). I think the rotating diffuser, or some vibrating diffuser, or a 'fiber shaker' will mainly break down the spatial coherence, but at the same time degrade the temporal coherence a bit.

Back to the SIM topic, supposed that you have very nice narrow linewidth single frequency laser to generate very fine interference pattern at the sample plane. However, as you record the image, this grating pattern will be, as zdenek mentioned, practical limited by the MTF of the lens. So does this mean there should be a limit to the grating pattern on sample? Or what is an optimal pattern dimensions at the sample plane?

Thanks,
Lu

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Friday, July 28, 2017 3:34:12 PM
To: [hidden email]
Subject: Re: Illumination source coherence requirement for SIM

*****
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.
*****

Let me just add, that it depends a lot on the way you create your pattern.
If you use fancy beamsplitters to get your two or three spots in the
objective BFP (the "GE OMX SIM" arrangement, for example), then the temporal
coherence (and, conversely, the spectral width) is of utmost importance. In
this case the spatial frequency of the pattern depends linearly on
wavelength, so if you want a good pattern contrast across, lets say, 1000
lines in your field of view, you need linewidth much smaller than 1/1000 of
the central wavelength. Easy with lasers, impossible with LEDs.

On the other hand, in the more traditional setup (e.g. Nikon N-SIM), where
you are imaging the pattern into the sample (as Lu and Marco mentioned) the
frequency of the pattern is given by the grating (or SLM patter) period, so
the contrast is high even with broadband source (note that for the shorter-
wavelength components the pattern frequency is less than the maximum
possible, and the long-wavelength components may miss the BFP aperture
completely, leading to zero contrast). Or, from another point of view: the
path difference between the two (or three) beams in the BFP is always zero
in this case. Also, the +/-1 "spots" in the BFP are no longer spots, but
little patches of rainbow...

Hope it's not too confusing.

Disclaimer: no commercial interest.

Disclaimer: I have not confirmed this experimentally. I still may be wrong.
Has anyone tried "broadband" SIM, e.g. with superluminescent diodes (not
really useful for fluorescence excitation due to red-NIR wavelengths), or
supercontinuum lasers?
Best, zdenek




---------- Původní e-mail ----------
Od: Marc Reinig <[hidden email]>
Komu: [hidden email]
Datum: 28. 7. 2017 14:28:27
Předmět: Re: Illumination source coherence requirement for SIM
"*****
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 Lu,

If you were able to only temporally decorrelate the light over time,
the relative phases of the light interacting at different places would
stay the same. The speckles, the places where they constructively and
destructively interfere, would stay the same because the path lengths
would stay constant.

When you spatially decorrelate over time, the light at different
places is changing phase relative to each other over time and you do
not get a stable speckle field because the places of constructive and
destructive interference are changing.

You need them both.

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 5:27 PM, Yan, Lu <[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 Marco,
>
>
> Agreed. But isn't the two beam interference depending on the temporal

coherence instead of spatial? Please correct me if I am wrong.
>
>
> Thanks,
>
> Lu
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
behalf of Marc Reinig <[hidden email]>

> Sent: Thursday, July 27, 2017 1:59:34 PM
> To: [hidden email]
> Subject: Re: Illumination source coherence requirement for SIM
>
> *****
> 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.
> *****
>
> Coherent light will produce speckled images. You need the coherence
> length to be considerably less than the exposure time. We decorrelate
> our laser with a spinning disk. That results in both spatial and
> temporal decorrelation.
>
> Marco
>
> Marco
>
> Marc R. Reinig
> W. M. Keck Center for Adaptive Optical Microscopy
> University of California Santa Cruz
>
>
>
> On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order
to generate the grating/grid/structured pattern on the sample plane by
interfering two beams would require coherent light sources like a laser with
reasonable coherent length (or temporal coherence).
>>
>>
>> But then I realized there are also SIM using low-coherence light source
like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also
sometimes used. So I got a little confused. What is the fundamental
difference bewteen these implementation? Is it just structured pattern
contrast difference? What is the coherence requirement for SIM or similar
tech. or is there any?
>>
>>
>> Thanks much in advance,
>>
>> Lu
"

[Marc Reinig]

*****
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.
*****

If the spatial cutoff frequency of your lens is F, you cannot project
a pattern at a spatial frequency, F, and a pattern with a spatial
frequency of F will not appear on the object plane.  Generally, a
pattern with a spatial frequency of 0.8 to 0.9 is the best you can
hope for.  This will give you a 1.8 to 1.9 theoretical increase in
resolution.

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Fri, Jul 28, 2017 at 2:51 PM, Yan, Lu <[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.
> *****
>
> zdenek, Marco,
>
> Thanks for your input. I found a pretty neat picture of explaining spatial and temporal coherence here https://www.rp-photonics.com/coherence.html (no commercial interest 😊). I think the rotating diffuser, or some vibrating diffuser, or a 'fiber shaker' will mainly break down the spatial coherence, but at the same time degrade the temporal coherence a bit.
>
> Back to the SIM topic, supposed that you have very nice narrow linewidth single frequency laser to generate very fine interference pattern at the sample plane. However, as you record the image, this grating pattern will be, as zdenek mentioned, practical limited by the MTF of the lens. So does this mean there should be a limit to the grating pattern on sample? Or what is an optimal pattern dimensions at the sample plane?
>
> Thanks,
> Lu
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
> Sent: Friday, July 28, 2017 3:34:12 PM
> To: [hidden email]
> Subject: Re: Illumination source coherence requirement for SIM
>
> *****
> 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.
> *****
>
> Let me just add, that it depends a lot on the way you create your pattern.
> If you use fancy beamsplitters to get your two or three spots in the
> objective BFP (the "GE OMX SIM" arrangement, for example), then the temporal
> coherence (and, conversely, the spectral width) is of utmost importance. In
> this case the spatial frequency of the pattern depends linearly on
> wavelength, so if you want a good pattern contrast across, lets say, 1000
> lines in your field of view, you need linewidth much smaller than 1/1000 of
> the central wavelength. Easy with lasers, impossible with LEDs.
>
> On the other hand, in the more traditional setup (e.g. Nikon N-SIM), where
> you are imaging the pattern into the sample (as Lu and Marco mentioned) the
> frequency of the pattern is given by the grating (or SLM patter) period, so
> the contrast is high even with broadband source (note that for the shorter-
> wavelength components the pattern frequency is less than the maximum
> possible, and the long-wavelength components may miss the BFP aperture
> completely, leading to zero contrast). Or, from another point of view: the
> path difference between the two (or three) beams in the BFP is always zero
> in this case. Also, the +/-1 "spots" in the BFP are no longer spots, but
> little patches of rainbow...
>
> Hope it's not too confusing.
>
> Disclaimer: no commercial interest.
>
> Disclaimer: I have not confirmed this experimentally. I still may be wrong.
> Has anyone tried "broadband" SIM, e.g. with superluminescent diodes (not
> really useful for fluorescence excitation due to red-NIR wavelengths), or
> supercontinuum lasers?
> Best, zdenek
>
>
>
>
> ---------- Původní e-mail ----------
> Od: Marc Reinig <[hidden email]>
> Komu: [hidden email]
> Datum: 28. 7. 2017 14:28:27
> Předmět: Re: Illumination source coherence requirement for SIM
> "*****
> 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 Lu,
>
> If you were able to only temporally decorrelate the light over time,
> the relative phases of the light interacting at different places would
> stay the same. The speckles, the places where they constructively and
> destructively interfere, would stay the same because the path lengths
> would stay constant.
>
> When you spatially decorrelate over time, the light at different
> places is changing phase relative to each other over time and you do
> not get a stable speckle field because the places of constructive and
> destructive interference are changing.
>
> You need them both.
>
> Marco
>
> Marc R. Reinig
> W. M. Keck Center for Adaptive Optical Microscopy
> University of California Santa Cruz
>
>
>
> On Thu, Jul 27, 2017 at 5:27 PM, Yan, Lu <[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 Marco,
>>
>>
>> Agreed. But isn't the two beam interference depending on the temporal
> coherence instead of spatial? Please correct me if I am wrong.
>>
>>
>> Thanks,
>>
>> Lu
>>
>> ________________________________
>> From: Confocal Microscopy List <[hidden email]> on
> behalf of Marc Reinig <[hidden email]>
>> Sent: Thursday, July 27, 2017 1:59:34 PM
>> To: [hidden email]
>> Subject: Re: Illumination source coherence requirement for SIM
>>
>> *****
>> 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.
>> *****
>>
>> Coherent light will produce speckled images. You need the coherence
>> length to be considerably less than the exposure time. We decorrelate
>> our laser with a spinning disk. That results in both spatial and
>> temporal decorrelation.
>>
>> Marco
>>
>> Marco
>>
>> Marc R. Reinig
>> W. M. Keck Center for Adaptive Optical Microscopy
>> University of California Santa Cruz
>>
>>
>>
>> On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order
> to generate the grating/grid/structured pattern on the sample plane by
> interfering two beams would require coherent light sources like a laser with
> reasonable coherent length (or temporal coherence).
>>>
>>>
>>> But then I realized there are also SIM using low-coherence light source
> like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also
> sometimes used. So I got a little confused. What is the fundamental
> difference bewteen these implementation? Is it just structured pattern
> contrast difference? What is the coherence requirement for SIM or similar
> tech. or is there any?
>>>
>>>
>>> Thanks much in advance,
>>>
>>> Lu
> "

[Zdenek Svindrych-2]

*****
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 Lu, 
to clarify, the point I was trying to make is that with LED illumination the
contrast of the high-frequency pattern will be vanishingly small (due to
nearly zero MTF near the highest spatial frequency).
With laser-based illumination you can get very close to the ultimate spatial
frequency limit with almost perfect contrast. The standard MTF does not
apply here (tried to explain this one of the previous posts).  Now, if you
place a thin fluorescent layer into the focus, you will not be able to see
that pattern on your camera! Here, again, you are limited by the MTF. But
the pattern is there, and that's what's important. The SIM algorithm can
extract the superresolution information from the variations of image
intensity as the pattern wipes across your sample (of course, there is no
superresolution information coming from an infinite fluorescent layer, but
if you put there subresolution fluorescent beads instead, their intensities
will vary periodically with pattern phase).

Note, the above holds for 2D SIM (+/-1st order) only, if you introduce the 0
th order beam, another pattern with half the spatial frequency will appear,
that frequency is low enough so you can actually see the pattern on your
camera (with the thin fluorescent layer sample in place).

So generally you want to use as high a spatial frequency as possible, so
that the +/-1st order just passes through the BFP aperture of the objective
lens. You can get very close to the NA limit of the lens (with a factor of
0.9 as mentioned by Marco, be also cautious about total internal
reflection!). 

The ultimate (shortest) pattern period in the sample plane is lambda/(2*NA).
It follows quite naturally from the fact, that a standing intensity pattern
of two counterpropagating beams has a period of lambda/2 (the factor of 1/2
comes from the important fact that intensity = amplitude^2).
Best, zdenek


---------- Původní e-mail ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 28. 7. 2017 17:53:33
Předmět: Re: Illumination source coherence requirement for SIM
"*****
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.
*****

zdenek, Marco,

Thanks for your input. I found a pretty neat picture of explaining spatial
and temporal coherence here https://www.rp-photonics.com/coherence.html (no
commercial interest 😊). I think the rotating diffuser, or some vibrating
diffuser, or a 'fiber shaker' will mainly break down the spatial coherence,
but at the same time degrade the temporal coherence a bit.

Back to the SIM topic, supposed that you have very nice narrow linewidth
single frequency laser to generate very fine interference pattern at the
sample plane. However, as you record the image, this grating pattern will
be, as zdenek mentioned, practical limited by the MTF of the lens. So does
this mean there should be a limit to the grating pattern on sample? Or what
is an optimal pattern dimensions at the sample plane?

Thanks,
Lu

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf
of [hidden email] <[hidden email]>
Sent: Friday, July 28, 2017 3:34:12 PM
To: [hidden email]
Subject: Re: Illumination source coherence requirement for SIM

*****
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.
*****

Let me just add, that it depends a lot on the way you create your pattern.
If you use fancy beamsplitters to get your two or three spots in the
objective BFP (the "GE OMX SIM" arrangement, for example), then the temporal

coherence (and, conversely, the spectral width) is of utmost importance. In
this case the spatial frequency of the pattern depends linearly on
wavelength, so if you want a good pattern contrast across, lets say, 1000
lines in your field of view, you need linewidth much smaller than 1/1000 of
the central wavelength. Easy with lasers, impossible with LEDs.

On the other hand, in the more traditional setup (e.g. Nikon N-SIM), where
you are imaging the pattern into the sample (as Lu and Marco mentioned) the
frequency of the pattern is given by the grating (or SLM patter) period, so
the contrast is high even with broadband source (note that for the shorter-
wavelength components the pattern frequency is less than the maximum
possible, and the long-wavelength components may miss the BFP aperture
completely, leading to zero contrast). Or, from another point of view: the
path difference between the two (or three) beams in the BFP is always zero
in this case. Also, the +/-1 "spots" in the BFP are no longer spots, but
little patches of rainbow...

Hope it's not too confusing.

Disclaimer: no commercial interest.

Disclaimer: I have not confirmed this experimentally. I still may be wrong.
Has anyone tried "broadband" SIM, e.g. with superluminescent diodes (not
really useful for fluorescence excitation due to red-NIR wavelengths), or
supercontinuum lasers?
Best, zdenek




---------- Původní e-mail ----------
Od: Marc Reinig <[hidden email]>
Komu: [hidden email]
Datum: 28. 7. 2017 14:28:27
Předmět: Re: Illumination source coherence requirement for SIM
"*****
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 Lu,

If you were able to only temporally decorrelate the light over time,
the relative phases of the light interacting at different places would
stay the same. The speckles, the places where they constructively and
destructively interfere, would stay the same because the path lengths
would stay constant.

When you spatially decorrelate over time, the light at different
places is changing phase relative to each other over time and you do
not get a stable speckle field because the places of constructive and
destructive interference are changing.

You need them both.

Marco

Marc R. Reinig
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz



On Thu, Jul 27, 2017 at 5:27 PM, Yan, Lu <[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 Marco,
>
>
> Agreed. But isn't the two beam interference depending on the temporal

coherence instead of spatial? Please correct me if I am wrong.
>
>
> Thanks,
>
> Lu
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
behalf of Marc Reinig <[hidden email]>

> Sent: Thursday, July 27, 2017 1:59:34 PM
> To: [hidden email]
> Subject: Re: Illumination source coherence requirement for SIM
>
> *****
> 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.
> *****
>
> Coherent light will produce speckled images. You need the coherence
> length to be considerably less than the exposure time. We decorrelate
> our laser with a spinning disk. That results in both spatial and
> temporal decorrelation.
>
> Marco
>
> Marco
>
> Marc R. Reinig
> W. M. Keck Center for Adaptive Optical Microscopy
> University of California Santa Cruz
>
>
>
> On Thu, Jul 27, 2017 at 10:22 AM, Yan, Lu <[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 have no SIM experience so far. But I had always thought that in order
to generate the grating/grid/structured pattern on the sample plane by
interfering two beams would require coherent light sources like a laser with

reasonable coherent length (or temporal coherence).
>>
>>
>> But then I realized there are also SIM using low-coherence light source
like diode lasers, LED or lamp to do SIM, and a Ronchi grating is also
sometimes used. So I got a little confused. What is the fundamental
difference bewteen these implementation? Is it just structured pattern
contrast difference? What is the coherence requirement for SIM or similar
tech. or is there any?
>>
>>
>> Thanks much in advance,
>>
>> Lu
"
"