*****
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,
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 <[log in to unmask]>
> 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 <
>> [log in to unmask]>
>> 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*
>>>
>>> *[log in to unmask] <[log in to unmask]>*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 <[log in to unmask]>
>>> 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
|
