Reflected image -> astigmatism

[Kyle Michael Douglass]

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Hi everyone,

A couple years ago there was a post on this forum about significant
astigmatism appearing in the reflected path of a custom-built, two
camera TIRF setup.
(http://confocal-microscopy-list.588098.n2.nabble.com/Reflected-image-gt-astigmatism-td7584402.html)
The original poster had placed a dichroic before a pair of matched tube
lenses to split the two color channels onto two separate cameras. The
general consensus was that you could "buy your way out of the problem"
by buying a thick, ultraflat dichroic.

I am trying a similar approach on a setup in our lab but, due to space
constraints that are not easily overcome, have tried first placing the
channel-splitting dichroic in the image space after the final tube lens
instead of in the infinity space between the objective and tube lens.
Even with a 3 mm thick ultraflat dichroic, I see moderately bad
astigmatism in the reflected channel. I have ruled out other possible
sources of the astigmatism, like an additional dichroic for the
excitation light and filters that already lie in the infinity space
between the objective and tube lens.

My question is: is it even worth trying to eliminate the astigmatism in
the reflected path of a split-channel setup if the dichroic does not lie
in the infinity space? Or is it pretty much always a bad idea to put the
splitter dichroic in the image space?

My current suspicion is that the tolerances in the alignment have to be
very, very tight to avoid astigmatism with the splitter in the image space.

Thanks for your feedback.

Kyle

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Zdenek Svindrych-2]

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Hi Kyle,
if your transmitted image is free of astigmatism, then the dichroic must be
responsible for the astigmatism in the reflected path (assuming there are no
other components between the dichoic and the cameras). Try a flat mirror (I
mean really flat, 6mm substrate, lambda/10 specs) instead of the dichroic
and see if the astigmatism is gone.

The new 3mm dichroics have similar specs (lambda/4 to lambda/10), so if it
is mounted properly (it's not about exact angles, but you must not introduce
any stress that may lead to bending), it should work OK. Try another
dichroic, maybe one factory-mounted in a cube.

Also, the closer the dichroic to the camera, the smaller the effects of
curvature. I don't think infinty space is less 'sensitive' to astigmatism.
Quite the opposite - least sensitive should be a position conjugate to image
plane (but then, every piece of dust will be in focus on the camera...).

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]

Annual FRET Workshop (March 6-10, 2017): http://kcci.virginia.edu/workshop-
2017


---------- Původní zpráva ----------
Od: Kyle Douglass <[hidden email]>
Komu: [hidden email]
Datum: 13. 2. 2017 9:29:31
Předmět: Reflected image -> astigmatism

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Hi everyone,

A couple years ago there was a post on this forum about significant
astigmatism appearing in the reflected path of a custom-built, two
camera TIRF setup.
(http://confocal-microscopy-list.588098.n2.nabble.com/Reflected-image-gt-
astigmatism-td7584402.html)
The original poster had placed a dichroic before a pair of matched tube
lenses to split the two color channels onto two separate cameras. The
general consensus was that you could "buy your way out of the problem"
by buying a thick, ultraflat dichroic.

I am trying a similar approach on a setup in our lab but, due to space
constraints that are not easily overcome, have tried first placing the
channel-splitting dichroic in the image space after the final tube lens
instead of in the infinity space between the objective and tube lens.
Even with a 3 mm thick ultraflat dichroic, I see moderately bad
astigmatism in the reflected channel. I have ruled out other possible
sources of the astigmatism, like an additional dichroic for the
excitation light and filters that already lie in the infinity space
between the objective and tube lens.

My question is: is it even worth trying to eliminate the astigmatism in
the reflected path of a split-channel setup if the dichroic does not lie
in the infinity space? Or is it pretty much always a bad idea to put the
splitter dichroic in the image space?

My current suspicion is that the tolerances in the alignment have to be
very, very tight to avoid astigmatism with the splitter in the image space.

Thanks for your feedback.

Kyle

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io 
http://leb.epfl.ch 
"

[Jeffrey Carmichael]

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COMMERCIAL RESPONSE

Hi Kyle,

We've found that it's most often the mounting of the dichroic itself that
causes the most distortion in an image reflected off of a dichroic.  You
could theoretically have an "ideal" dichroic with no curvature, but once it
is affixed, it will be torqued out of flatness to some extent unless you
can completely avoid any mounting stress.

If held by means that apply pressure on top or bottom of the large surface,
you will get various forms of non-spherical astigmatism.  Even light
pressure on the sides of the dichroic will have some effect.

As Zdenek suggested, thicker dichroics are stiffer and will better resist
deformation.

If possible, try using something like silicone RTV applied very sparingly
in small beads around the outside edges of the dichroic, but never under
it.  This serves to prevent the dichroic from moving around without
applying stress.  If the "holder" is removable, then you can swap out these
mounted dichroics and not fiddle with the dichroic itself once affixed.

Jeff


*Jeff Carmichael*

*Technical and Product Marketing Manager*

*[hidden email] <[hidden email]>*Chroma Technology Corp.

*an employee owned company*
*10 Imtec Lane*
*Bellows Falls, VT  05301*
*802-428-2528 Office*
*802-428-2528 Fax**800-824-7662 Toll Free*

On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
>
> A couple years ago there was a post on this forum about significant
> astigmatism appearing in the reflected path of a custom-built, two camera
> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/Reflec
> ted-image-gt-astigmatism-td7584402.html) The original poster had placed a
> dichroic before a pair of matched tube lenses to split the two color
> channels onto two separate cameras. The general consensus was that you
> could "buy your way out of the problem" by buying a thick, ultraflat
> dichroic.
>
> I am trying a similar approach on a setup in our lab but, due to space
> constraints that are not easily overcome, have tried first placing the
> channel-splitting dichroic in the image space after the final tube lens
> instead of in the infinity space between the objective and tube lens. Even
> with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism in
> the reflected channel. I have ruled out other possible sources of the
> astigmatism, like an additional dichroic for the excitation light and
> filters that already lie in the infinity space between the objective and
> tube lens.
>
> My question is: is it even worth trying to eliminate the astigmatism in
> the reflected path of a split-channel setup if the dichroic does not lie in
> the infinity space? Or is it pretty much always a bad idea to put the
> splitter dichroic in the image space?
>
> My current suspicion is that the tolerances in the alignment have to be
> very, very tight to avoid astigmatism with the splitter in the image space.
>
> Thanks for your feedback.
>
> Kyle
>
> --
> Kyle M. Douglass, PhD
> Post-doctoral researcher
> The Laboratory of Experimental Biophysics
> EPFL, Lausanne, Switzerland
> http://kmdouglass.github.io
> http://leb.epfl.ch
>

[Rusty Nicovich]

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*****

Kyle,

The other poster was me.  I ultimately fixed the issue to my satisfaction
by carefully mounting the dichroic to the cube with double-sided tape
rather than the provided clamp.  That method was stress-free enough to not
induce any undue curvature in the dichroic, at least qualitatively as no
astigmatism immediately obvious in the reflected image.  This was even with
a relatively thin (1.1 mm) substrate for the mirror. This solution is
similar to what Jeff suggested using silicone adhesive instead of tape.

Another fun part of that system is that the camera fans would induce quite
a large vibration and only along one axis.  The vibration period was 10-20
ms, which meant any longer exposures would yield a PSF elongated on that
axis.  Astigmatism would vary along the focal axis and this didn't, but it
still gave an asymmetric PSF at the focal plane.

Thanks,
Rusty

On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <[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.
> *****
>
> COMMERCIAL RESPONSE
>
> Hi Kyle,
>
> We've found that it's most often the mounting of the dichroic itself that
> causes the most distortion in an image reflected off of a dichroic.  You
> could theoretically have an "ideal" dichroic with no curvature, but once it
> is affixed, it will be torqued out of flatness to some extent unless you
> can completely avoid any mounting stress.
>
> If held by means that apply pressure on top or bottom of the large surface,
> you will get various forms of non-spherical astigmatism.  Even light
> pressure on the sides of the dichroic will have some effect.
>
> As Zdenek suggested, thicker dichroics are stiffer and will better resist
> deformation.
>
> If possible, try using something like silicone RTV applied very sparingly
> in small beads around the outside edges of the dichroic, but never under
> it.  This serves to prevent the dichroic from moving around without
> applying stress.  If the "holder" is removable, then you can swap out these
> mounted dichroics and not fiddle with the dichroic itself once affixed.
>
> Jeff
>
>
> *Jeff Carmichael*
>
> *Technical and Product Marketing Manager*
>
> *[hidden email] <[hidden email]>*Chroma Technology Corp.
>
> *an employee owned company*
> *10 Imtec Lane*
> *Bellows Falls, VT  05301*
> *802-428-2528 Office*
> *802-428-2528 Fax**800-824-7662 Toll Free*
>
> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
> >
> > A couple years ago there was a post on this forum about significant
> > astigmatism appearing in the reflected path of a custom-built, two camera
> > TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/Reflec
> > ted-image-gt-astigmatism-td7584402.html) The original poster had placed
> a
> > dichroic before a pair of matched tube lenses to split the two color
> > channels onto two separate cameras. The general consensus was that you
> > could "buy your way out of the problem" by buying a thick, ultraflat
> > dichroic.
> >
> > I am trying a similar approach on a setup in our lab but, due to space
> > constraints that are not easily overcome, have tried first placing the
> > channel-splitting dichroic in the image space after the final tube lens
> > instead of in the infinity space between the objective and tube lens.
> Even
> > with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism in
> > the reflected channel. I have ruled out other possible sources of the
> > astigmatism, like an additional dichroic for the excitation light and
> > filters that already lie in the infinity space between the objective and
> > tube lens.
> >
> > My question is: is it even worth trying to eliminate the astigmatism in
> > the reflected path of a split-channel setup if the dichroic does not lie
> in
> > the infinity space? Or is it pretty much always a bad idea to put the
> > splitter dichroic in the image space?
> >
> > My current suspicion is that the tolerances in the alignment have to be
> > very, very tight to avoid astigmatism with the splitter in the image
> space.
> >
> > Thanks for your feedback.
> >
> > Kyle
> >
> > --
> > Kyle M. Douglass, PhD
> > Post-doctoral researcher
> > The Laboratory of Experimental Biophysics
> > EPFL, Lausanne, Switzerland
> > http://kmdouglass.github.io
> > http://leb.epfl.ch
> >
>

[Andrew York]

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*****

 I'm worried that your transmission path will also have astigmatism. Even a
perfectly flat piece of glass yields astigmatism in transmission if it's
tilted in a non-infinity space, right?

(Warning: lousy advice below)
 I've encountered this myself, and "fixed" it with a second piece of glass,
the same thickness and material as the first, tilted equally about an
orthogonal axis, to introduce "equal and opposite" astigmatism. This was a
hack, but it was good enough for my needs at the time. Depending on your
needs, perhaps you could use tilted flat glass as a tunable astigmatism
compensation.

On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <[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.
> *****
>
> Kyle,
>
> The other poster was me.  I ultimately fixed the issue to my satisfaction
> by carefully mounting the dichroic to the cube with double-sided tape
> rather than the provided clamp.  That method was stress-free enough to not
> induce any undue curvature in the dichroic, at least qualitatively as no
> astigmatism immediately obvious in the reflected image.  This was even with
> a relatively thin (1.1 mm) substrate for the mirror. This solution is
> similar to what Jeff suggested using silicone adhesive instead of tape.
>
> Another fun part of that system is that the camera fans would induce quite
> a large vibration and only along one axis.  The vibration period was 10-20
> ms, which meant any longer exposures would yield a PSF elongated on that
> axis.  Astigmatism would vary along the focal axis and this didn't, but it
> still gave an asymmetric PSF at the focal plane.
>
> Thanks,
> Rusty
>
> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
> [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.
> > *****
> >
> > COMMERCIAL RESPONSE
> >
> > Hi Kyle,
> >
> > We've found that it's most often the mounting of the dichroic itself that
> > causes the most distortion in an image reflected off of a dichroic.  You
> > could theoretically have an "ideal" dichroic with no curvature, but once
> it
> > is affixed, it will be torqued out of flatness to some extent unless you
> > can completely avoid any mounting stress.
> >
> > If held by means that apply pressure on top or bottom of the large
> surface,
> > you will get various forms of non-spherical astigmatism.  Even light
> > pressure on the sides of the dichroic will have some effect.
> >
> > As Zdenek suggested, thicker dichroics are stiffer and will better resist
> > deformation.
> >
> > If possible, try using something like silicone RTV applied very sparingly
> > in small beads around the outside edges of the dichroic, but never under
> > it.  This serves to prevent the dichroic from moving around without
> > applying stress.  If the "holder" is removable, then you can swap out
> these
> > mounted dichroics and not fiddle with the dichroic itself once affixed.
> >
> > Jeff
> >
> >
> > *Jeff Carmichael*
> >
> > *Technical and Product Marketing Manager*
> >
> > *[hidden email] <[hidden email]>*Chroma Technology Corp.
> >
> > *an employee owned company*
> > *10 Imtec Lane*
> > *Bellows Falls, VT  05301*
> > *802-428-2528 Office*
> > *802-428-2528 Fax**800-824-7662 Toll Free*
> >
> > On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
> > >
> > > A couple years ago there was a post on this forum about significant
> > > astigmatism appearing in the reflected path of a custom-built, two
> camera
> > > TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/
> Reflec
> > > ted-image-gt-astigmatism-td7584402.html) The original poster had
> placed
> > a
> > > dichroic before a pair of matched tube lenses to split the two color
> > > channels onto two separate cameras. The general consensus was that you
> > > could "buy your way out of the problem" by buying a thick, ultraflat
> > > dichroic.
> > >
> > > I am trying a similar approach on a setup in our lab but, due to space
> > > constraints that are not easily overcome, have tried first placing the
> > > channel-splitting dichroic in the image space after the final tube lens
> > > instead of in the infinity space between the objective and tube lens.
> > Even
> > > with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism
> in
> > > the reflected channel. I have ruled out other possible sources of the
> > > astigmatism, like an additional dichroic for the excitation light and
> > > filters that already lie in the infinity space between the objective
> and
> > > tube lens.
> > >
> > > My question is: is it even worth trying to eliminate the astigmatism in
> > > the reflected path of a split-channel setup if the dichroic does not
> lie
> > in
> > > the infinity space? Or is it pretty much always a bad idea to put the
> > > splitter dichroic in the image space?
> > >
> > > My current suspicion is that the tolerances in the alignment have to be
> > > very, very tight to avoid astigmatism with the splitter in the image
> > space.
> > >
> > > Thanks for your feedback.
> > >
> > > Kyle
> > >
> > > --
> > > Kyle M. Douglass, PhD
> > > Post-doctoral researcher
> > > The Laboratory of Experimental Biophysics
> > > EPFL, Lausanne, Switzerland
> > > http://kmdouglass.github.io
> > > http://leb.epfl.ch
> > >
> >
>

[Kyle Michael Douglass]

*****
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*****

Hi everyone,

Thanks a lot for your replies. To summarize your responses as I
understand them so far:

1) Mounting of the dichroic is an absolutely critical step and first
thing to check when troubleshooting astigmatism. I was taking care to
tighten the set screws in the mount only to the extent required to
prevent the dichroic from falling out. However, it seems that even this
is suspect and can warp the dichroic surface enough to induce
astigmatism upon reflection.

2) Using double-sided tape or a silicone adhesive applied to the outside
edges of the dichroic is a better way to mount it; avoid applying any
pressure to the dichroics large front and back surfaces.

3) Using a flat mirror in place of the dichroic is one way to check
whether it's really the dichroic causing the astigmatism or something else.

4) Based on differing responses, it's still not clear to me whether a
splitter dichroic in the image space (as opposed to the infinity space)
is a workable solution or something to be avoided at all costs. I can
perhaps partially correct for the astigmatism with a flat compensator plate.

Now that I think about it, I had already known that a tilted coverslip
can induce coma, especially in water-immersion objectives
(http://onlinelibrary.wiley.com/doi/10.1111/j.0022-2720.2004.01383.x/abstract 
<http://onlinelibrary.wiley.com/doi/10.1111/j.0022-2720.2004.01383.x/pdf>).
In general, coma (and astigmatism too, I think) originates from any
element where rays from common object points but traveling at different
azimuthal angles see different path lengths. I don't see why this logic
couldn't extend to a tilted dichroic in the image space as well.

Best regards,
Kyle

On 02/13/2017 10:34 PM, Andrew York 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'm worried that your transmission path will also have astigmatism. Even a
> perfectly flat piece of glass yields astigmatism in transmission if it's
> tilted in a non-infinity space, right?
>
> (Warning: lousy advice below)
>   I've encountered this myself, and "fixed" it with a second piece of glass,
> the same thickness and material as the first, tilted equally about an
> orthogonal axis, to introduce "equal and opposite" astigmatism. This was a
> hack, but it was good enough for my needs at the time. Depending on your
> needs, perhaps you could use tilted flat glass as a tunable astigmatism
> compensation.
>
> On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <[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.
>> *****
>>
>> Kyle,
>>
>> The other poster was me.  I ultimately fixed the issue to my satisfaction
>> by carefully mounting the dichroic to the cube with double-sided tape
>> rather than the provided clamp.  That method was stress-free enough to not
>> induce any undue curvature in the dichroic, at least qualitatively as no
>> astigmatism immediately obvious in the reflected image.  This was even with
>> a relatively thin (1.1 mm) substrate for the mirror. This solution is
>> similar to what Jeff suggested using silicone adhesive instead of tape.
>>
>> Another fun part of that system is that the camera fans would induce quite
>> a large vibration and only along one axis.  The vibration period was 10-20
>> ms, which meant any longer exposures would yield a PSF elongated on that
>> axis.  Astigmatism would vary along the focal axis and this didn't, but it
>> still gave an asymmetric PSF at the focal plane.
>>
>> Thanks,
>> Rusty
>>
>> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
>> [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.
>>> *****
>>>
>>> COMMERCIAL RESPONSE
>>>
>>> Hi Kyle,
>>>
>>> We've found that it's most often the mounting of the dichroic itself that
>>> causes the most distortion in an image reflected off of a dichroic.  You
>>> could theoretically have an "ideal" dichroic with no curvature, but once
>> it
>>> is affixed, it will be torqued out of flatness to some extent unless you
>>> can completely avoid any mounting stress.
>>>
>>> If held by means that apply pressure on top or bottom of the large
>> surface,
>>> you will get various forms of non-spherical astigmatism.  Even light
>>> pressure on the sides of the dichroic will have some effect.
>>>
>>> As Zdenek suggested, thicker dichroics are stiffer and will better resist
>>> deformation.
>>>
>>> If possible, try using something like silicone RTV applied very sparingly
>>> in small beads around the outside edges of the dichroic, but never under
>>> it.  This serves to prevent the dichroic from moving around without
>>> applying stress.  If the "holder" is removable, then you can swap out
>> these
>>> mounted dichroics and not fiddle with the dichroic itself once affixed.
>>>
>>> Jeff
>>>
>>>
>>> *Jeff Carmichael*
>>>
>>> *Technical and Product Marketing Manager*
>>>
>>> *[hidden email] <[hidden email]>*Chroma Technology Corp.
>>>
>>> *an employee owned company*
>>> *10 Imtec Lane*
>>> *Bellows Falls, VT  05301*
>>> *802-428-2528 Office*
>>> *802-428-2528 Fax**800-824-7662 Toll Free*
>>>
>>> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
>>>>
>>>> A couple years ago there was a post on this forum about significant
>>>> astigmatism appearing in the reflected path of a custom-built, two
>> camera
>>>> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/
>> Reflec
>>>> ted-image-gt-astigmatism-td7584402.html) The original poster had
>> placed
>>> a
>>>> dichroic before a pair of matched tube lenses to split the two color
>>>> channels onto two separate cameras. The general consensus was that you
>>>> could "buy your way out of the problem" by buying a thick, ultraflat
>>>> dichroic.
>>>>
>>>> I am trying a similar approach on a setup in our lab but, due to space
>>>> constraints that are not easily overcome, have tried first placing the
>>>> channel-splitting dichroic in the image space after the final tube lens
>>>> instead of in the infinity space between the objective and tube lens.
>>> Even
>>>> with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism
>> in
>>>> the reflected channel. I have ruled out other possible sources of the
>>>> astigmatism, like an additional dichroic for the excitation light and
>>>> filters that already lie in the infinity space between the objective
>> and
>>>> tube lens.
>>>>
>>>> My question is: is it even worth trying to eliminate the astigmatism in
>>>> the reflected path of a split-channel setup if the dichroic does not
>> lie
>>> in
>>>> the infinity space? Or is it pretty much always a bad idea to put the
>>>> splitter dichroic in the image space?
>>>>
>>>> My current suspicion is that the tolerances in the alignment have to be
>>>> very, very tight to avoid astigmatism with the splitter in the image
>>> space.
>>>> Thanks for your feedback.
>>>>
>>>> Kyle
>>>>
>>>> --
>>>> Kyle M. Douglass, PhD
>>>> Post-doctoral researcher
>>>> The Laboratory of Experimental Biophysics
>>>> EPFL, Lausanne, Switzerland
>>>> http://kmdouglass.github.io
>>>> http://leb.epfl.ch
>>>>

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Zdenek Svindrych-2]

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Hi Kyle,
First, reflection off of a flat mirror does not introduce any aberrations.
That's clear. So as long as it's the first surface that is reflective, the
reflected path should be perfect.


Second, transmission through a flat glass slab introduces spherical
aberration (and other aberrations if it's tilted). But the camera space
numerical aperture is so low that you can ignore them.

So, the main reason against putting filters and dichroics into the camera
image space (as I see it) is image shift and defocus as you change the
thickness and angle of the filters - this only applies to filter wheels, not
to rigidly fixed setup. Secondary reasons are blemishes (dirt on the filters
may be visible on the camera with very low NA illumination, especially when
observing specimens with transmitted light) and filter size (usually the
camera chip is larger than the back focal aperture of the objective lens, so
in principle you could use smaller filter there - not a concern in modern
microscopes where all filters are 1 inch dia...).

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]
Annual FRET Workshop (March 6-10, 2017): http://kcci.virginia.edu/workshop-
2017


---------- Původní zpráva ----------
Od: Kyle Douglass <[hidden email]>
Komu: [hidden email]
Datum: 14. 2. 2017 3:21:53
Předmět: Re: Reflected image -> astigmatism

"*****
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*****

Hi everyone,

Thanks a lot for your replies. To summarize your responses as I
understand them so far:

1) Mounting of the dichroic is an absolutely critical step and first
thing to check when troubleshooting astigmatism. I was taking care to
tighten the set screws in the mount only to the extent required to
prevent the dichroic from falling out. However, it seems that even this
is suspect and can warp the dichroic surface enough to induce
astigmatism upon reflection.

2) Using double-sided tape or a silicone adhesive applied to the outside
edges of the dichroic is a better way to mount it; avoid applying any
pressure to the dichroics large front and back surfaces.

3) Using a flat mirror in place of the dichroic is one way to check
whether it's really the dichroic causing the astigmatism or something else.

4) Based on differing responses, it's still not clear to me whether a
splitter dichroic in the image space (as opposed to the infinity space)
is a workable solution or something to be avoided at all costs. I can
perhaps partially correct for the astigmatism with a flat compensator plate.


Now that I think about it, I had already known that a tilted coverslip
can induce coma, especially in water-immersion objectives
(http://onlinelibrary.wiley.com/doi/10.1111/j.0022-2720.2004.01383.x/
abstract
<http://onlinelibrary.wiley.com/doi/10.1111/j.0022-2720.2004.01383.x/pdf>).

In general, coma (and astigmatism too, I think) originates from any
element where rays from common object points but traveling at different
azimuthal angles see different path lengths. I don't see why this logic
couldn't extend to a tilted dichroic in the image space as well.

Best regards,
Kyle

On 02/13/2017 10:34 PM, Andrew York 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'm worried that your transmission path will also have astigmatism. Even a

> perfectly flat piece of glass yields astigmatism in transmission if it's
> tilted in a non-infinity space, right?
>
> (Warning: lousy advice below)
> I've encountered this myself, and "fixed" it with a second piece of glass,

> the same thickness and material as the first, tilted equally about an
> orthogonal axis, to introduce "equal and opposite" astigmatism. This was a

> hack, but it was good enough for my needs at the time. Depending on your
> needs, perhaps you could use tilted flat glass as a tunable astigmatism
> compensation.
>
> On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <[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.

>> *****
>>
>> Kyle,
>>
>> The other poster was me. I ultimately fixed the issue to my satisfaction
>> by carefully mounting the dichroic to the cube with double-sided tape
>> rather than the provided clamp. That method was stress-free enough to not

>> induce any undue curvature in the dichroic, at least qualitatively as no
>> astigmatism immediately obvious in the reflected image. This was even
with
>> a relatively thin (1.1 mm) substrate for the mirror. This solution is
>> similar to what Jeff suggested using silicone adhesive instead of tape.
>>
>> Another fun part of that system is that the camera fans would induce
quite
>> a large vibration and only along one axis. The vibration period was 10-20

>> ms, which meant any longer exposures would yield a PSF elongated on that
>> axis. Astigmatism would vary along the focal axis and this didn't, but it

>> still gave an asymmetric PSF at the focal plane.
>>
>> Thanks,
>> Rusty
>>
>> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
>> [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.
>>> *****
>>>
>>> COMMERCIAL RESPONSE
>>>
>>> Hi Kyle,
>>>
>>> We've found that it's most often the mounting of the dichroic itself

that
>>> causes the most distortion in an image reflected off of a dichroic. You
>>> could theoretically have an "ideal" dichroic with no curvature, but once

>> it
>>> is affixed, it will be torqued out of flatness to some extent unless you

>>> can completely avoid any mounting stress.
>>>
>>> If held by means that apply pressure on top or bottom of the large
>> surface,
>>> you will get various forms of non-spherical astigmatism. Even light
>>> pressure on the sides of the dichroic will have some effect.
>>>
>>> As Zdenek suggested, thicker dichroics are stiffer and will better
resist
>>> deformation.
>>>
>>> If possible, try using something like silicone RTV applied very
sparingly
>>> in small beads around the outside edges of the dichroic, but never under

>>> it. This serves to prevent the dichroic from moving around without
>>> applying stress. If the "holder" is removable, then you can swap out
>> these
>>> mounted dichroics and not fiddle with the dichroic itself once affixed.
>>>
>>> Jeff
>>>
>>>
>>> *Jeff Carmichael*
>>>
>>> *Technical and Product Marketing Manager*
>>>
>>> *[hidden email] <[hidden email]>*Chroma Technology Corp.

>>>
>>> *an employee owned company*
>>> *10 Imtec Lane*
>>> *Bellows Falls, VT 05301*
>>> *802-428-2528 Office*
>>> *802-428-2528 Fax**800-824-7662 Toll Free*
>>>
>>> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
>>>>
>>>> A couple years ago there was a post on this forum about significant
>>>> astigmatism appearing in the reflected path of a custom-built, two
>> camera
>>>> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/ 
>> Reflec
>>>> ted-image-gt-astigmatism-td7584402.html) The original poster had
>> placed
>>> a
>>>> dichroic before a pair of matched tube lenses to split the two color
>>>> channels onto two separate cameras. The general consensus was that you
>>>> could "buy your way out of the problem" by buying a thick, ultraflat
>>>> dichroic.
>>>>
>>>> I am trying a similar approach on a setup in our lab but, due to space
>>>> constraints that are not easily overcome, have tried first placing the
>>>> channel-splitting dichroic in the image space after the final tube lens

>>>> instead of in the infinity space between the objective and tube lens.
>>> Even
>>>> with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism
>> in
>>>> the reflected channel. I have ruled out other possible sources of the
>>>> astigmatism, like an additional dichroic for the excitation light and
>>>> filters that already lie in the infinity space between the objective
>> and
>>>> tube lens.
>>>>
>>>> My question is: is it even worth trying to eliminate the astigmatism in

>>>> the reflected path of a split-channel setup if the dichroic does not
>> lie
>>> in
>>>> the infinity space? Or is it pretty much always a bad idea to put the
>>>> splitter dichroic in the image space?
>>>>
>>>> My current suspicion is that the tolerances in the alignment have to be

>>>> very, very tight to avoid astigmatism with the splitter in the image
>>> space.
>>>> Thanks for your feedback.
>>>>
>>>> Kyle
>>>>
>>>> --
>>>> Kyle M. Douglass, PhD
>>>> Post-doctoral researcher
>>>> The Laboratory of Experimental Biophysics
>>>> EPFL, Lausanne, Switzerland
>>>> http://kmdouglass.github.io 
>>>> http://leb.epfl.ch 
>>>>

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io 
http://leb.epfl.ch 
"

[James Kerin]

*****
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.
*****

COMMERCIAL RESPONSE

Our President and Managing Director have prepared the following response:

Hi Kyle

Based on our experience of designing image splitters and multicamera
adapters, we can strongly underline the points made by Jeff Carmichael.  
As he says, to avoid any aberrations in the reflected channel, the
dichroic must be “softly” mounted against a truly flat surface.  
Curvature in only one direction will indeed give astigmatism, but more
complex curvatures, especially if there is a bit of twist as well, can
give significant image distortion too – think fairground mirrors!  Even
if the images are more or less in focus over the field, this will
clearly show up when (as is so often required) the transmitted and
reflected images are superimposed for analysis.  As camera resolution
has improved, such effects have become ever more obvious, but even with
megapixel cameras it should be possible to overlay the images to within
a just a pixel or two over the field.  However that does require similar
detail to the design of the rest of the optical system, which should be
as symmetrical as possible between the transmitted and reflected
pathways, as it is in our image splitters as well as our camera adapters.

The flatness issue applies whether the reflection occurs in an infinity
space or an imaging space, but there is another problem if the dichroic
is in imaging space, namely that of astigmatism in the transmitted
channel.  This is because the dichroic is at an angle (typically 45
degrees of course) with respect to the light path. Optical modelling
shows the following effects, some of which may be counterintuitive.  As
might be expected though, the astigmatism, which is uniform over the
field, increases with the refractive index and thickness of the
dichroic, as well as with its angle.  (By the way, that's probably why
the dichroics used in microscopes to introduce an epi-illumination beam
are traditionally “thin”, typically just a millimetre, as in the older
(noninfinity) microscopes the dichroic was in the imaging space from the
objective).

We wonder if this effect might be relevant to Kyle's problem, especially
if the dichroic is relatively thick.  Relating to that, we have an
observation and a suggestion.  The optical modelling shows that the
astigmatism is independent of the dichroic's position within the imaging
space, so moving it closer to either the camera or the focussing lens
won't help.

We are also pleased from our modelling to confirm that Andrew York's
solution to use a second piece of glass mounted at an orthogonal angle
is a correct one rather than just a “hack”, even though it may “sound”
wrong.  The astigmatism arises because the effective thickness of the
glass is different in the x and y directions because of it being at an
angle.  The sign of the angle doesn't matter, so adding another piece of
glass angled oppositely would make the problem worse rather than
better.  But it you rotate by 90 degrees, the directional differences
now cancel, and you're just left with a small and probably negligible
(according to our modelling at least!) chromatic effect from the
dispersion of the glass.

Dr MV Thomas & Jez Graham
Cairn Research Ltd
Graveney Road
Faversham
Kent, ME13 8UP
UK
Cairn Research is a European scientific instruments manufacturer based
in Kent, UK.
www.cairn-research.co.uk




On 2/13/2017 9:34 PM, Andrew York 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'm worried that your transmission path will also have astigmatism. Even a
> perfectly flat piece of glass yields astigmatism in transmission if it's
> tilted in a non-infinity space, right?
>
> (Warning: lousy advice below)
>   I've encountered this myself, and "fixed" it with a second piece of glass,
> the same thickness and material as the first, tilted equally about an
> orthogonal axis, to introduce "equal and opposite" astigmatism. This was a
> hack, but it was good enough for my needs at the time. Depending on your
> needs, perhaps you could use tilted flat glass as a tunable astigmatism
> compensation.
>
> On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <[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.
>> *****
>>
>> Kyle,
>>
>> The other poster was me.  I ultimately fixed the issue to my satisfaction
>> by carefully mounting the dichroic to the cube with double-sided tape
>> rather than the provided clamp.  That method was stress-free enough to not
>> induce any undue curvature in the dichroic, at least qualitatively as no
>> astigmatism immediately obvious in the reflected image.  This was even with
>> a relatively thin (1.1 mm) substrate for the mirror. This solution is
>> similar to what Jeff suggested using silicone adhesive instead of tape.
>>
>> Another fun part of that system is that the camera fans would induce quite
>> a large vibration and only along one axis.  The vibration period was 10-20
>> ms, which meant any longer exposures would yield a PSF elongated on that
>> axis.  Astigmatism would vary along the focal axis and this didn't, but it
>> still gave an asymmetric PSF at the focal plane.
>>
>> Thanks,
>> Rusty
>>
>> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
>> [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.
>>> *****
>>>
>>> COMMERCIAL RESPONSE
>>>
>>> Hi Kyle,
>>>
>>> We've found that it's most often the mounting of the dichroic itself that
>>> causes the most distortion in an image reflected off of a dichroic.  You
>>> could theoretically have an "ideal" dichroic with no curvature, but once
>> it
>>> is affixed, it will be torqued out of flatness to some extent unless you
>>> can completely avoid any mounting stress.
>>>
>>> If held by means that apply pressure on top or bottom of the large
>> surface,
>>> you will get various forms of non-spherical astigmatism.  Even light
>>> pressure on the sides of the dichroic will have some effect.
>>>
>>> As Zdenek suggested, thicker dichroics are stiffer and will better resist
>>> deformation.
>>>
>>> If possible, try using something like silicone RTV applied very sparingly
>>> in small beads around the outside edges of the dichroic, but never under
>>> it.  This serves to prevent the dichroic from moving around without
>>> applying stress.  If the "holder" is removable, then you can swap out
>> these
>>> mounted dichroics and not fiddle with the dichroic itself once affixed.
>>>
>>> Jeff
>>>
>>>
>>> *Jeff Carmichael*
>>>
>>> *Technical and Product Marketing Manager*
>>>
>>> *[hidden email] <[hidden email]>*Chroma Technology Corp.
>>>
>>> *an employee owned company*
>>> *10 Imtec Lane*
>>> *Bellows Falls, VT  05301*
>>> *802-428-2528 Office*
>>> *802-428-2528 Fax**800-824-7662 Toll Free*
>>>
>>> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
>>>>
>>>> A couple years ago there was a post on this forum about significant
>>>> astigmatism appearing in the reflected path of a custom-built, two
>> camera
>>>> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/
>> Reflec
>>>> ted-image-gt-astigmatism-td7584402.html) The original poster had
>> placed
>>> a
>>>> dichroic before a pair of matched tube lenses to split the two color
>>>> channels onto two separate cameras. The general consensus was that you
>>>> could "buy your way out of the problem" by buying a thick, ultraflat
>>>> dichroic.
>>>>
>>>> I am trying a similar approach on a setup in our lab but, due to space
>>>> constraints that are not easily overcome, have tried first placing the
>>>> channel-splitting dichroic in the image space after the final tube lens
>>>> instead of in the infinity space between the objective and tube lens.
>>> Even
>>>> with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism
>> in
>>>> the reflected channel. I have ruled out other possible sources of the
>>>> astigmatism, like an additional dichroic for the excitation light and
>>>> filters that already lie in the infinity space between the objective
>> and
>>>> tube lens.
>>>>
>>>> My question is: is it even worth trying to eliminate the astigmatism in
>>>> the reflected path of a split-channel setup if the dichroic does not
>> lie
>>> in
>>>> the infinity space? Or is it pretty much always a bad idea to put the
>>>> splitter dichroic in the image space?
>>>>
>>>> My current suspicion is that the tolerances in the alignment have to be
>>>> very, very tight to avoid astigmatism with the splitter in the image
>>> space.
>>>> Thanks for your feedback.
>>>>
>>>> Kyle
>>>>
>>>> --
>>>> Kyle M. Douglass, PhD
>>>> Post-doctoral researcher
>>>> The Laboratory of Experimental Biophysics
>>>> EPFL, Lausanne, Switzerland
>>>> http://kmdouglass.github.io
>>>> http://leb.epfl.ch
>>>>

--

*James Kerin
*

*Marketing Director*
Cairn Research Ltd
Graveney Road
Faversham
Kent, ME13 8UP
UK

Direct: + 44 (0)1795 594507
Fax: + 44 (0) 1795 594510

[Alfred Millett-Sikking]

*****
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 Kyle,

Our friends and colleagues have done an excellent job of covering this
topic, and I very much agree with the contents and summaries provided. I
would like to add one further minor point which may or may not be
applicable depending on your setup:

WFE of any type (including astigmatism) is additive and you mentioned
upstream dichroics and filters (and mirrors?). If you're unlucky then it
may be that these previous optics have already put you 'on the edge' of an
acceptable/perceptible astigmatism and that the last guy is simply pushing
you over. If you can't fix it with careful mounting of the last guy then it
may be worth giving the upstream optics an equal measure of care
(especially if you have reflectors). Without a TWE measurement of the
system optic by optic you cannot tell what's going on, so just be
suspicious of everyone!

Regards,

Alfred Millett-Sikking.


On 14 February 2017 at 06:35, James Kerin <[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.
> *****
>
> COMMERCIAL RESPONSE
>
> Our President and Managing Director have prepared the following response:
>
> Hi Kyle
>
> Based on our experience of designing image splitters and multicamera
> adapters, we can strongly underline the points made by Jeff Carmichael.  As
> he says, to avoid any aberrations in the reflected channel, the dichroic
> must be “softly” mounted against a truly flat surface.  Curvature in only
> one direction will indeed give astigmatism, but more complex curvatures,
> especially if there is a bit of twist as well, can give significant image
> distortion too – think fairground mirrors!  Even if the images are more or
> less in focus over the field, this will clearly show up when (as is so
> often required) the transmitted and reflected images are superimposed for
> analysis.  As camera resolution has improved, such effects have become ever
> more obvious, but even with megapixel cameras it should be possible to
> overlay the images to within a just a pixel or two over the field.  However
> that does require similar detail to the design of the rest of the optical
> system, which should be as symmetrical as possible between the transmitted
> and reflected pathways, as it is in our image splitters as well as our
> camera adapters.
>
> The flatness issue applies whether the reflection occurs in an infinity
> space or an imaging space, but there is another problem if the dichroic is
> in imaging space, namely that of astigmatism in the transmitted channel.
> This is because the dichroic is at an angle (typically 45 degrees of
> course) with respect to the light path. Optical modelling shows the
> following effects, some of which may be counterintuitive.  As might be
> expected though, the astigmatism, which is uniform over the field,
> increases with the refractive index and thickness of the dichroic, as well
> as with its angle.  (By the way, that's probably why the dichroics used in
> microscopes to introduce an epi-illumination beam are traditionally “thin”,
> typically just a millimetre, as in the older (noninfinity) microscopes the
> dichroic was in the imaging space from the objective).
>
> We wonder if this effect might be relevant to Kyle's problem, especially
> if the dichroic is relatively thick.  Relating to that, we have an
> observation and a suggestion.  The optical modelling shows that the
> astigmatism is independent of the dichroic's position within the imaging
> space, so moving it closer to either the camera or the focussing lens won't
> help.
>
> We are also pleased from our modelling to confirm that Andrew York's
> solution to use a second piece of glass mounted at an orthogonal angle is a
> correct one rather than just a “hack”, even though it may “sound” wrong.
> The astigmatism arises because the effective thickness of the glass is
> different in the x and y directions because of it being at an angle.  The
> sign of the angle doesn't matter, so adding another piece of glass angled
> oppositely would make the problem worse rather than better.  But it you
> rotate by 90 degrees, the directional differences now cancel, and you're
> just left with a small and probably negligible (according to our modelling
> at least!) chromatic effect from the dispersion of the glass.
>
> Dr MV Thomas & Jez Graham
> Cairn Research Ltd
> Graveney Road
> Faversham
> Kent, ME13 8UP
> UK
> Cairn Research is a European scientific instruments manufacturer based in
> Kent, UK.
> www.cairn-research.co.uk
>
>
>
>
>
> On 2/13/2017 9:34 PM, Andrew York 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'm worried that your transmission path will also have astigmatism.
>> Even a
>> perfectly flat piece of glass yields astigmatism in transmission if it's
>> tilted in a non-infinity space, right?
>>
>> (Warning: lousy advice below)
>>   I've encountered this myself, and "fixed" it with a second piece of
>> glass,
>> the same thickness and material as the first, tilted equally about an
>> orthogonal axis, to introduce "equal and opposite" astigmatism. This was a
>> hack, but it was good enough for my needs at the time. Depending on your
>> needs, perhaps you could use tilted flat glass as a tunable astigmatism
>> compensation.
>>
>> On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <[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.
>>> *****
>>>
>>> Kyle,
>>>
>>> The other poster was me.  I ultimately fixed the issue to my satisfaction
>>> by carefully mounting the dichroic to the cube with double-sided tape
>>> rather than the provided clamp.  That method was stress-free enough to
>>> not
>>> induce any undue curvature in the dichroic, at least qualitatively as no
>>> astigmatism immediately obvious in the reflected image.  This was even
>>> with
>>> a relatively thin (1.1 mm) substrate for the mirror. This solution is
>>> similar to what Jeff suggested using silicone adhesive instead of tape.
>>>
>>> Another fun part of that system is that the camera fans would induce
>>> quite
>>> a large vibration and only along one axis.  The vibration period was
>>> 10-20
>>> ms, which meant any longer exposures would yield a PSF elongated on that
>>> axis.  Astigmatism would vary along the focal axis and this didn't, but
>>> it
>>> still gave an asymmetric PSF at the focal plane.
>>>
>>> Thanks,
>>> Rusty
>>>
>>> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
>>> [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.
>>>
>>>> *****
>>>>
>>>> COMMERCIAL RESPONSE
>>>>
>>>> Hi Kyle,
>>>>
>>>> We've found that it's most often the mounting of the dichroic itself
>>>> that
>>>> causes the most distortion in an image reflected off of a dichroic.  You
>>>> could theoretically have an "ideal" dichroic with no curvature, but once
>>>>
>>> it
>>>
>>>> is affixed, it will be torqued out of flatness to some extent unless you
>>>> can completely avoid any mounting stress.
>>>>
>>>> If held by means that apply pressure on top or bottom of the large
>>>>
>>> surface,
>>>
>>>> you will get various forms of non-spherical astigmatism.  Even light
>>>> pressure on the sides of the dichroic will have some effect.
>>>>
>>>> As Zdenek suggested, thicker dichroics are stiffer and will better
>>>> resist
>>>> deformation.
>>>>
>>>> If possible, try using something like silicone RTV applied very
>>>> sparingly
>>>> in small beads around the outside edges of the dichroic, but never under
>>>> it.  This serves to prevent the dichroic from moving around without
>>>> applying stress.  If the "holder" is removable, then you can swap out
>>>>
>>> these
>>>
>>>> mounted dichroics and not fiddle with the dichroic itself once affixed.
>>>>
>>>> Jeff
>>>>
>>>>
>>>> *Jeff Carmichael*
>>>>
>>>> *Technical and Product Marketing Manager*
>>>>
>>>> *[hidden email] <[hidden email]>*Chroma Technology
>>>> Corp.
>>>>
>>>> *an employee owned company*
>>>> *10 Imtec Lane*
>>>> *Bellows Falls, VT  05301*
>>>> *802-428-2528 Office*
>>>> *802-428-2528 Fax**800-824-7662 Toll Free*
>>>>
>>>> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
>>>>>
>>>>> A couple years ago there was a post on this forum about significant
>>>>> astigmatism appearing in the reflected path of a custom-built, two
>>>>>
>>>> camera
>>>
>>>> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/
>>>>>
>>>> Reflec
>>>
>>>> ted-image-gt-astigmatism-td7584402.html) The original poster had
>>>>>
>>>> placed
>>>
>>>> a
>>>>
>>>>> dichroic before a pair of matched tube lenses to split the two color
>>>>> channels onto two separate cameras. The general consensus was that you
>>>>> could "buy your way out of the problem" by buying a thick, ultraflat
>>>>> dichroic.
>>>>>
>>>>> I am trying a similar approach on a setup in our lab but, due to space
>>>>> constraints that are not easily overcome, have tried first placing the
>>>>> channel-splitting dichroic in the image space after the final tube lens
>>>>> instead of in the infinity space between the objective and tube lens.
>>>>>
>>>> Even
>>>>
>>>>> with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism
>>>>>
>>>> in
>>>
>>>> the reflected channel. I have ruled out other possible sources of the
>>>>> astigmatism, like an additional dichroic for the excitation light and
>>>>> filters that already lie in the infinity space between the objective
>>>>>
>>>> and
>>>
>>>> tube lens.
>>>>>
>>>>> My question is: is it even worth trying to eliminate the astigmatism in
>>>>> the reflected path of a split-channel setup if the dichroic does not
>>>>>
>>>> lie
>>>
>>>> in
>>>>
>>>>> the infinity space? Or is it pretty much always a bad idea to put the
>>>>> splitter dichroic in the image space?
>>>>>
>>>>> My current suspicion is that the tolerances in the alignment have to be
>>>>> very, very tight to avoid astigmatism with the splitter in the image
>>>>>
>>>> space.
>>>>
>>>>> Thanks for your feedback.
>>>>>
>>>>> Kyle
>>>>>
>>>>> --
>>>>> Kyle M. Douglass, PhD
>>>>> Post-doctoral researcher
>>>>> The Laboratory of Experimental Biophysics
>>>>> EPFL, Lausanne, Switzerland
>>>>> http://kmdouglass.github.io
>>>>> http://leb.epfl.ch
>>>>>
>>>>>
> --
>
> *James Kerin
> *
>
> *Marketing Director*
> Cairn Research Ltd
> Graveney Road
> Faversham
> Kent, ME13 8UP
> UK
>
> Direct: + 44 (0)1795 594507
> Fax: + 44 (0) 1795 594510
>

[Andrew York]

*****
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.
*****

Is the reflective side of the dichroic facing the incoming light? If not,
you might get astigmatism in reflection even from a perfectly flat dichroic
in a non-infinity space.

On Tue, Feb 14, 2017 at 10:51 AM, Alfred Millett-Sikking <
[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 Kyle,
>
> Our friends and colleagues have done an excellent job of covering this
> topic, and I very much agree with the contents and summaries provided. I
> would like to add one further minor point which may or may not be
> applicable depending on your setup:
>
> WFE of any type (including astigmatism) is additive and you mentioned
> upstream dichroics and filters (and mirrors?). If you're unlucky then it
> may be that these previous optics have already put you 'on the edge' of an
> acceptable/perceptible astigmatism and that the last guy is simply pushing
> you over. If you can't fix it with careful mounting of the last guy then it
> may be worth giving the upstream optics an equal measure of care
> (especially if you have reflectors). Without a TWE measurement of the
> system optic by optic you cannot tell what's going on, so just be
> suspicious of everyone!
>
> Regards,
>
> Alfred Millett-Sikking.
>
>
> On 14 February 2017 at 06:35, James Kerin <[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.
> > *****
> >
> > COMMERCIAL RESPONSE
> >
> > Our President and Managing Director have prepared the following response:
> >
> > Hi Kyle
> >
> > Based on our experience of designing image splitters and multicamera
> > adapters, we can strongly underline the points made by Jeff Carmichael.
> As
> > he says, to avoid any aberrations in the reflected channel, the dichroic
> > must be “softly” mounted against a truly flat surface.  Curvature in only
> > one direction will indeed give astigmatism, but more complex curvatures,
> > especially if there is a bit of twist as well, can give significant image
> > distortion too – think fairground mirrors!  Even if the images are more
> or
> > less in focus over the field, this will clearly show up when (as is so
> > often required) the transmitted and reflected images are superimposed for
> > analysis.  As camera resolution has improved, such effects have become
> ever
> > more obvious, but even with megapixel cameras it should be possible to
> > overlay the images to within a just a pixel or two over the field.
> However
> > that does require similar detail to the design of the rest of the optical
> > system, which should be as symmetrical as possible between the
> transmitted
> > and reflected pathways, as it is in our image splitters as well as our
> > camera adapters.
> >
> > The flatness issue applies whether the reflection occurs in an infinity
> > space or an imaging space, but there is another problem if the dichroic
> is
> > in imaging space, namely that of astigmatism in the transmitted channel.
> > This is because the dichroic is at an angle (typically 45 degrees of
> > course) with respect to the light path. Optical modelling shows the
> > following effects, some of which may be counterintuitive.  As might be
> > expected though, the astigmatism, which is uniform over the field,
> > increases with the refractive index and thickness of the dichroic, as
> well
> > as with its angle.  (By the way, that's probably why the dichroics used
> in
> > microscopes to introduce an epi-illumination beam are traditionally
> “thin”,
> > typically just a millimetre, as in the older (noninfinity) microscopes
> the
> > dichroic was in the imaging space from the objective).
> >
> > We wonder if this effect might be relevant to Kyle's problem, especially
> > if the dichroic is relatively thick.  Relating to that, we have an
> > observation and a suggestion.  The optical modelling shows that the
> > astigmatism is independent of the dichroic's position within the imaging
> > space, so moving it closer to either the camera or the focussing lens
> won't
> > help.
> >
> > We are also pleased from our modelling to confirm that Andrew York's
> > solution to use a second piece of glass mounted at an orthogonal angle
> is a
> > correct one rather than just a “hack”, even though it may “sound” wrong.
> > The astigmatism arises because the effective thickness of the glass is
> > different in the x and y directions because of it being at an angle.  The
> > sign of the angle doesn't matter, so adding another piece of glass angled
> > oppositely would make the problem worse rather than better.  But it you
> > rotate by 90 degrees, the directional differences now cancel, and you're
> > just left with a small and probably negligible (according to our
> modelling
> > at least!) chromatic effect from the dispersion of the glass.
> >
> > Dr MV Thomas & Jez Graham
> > Cairn Research Ltd
> > Graveney Road
> > Faversham
> > Kent, ME13 8UP
> > UK
> > Cairn Research is a European scientific instruments manufacturer based in
> > Kent, UK.
> > www.cairn-research.co.uk
> >
> >
> >
> >
> >
> > On 2/13/2017 9:34 PM, Andrew York 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'm worried that your transmission path will also have astigmatism.
> >> Even a
> >> perfectly flat piece of glass yields astigmatism in transmission if it's
> >> tilted in a non-infinity space, right?
> >>
> >> (Warning: lousy advice below)
> >>   I've encountered this myself, and "fixed" it with a second piece of
> >> glass,
> >> the same thickness and material as the first, tilted equally about an
> >> orthogonal axis, to introduce "equal and opposite" astigmatism. This
> was a
> >> hack, but it was good enough for my needs at the time. Depending on your
> >> needs, perhaps you could use tilted flat glass as a tunable astigmatism
> >> compensation.
> >>
> >> On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <
> [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.
> >>> *****
> >>>
> >>> Kyle,
> >>>
> >>> The other poster was me.  I ultimately fixed the issue to my
> satisfaction
> >>> by carefully mounting the dichroic to the cube with double-sided tape
> >>> rather than the provided clamp.  That method was stress-free enough to
> >>> not
> >>> induce any undue curvature in the dichroic, at least qualitatively as
> no
> >>> astigmatism immediately obvious in the reflected image.  This was even
> >>> with
> >>> a relatively thin (1.1 mm) substrate for the mirror. This solution is
> >>> similar to what Jeff suggested using silicone adhesive instead of tape.
> >>>
> >>> Another fun part of that system is that the camera fans would induce
> >>> quite
> >>> a large vibration and only along one axis.  The vibration period was
> >>> 10-20
> >>> ms, which meant any longer exposures would yield a PSF elongated on
> that
> >>> axis.  Astigmatism would vary along the focal axis and this didn't, but
> >>> it
> >>> still gave an asymmetric PSF at the focal plane.
> >>>
> >>> Thanks,
> >>> Rusty
> >>>
> >>> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
> >>> [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.
> >>>
> >>>> *****
> >>>>
> >>>> COMMERCIAL RESPONSE
> >>>>
> >>>> Hi Kyle,
> >>>>
> >>>> We've found that it's most often the mounting of the dichroic itself
> >>>> that
> >>>> causes the most distortion in an image reflected off of a dichroic.
> You
> >>>> could theoretically have an "ideal" dichroic with no curvature, but
> once
> >>>>
> >>> it
> >>>
> >>>> is affixed, it will be torqued out of flatness to some extent unless
> you
> >>>> can completely avoid any mounting stress.
> >>>>
> >>>> If held by means that apply pressure on top or bottom of the large
> >>>>
> >>> surface,
> >>>
> >>>> you will get various forms of non-spherical astigmatism.  Even light
> >>>> pressure on the sides of the dichroic will have some effect.
> >>>>
> >>>> As Zdenek suggested, thicker dichroics are stiffer and will better
> >>>> resist
> >>>> deformation.
> >>>>
> >>>> If possible, try using something like silicone RTV applied very
> >>>> sparingly
> >>>> in small beads around the outside edges of the dichroic, but never
> under
> >>>> it.  This serves to prevent the dichroic from moving around without
> >>>> applying stress.  If the "holder" is removable, then you can swap out
> >>>>
> >>> these
> >>>
> >>>> mounted dichroics and not fiddle with the dichroic itself once
> affixed.
> >>>>
> >>>> Jeff
> >>>>
> >>>>
> >>>> *Jeff Carmichael*
> >>>>
> >>>> *Technical and Product Marketing Manager*
> >>>>
> >>>> *[hidden email] <[hidden email]>*Chroma Technology
> >>>> Corp.
> >>>>
> >>>> *an employee owned company*
> >>>> *10 Imtec Lane*
> >>>> *Bellows Falls, VT  05301*
> >>>> *802-428-2528 Office*
> >>>> *802-428-2528 Fax**800-824-7662 Toll Free*
> >>>>
> >>>> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
> >>>>>
> >>>>> A couple years ago there was a post on this forum about significant
> >>>>> astigmatism appearing in the reflected path of a custom-built, two
> >>>>>
> >>>> camera
> >>>
> >>>> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/
> >>>>>
> >>>> Reflec
> >>>
> >>>> ted-image-gt-astigmatism-td7584402.html) The original poster had
> >>>>>
> >>>> placed
> >>>
> >>>> a
> >>>>
> >>>>> dichroic before a pair of matched tube lenses to split the two color
> >>>>> channels onto two separate cameras. The general consensus was that
> you
> >>>>> could "buy your way out of the problem" by buying a thick, ultraflat
> >>>>> dichroic.
> >>>>>
> >>>>> I am trying a similar approach on a setup in our lab but, due to
> space
> >>>>> constraints that are not easily overcome, have tried first placing
> the
> >>>>> channel-splitting dichroic in the image space after the final tube
> lens
> >>>>> instead of in the infinity space between the objective and tube lens.
> >>>>>
> >>>> Even
> >>>>
> >>>>> with a 3 mm thick ultraflat dichroic, I see moderately bad
> astigmatism
> >>>>>
> >>>> in
> >>>
> >>>> the reflected channel. I have ruled out other possible sources of the
> >>>>> astigmatism, like an additional dichroic for the excitation light and
> >>>>> filters that already lie in the infinity space between the objective
> >>>>>
> >>>> and
> >>>
> >>>> tube lens.
> >>>>>
> >>>>> My question is: is it even worth trying to eliminate the astigmatism
> in
> >>>>> the reflected path of a split-channel setup if the dichroic does not
> >>>>>
> >>>> lie
> >>>
> >>>> in
> >>>>
> >>>>> the infinity space? Or is it pretty much always a bad idea to put the
> >>>>> splitter dichroic in the image space?
> >>>>>
> >>>>> My current suspicion is that the tolerances in the alignment have to
> be
> >>>>> very, very tight to avoid astigmatism with the splitter in the image
> >>>>>
> >>>> space.
> >>>>
> >>>>> Thanks for your feedback.
> >>>>>
> >>>>> Kyle
> >>>>>
> >>>>> --
> >>>>> Kyle M. Douglass, PhD
> >>>>> Post-doctoral researcher
> >>>>> The Laboratory of Experimental Biophysics
> >>>>> EPFL, Lausanne, Switzerland
> >>>>> http://kmdouglass.github.io
> >>>>> http://leb.epfl.ch
> >>>>>
> >>>>>
> > --
> >
> > *James Kerin
> > *
> >
> > *Marketing Director*
> > Cairn Research Ltd
> > Graveney Road
> > Faversham
> > Kent, ME13 8UP
> > UK
> >
> > Direct: + 44 (0)1795 594507
> > Fax: + 44 (0) 1795 594510
> >
>

[Kyle Michael Douglass]

*****
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.
*****

Thanks everyone for your replies. I should have enough info to better
troubleshoot the problem.

If I manage to find a solution, I'll post it to this same thread so
future listers can benefit.

Cheers,
Kyle

On 02/14/2017 07:51 PM, Alfred Millett-Sikking 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 Kyle,
>
> Our friends and colleagues have done an excellent job of covering this
> topic, and I very much agree with the contents and summaries provided. I
> would like to add one further minor point which may or may not be
> applicable depending on your setup:
>
> WFE of any type (including astigmatism) is additive and you mentioned
> upstream dichroics and filters (and mirrors?). If you're unlucky then it
> may be that these previous optics have already put you 'on the edge' of an
> acceptable/perceptible astigmatism and that the last guy is simply pushing
> you over. If you can't fix it with careful mounting of the last guy then it
> may be worth giving the upstream optics an equal measure of care
> (especially if you have reflectors). Without a TWE measurement of the
> system optic by optic you cannot tell what's going on, so just be
> suspicious of everyone!
>
> Regards,
>
> Alfred Millett-Sikking.
>
>
> On 14 February 2017 at 06:35, James Kerin <[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.
>> *****
>>
>> COMMERCIAL RESPONSE
>>
>> Our President and Managing Director have prepared the following response:
>>
>> Hi Kyle
>>
>> Based on our experience of designing image splitters and multicamera
>> adapters, we can strongly underline the points made by Jeff Carmichael.  As
>> he says, to avoid any aberrations in the reflected channel, the dichroic
>> must be “softly” mounted against a truly flat surface.  Curvature in only
>> one direction will indeed give astigmatism, but more complex curvatures,
>> especially if there is a bit of twist as well, can give significant image
>> distortion too – think fairground mirrors!  Even if the images are more or
>> less in focus over the field, this will clearly show up when (as is so
>> often required) the transmitted and reflected images are superimposed for
>> analysis.  As camera resolution has improved, such effects have become ever
>> more obvious, but even with megapixel cameras it should be possible to
>> overlay the images to within a just a pixel or two over the field.  However
>> that does require similar detail to the design of the rest of the optical
>> system, which should be as symmetrical as possible between the transmitted
>> and reflected pathways, as it is in our image splitters as well as our
>> camera adapters.
>>
>> The flatness issue applies whether the reflection occurs in an infinity
>> space or an imaging space, but there is another problem if the dichroic is
>> in imaging space, namely that of astigmatism in the transmitted channel.
>> This is because the dichroic is at an angle (typically 45 degrees of
>> course) with respect to the light path. Optical modelling shows the
>> following effects, some of which may be counterintuitive.  As might be
>> expected though, the astigmatism, which is uniform over the field,
>> increases with the refractive index and thickness of the dichroic, as well
>> as with its angle.  (By the way, that's probably why the dichroics used in
>> microscopes to introduce an epi-illumination beam are traditionally “thin”,
>> typically just a millimetre, as in the older (noninfinity) microscopes the
>> dichroic was in the imaging space from the objective).
>>
>> We wonder if this effect might be relevant to Kyle's problem, especially
>> if the dichroic is relatively thick.  Relating to that, we have an
>> observation and a suggestion.  The optical modelling shows that the
>> astigmatism is independent of the dichroic's position within the imaging
>> space, so moving it closer to either the camera or the focussing lens won't
>> help.
>>
>> We are also pleased from our modelling to confirm that Andrew York's
>> solution to use a second piece of glass mounted at an orthogonal angle is a
>> correct one rather than just a “hack”, even though it may “sound” wrong.
>> The astigmatism arises because the effective thickness of the glass is
>> different in the x and y directions because of it being at an angle.  The
>> sign of the angle doesn't matter, so adding another piece of glass angled
>> oppositely would make the problem worse rather than better.  But it you
>> rotate by 90 degrees, the directional differences now cancel, and you're
>> just left with a small and probably negligible (according to our modelling
>> at least!) chromatic effect from the dispersion of the glass.
>>
>> Dr MV Thomas & Jez Graham
>> Cairn Research Ltd
>> Graveney Road
>> Faversham
>> Kent, ME13 8UP
>> UK
>> Cairn Research is a European scientific instruments manufacturer based in
>> Kent, UK.
>> www.cairn-research.co.uk
>>
>>
>>
>>
>>
>> On 2/13/2017 9:34 PM, Andrew York 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'm worried that your transmission path will also have astigmatism.
>>> Even a
>>> perfectly flat piece of glass yields astigmatism in transmission if it's
>>> tilted in a non-infinity space, right?
>>>
>>> (Warning: lousy advice below)
>>>    I've encountered this myself, and "fixed" it with a second piece of
>>> glass,
>>> the same thickness and material as the first, tilted equally about an
>>> orthogonal axis, to introduce "equal and opposite" astigmatism. This was a
>>> hack, but it was good enough for my needs at the time. Depending on your
>>> needs, perhaps you could use tilted flat glass as a tunable astigmatism
>>> compensation.
>>>
>>> On Mon, Feb 13, 2017 at 8:45 AM, Rusty Nicovich <[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.
>>>> *****
>>>>
>>>> Kyle,
>>>>
>>>> The other poster was me.  I ultimately fixed the issue to my satisfaction
>>>> by carefully mounting the dichroic to the cube with double-sided tape
>>>> rather than the provided clamp.  That method was stress-free enough to
>>>> not
>>>> induce any undue curvature in the dichroic, at least qualitatively as no
>>>> astigmatism immediately obvious in the reflected image.  This was even
>>>> with
>>>> a relatively thin (1.1 mm) substrate for the mirror. This solution is
>>>> similar to what Jeff suggested using silicone adhesive instead of tape.
>>>>
>>>> Another fun part of that system is that the camera fans would induce
>>>> quite
>>>> a large vibration and only along one axis.  The vibration period was
>>>> 10-20
>>>> ms, which meant any longer exposures would yield a PSF elongated on that
>>>> axis.  Astigmatism would vary along the focal axis and this didn't, but
>>>> it
>>>> still gave an asymmetric PSF at the focal plane.
>>>>
>>>> Thanks,
>>>> Rusty
>>>>
>>>> On Mon, Feb 13, 2017 at 7:49 AM, Jeffrey Carmichael <
>>>> [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.
>>>>
>>>>> *****
>>>>>
>>>>> COMMERCIAL RESPONSE
>>>>>
>>>>> Hi Kyle,
>>>>>
>>>>> We've found that it's most often the mounting of the dichroic itself
>>>>> that
>>>>> causes the most distortion in an image reflected off of a dichroic.  You
>>>>> could theoretically have an "ideal" dichroic with no curvature, but once
>>>>>
>>>> it
>>>>
>>>>> is affixed, it will be torqued out of flatness to some extent unless you
>>>>> can completely avoid any mounting stress.
>>>>>
>>>>> If held by means that apply pressure on top or bottom of the large
>>>>>
>>>> surface,
>>>>
>>>>> you will get various forms of non-spherical astigmatism.  Even light
>>>>> pressure on the sides of the dichroic will have some effect.
>>>>>
>>>>> As Zdenek suggested, thicker dichroics are stiffer and will better
>>>>> resist
>>>>> deformation.
>>>>>
>>>>> If possible, try using something like silicone RTV applied very
>>>>> sparingly
>>>>> in small beads around the outside edges of the dichroic, but never under
>>>>> it.  This serves to prevent the dichroic from moving around without
>>>>> applying stress.  If the "holder" is removable, then you can swap out
>>>>>
>>>> these
>>>>
>>>>> mounted dichroics and not fiddle with the dichroic itself once affixed.
>>>>>
>>>>> Jeff
>>>>>
>>>>>
>>>>> *Jeff Carmichael*
>>>>>
>>>>> *Technical and Product Marketing Manager*
>>>>>
>>>>> *[hidden email] <[hidden email]>*Chroma Technology
>>>>> Corp.
>>>>>
>>>>> *an employee owned company*
>>>>> *10 Imtec Lane*
>>>>> *Bellows Falls, VT  05301*
>>>>> *802-428-2528 Office*
>>>>> *802-428-2528 Fax**800-824-7662 Toll Free*
>>>>>
>>>>> On Mon, Feb 13, 2017 at 9:27 AM, Kyle Douglass <[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 everyone,
>>>>>>
>>>>>> A couple years ago there was a post on this forum about significant
>>>>>> astigmatism appearing in the reflected path of a custom-built, two
>>>>>>
>>>>> camera
>>>>> TIRF setup. (http://confocal-microscopy-list.588098.n2.nabble.com/
>>>>> Reflec
>>>>> ted-image-gt-astigmatism-td7584402.html) The original poster had
>>>>> placed
>>>>> a
>>>>>
>>>>>> dichroic before a pair of matched tube lenses to split the two color
>>>>>> channels onto two separate cameras. The general consensus was that you
>>>>>> could "buy your way out of the problem" by buying a thick, ultraflat
>>>>>> dichroic.
>>>>>>
>>>>>> I am trying a similar approach on a setup in our lab but, due to space
>>>>>> constraints that are not easily overcome, have tried first placing the
>>>>>> channel-splitting dichroic in the image space after the final tube lens
>>>>>> instead of in the infinity space between the objective and tube lens.
>>>>>>
>>>>> Even
>>>>>
>>>>>> with a 3 mm thick ultraflat dichroic, I see moderately bad astigmatism
>>>>>>
>>>>> in
>>>>> the reflected channel. I have ruled out other possible sources of the
>>>>>> astigmatism, like an additional dichroic for the excitation light and
>>>>>> filters that already lie in the infinity space between the objective
>>>>>>
>>>>> and
>>>>> tube lens.
>>>>>> My question is: is it even worth trying to eliminate the astigmatism in
>>>>>> the reflected path of a split-channel setup if the dichroic does not
>>>>>>
>>>>> lie
>>>>> in
>>>>>
>>>>>> the infinity space? Or is it pretty much always a bad idea to put the
>>>>>> splitter dichroic in the image space?
>>>>>>
>>>>>> My current suspicion is that the tolerances in the alignment have to be
>>>>>> very, very tight to avoid astigmatism with the splitter in the image
>>>>>>
>>>>> space.
>>>>>
>>>>>> Thanks for your feedback.
>>>>>>
>>>>>> Kyle
>>>>>>
>>>>>> --
>>>>>> Kyle M. Douglass, PhD
>>>>>> Post-doctoral researcher
>>>>>> The Laboratory of Experimental Biophysics
>>>>>> EPFL, Lausanne, Switzerland
>>>>>> http://kmdouglass.github.io
>>>>>> http://leb.epfl.ch
>>>>>>
>>>>>>
>> --
>>
>> *James Kerin
>> *
>>
>> *Marketing Director*
>> Cairn Research Ltd
>> Graveney Road
>> Faversham
>> Kent, ME13 8UP
>> UK
>>
>> Direct: + 44 (0)1795 594507
>> Fax: + 44 (0) 1795 594510
>>

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch