incomplete airy disks

[berkserk]

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Hello,

I am observing some sort of an aberration (looks like coma) when imaging
sub-resolution sized fluorescent beads under epifluorescence. I am using a
Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water Obj.
NA=1.2.

I match the collar correction to the coverslip thickness but the airy disks
are not complete. I uploaded one of the images here
(https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).

Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0) attached to
the side-port of the microscope. Particles are excited with SpectraX light
engine from Lumencor through a GFP filter set from Semrock.

Has anyone come across such problem? Let me know if you have any ideas about
what might be the possible causes?

Thanks
Serkan Berk

University of Minnesota
116 Church St. SE
Minneapolis, MN 55414

[EricMarino]

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You really need to look at the whole Z stack to determine the problem. Would you put the TIF stack on Dropbox along with the capture metadata?


Eric Marino
Senior Imaging Specialist
Program in Cellular and Molecular Medicine
Boston Children's Hospital
200 Longwood Ave
WAB Room 133D
Boston, MA 02115
Lab: 617 713-8885
Cell: 617 913-9647
[hidden email]<mailto:[hidden email]>



On Sep 13, 2013, at 2:49 PM, Serkan Berk <[hidden email]<mailto:[hidden email]>> wrote:

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Hello,

I am observing some sort of an aberration (looks like coma) when imaging
sub-resolution sized fluorescent beads under epifluorescence. I am using a
Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water Obj.
NA=1.2.

I match the collar correction to the coverslip thickness but the airy disks
are not complete. I uploaded one of the images here
(https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).

Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0) attached to
the side-port of the microscope. Particles are excited with SpectraX light
engine from Lumencor through a GFP filter set from Semrock.

Has anyone come across such problem? Let me know if you have any ideas about
what might be the possible causes?

Thanks
Serkan Berk

University of Minnesota
116 Church St. SE
Minneapolis, MN 55414

[John Oreopoulos]

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Serkan,

That doesn't quite look like coma to me. Where in the field of view was this image taken? By any chance are those beads near the edge of the field of view? Does every bead, even those in the middle of the field of view look like the ones you've shown here? (Note to everyone else who tries to download this image, you have to really stretch the contrast to see the artifact Serkan is talking about. I used ImageJ to do this).

To me, it almost looks like there's something blocking part of the back-aperture of the objective since you see the same "bite" missing out of all the bead Airy disks, and on the same angle. You might want to check the back of the objective, the nose piece, or any other part of the infinity space of the optical train (any other conjugate plane if you have any other periferal devices between the camera and microscope).

Does the pattern change when you change the correction collar?

Just some ideas,

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-09-13, at 2:48 PM, Serkan Berk wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I am observing some sort of an aberration (looks like coma) when imaging
> sub-resolution sized fluorescent beads under epifluorescence. I am using a
> Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water Obj.
> NA=1.2.
>
> I match the collar correction to the coverslip thickness but the airy disks
> are not complete. I uploaded one of the images here
> (https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).
>
> Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0) attached to
> the side-port of the microscope. Particles are excited with SpectraX light
> engine from Lumencor through a GFP filter set from Semrock.
>
> Has anyone come across such problem? Let me know if you have any ideas about
> what might be the possible causes?
>
> Thanks
> Serkan Berk
>
> University of Minnesota
> 116 Church St. SE
> Minneapolis, MN 55414

[Craig Brideau]

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It looks to me like you have something misaligned in the optical path, or
that your sample is tilted.  Tilted samples cause problems with water
objectives with coverslips in particular, and will cause slants similar to
what you are seeing.

Craig


On Fri, Sep 13, 2013 at 1:34 PM, John Oreopoulos <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Serkan,
>
> That doesn't quite look like coma to me. Where in the field of view was
> this image taken? By any chance are those beads near the edge of the field
> of view? Does every bead, even those in the middle of the field of view
> look like the ones you've shown here? (Note to everyone else who tries to
> download this image, you have to really stretch the contrast to see the
> artifact Serkan is talking about. I used ImageJ to do this).
>
> To me, it almost looks like there's something blocking part of the
> back-aperture of the objective since you see the same "bite" missing out of
> all the bead Airy disks, and on the same angle. You might want to check the
> back of the objective, the nose piece, or any other part of the infinity
> space of the optical train (any other conjugate plane if you have any other
> periferal devices between the camera and microscope).
>
> Does the pattern change when you change the correction collar?
>
> Just some ideas,
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2013-09-13, at 2:48 PM, Serkan Berk wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hello,
> >
> > I am observing some sort of an aberration (looks like coma) when imaging
> > sub-resolution sized fluorescent beads under epifluorescence. I am using
> a
> > Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water
> Obj.
> > NA=1.2.
> >
> > I match the collar correction to the coverslip thickness but the airy
> disks
> > are not complete. I uploaded one of the images here
> > (https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).
> >
> > Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0) attached
> to
> > the side-port of the microscope. Particles are excited with SpectraX
> light
> > engine from Lumencor through a GFP filter set from Semrock.
> >
> > Has anyone come across such problem? Let me know if you have any ideas
> about
> > what might be the possible causes?
> >
> > Thanks
> > Serkan Berk
> >
> > University of Minnesota
> > 116 Church St. SE
> > Minneapolis, MN 55414
>

[John Oreopoulos]

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Yes,

Craig brings up a good point about the water-immersion lens artifact I sometimes forget about. It is documented here in this paper by John Murray:

Arimoto, R. and J.M. Murray, A common aberration with water-immersion objective lenses. Journal of Microscopy-Oxford, 2004. 216: p. 49-51.

Take a look and you'll see the same sort of images there. Check the tilt of the specimen on your stage as he suggests as well.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-09-13, at 3:50 PM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It looks to me like you have something misaligned in the optical path, or
> that your sample is tilted.  Tilted samples cause problems with water
> objectives with coverslips in particular, and will cause slants similar to
> what you are seeing.
>
> Craig
>
>
> On Fri, Sep 13, 2013 at 1:34 PM, John Oreopoulos <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Serkan,
>>
>> That doesn't quite look like coma to me. Where in the field of view was
>> this image taken? By any chance are those beads near the edge of the field
>> of view? Does every bead, even those in the middle of the field of view
>> look like the ones you've shown here? (Note to everyone else who tries to
>> download this image, you have to really stretch the contrast to see the
>> artifact Serkan is talking about. I used ImageJ to do this).
>>
>> To me, it almost looks like there's something blocking part of the
>> back-aperture of the objective since you see the same "bite" missing out of
>> all the bead Airy disks, and on the same angle. You might want to check the
>> back of the objective, the nose piece, or any other part of the infinity
>> space of the optical train (any other conjugate plane if you have any other
>> periferal devices between the camera and microscope).
>>
>> Does the pattern change when you change the correction collar?
>>
>> Just some ideas,
>>
>> John Oreopoulos
>> Staff Scientist
>> Spectral Applied Research
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca
>>
>>
>> On 2013-09-13, at 2:48 PM, Serkan Berk wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hello,
>>>
>>> I am observing some sort of an aberration (looks like coma) when imaging
>>> sub-resolution sized fluorescent beads under epifluorescence. I am using
>> a
>>> Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water
>> Obj.
>>> NA=1.2.
>>>
>>> I match the collar correction to the coverslip thickness but the airy
>> disks
>>> are not complete. I uploaded one of the images here
>>> (https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).
>>>
>>> Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0) attached
>> to
>>> the side-port of the microscope. Particles are excited with SpectraX
>> light
>>> engine from Lumencor through a GFP filter set from Semrock.
>>>
>>> Has anyone come across such problem? Let me know if you have any ideas
>> about
>>> what might be the possible causes?
>>>
>>> Thanks
>>> Serkan Berk
>>>
>>> University of Minnesota
>>> 116 Church St. SE
>>> Minneapolis, MN 55414
>>

[Craig Brideau]

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We use water lenses quite extensively.  I actually installed goniometers on
my stages to carefully adjust the angle of my slides to counteract this
issue.  You can line up with a sample by looking at the reflection off the
coverslip as you adjust the focus.  If all is aligned, the light will
exhibit a gaussian profile that fades in and out.  If you have tilt, you
will see a bar of light sweep across the coverslip.  Also, keep in mind
that even though your coverslip is aligned flat, your sample can be tilted
underneath the cover slip if you are working with cell layers, difficult
mounting media, etc.

Craig


On Fri, Sep 13, 2013 at 1:57 PM, John Oreopoulos <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes,
>
> Craig brings up a good point about the water-immersion lens artifact I
> sometimes forget about. It is documented here in this paper by John Murray:
>
> Arimoto, R. and J.M. Murray, A common aberration with water-immersion
> objective lenses. Journal of Microscopy-Oxford, 2004. 216: p. 49-51.
>
> Take a look and you'll see the same sort of images there. Check the tilt
> of the specimen on your stage as he suggests as well.
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2013-09-13, at 3:50 PM, Craig Brideau wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > It looks to me like you have something misaligned in the optical path, or
> > that your sample is tilted.  Tilted samples cause problems with water
> > objectives with coverslips in particular, and will cause slants similar
> to
> > what you are seeing.
> >
> > Craig
> >
> >
> > On Fri, Sep 13, 2013 at 1:34 PM, John Oreopoulos <
> > [hidden email]> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Serkan,
> >>
> >> That doesn't quite look like coma to me. Where in the field of view was
> >> this image taken? By any chance are those beads near the edge of the
> field
> >> of view? Does every bead, even those in the middle of the field of view
> >> look like the ones you've shown here? (Note to everyone else who tries
> to
> >> download this image, you have to really stretch the contrast to see the
> >> artifact Serkan is talking about. I used ImageJ to do this).
> >>
> >> To me, it almost looks like there's something blocking part of the
> >> back-aperture of the objective since you see the same "bite" missing
> out of
> >> all the bead Airy disks, and on the same angle. You might want to check
> the
> >> back of the objective, the nose piece, or any other part of the infinity
> >> space of the optical train (any other conjugate plane if you have any
> other
> >> periferal devices between the camera and microscope).
> >>
> >> Does the pattern change when you change the correction collar?
> >>
> >> Just some ideas,
> >>
> >> John Oreopoulos
> >> Staff Scientist
> >> Spectral Applied Research
> >> Richmond Hill, Ontario
> >> Canada
> >> www.spectral.ca
> >>
> >>
> >> On 2013-09-13, at 2:48 PM, Serkan Berk wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Hello,
> >>>
> >>> I am observing some sort of an aberration (looks like coma) when
> imaging
> >>> sub-resolution sized fluorescent beads under epifluorescence. I am
> using
> >> a
> >>> Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water
> >> Obj.
> >>> NA=1.2.
> >>>
> >>> I match the collar correction to the coverslip thickness but the airy
> >> disks
> >>> are not complete. I uploaded one of the images here
> >>> (https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).
> >>>
> >>> Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0)
> attached
> >> to
> >>> the side-port of the microscope. Particles are excited with SpectraX
> >> light
> >>> engine from Lumencor through a GFP filter set from Semrock.
> >>>
> >>> Has anyone come across such problem? Let me know if you have any ideas
> >> about
> >>> what might be the possible causes?
> >>>
> >>> Thanks
> >>> Serkan Berk
> >>>
> >>> University of Minnesota
> >>> 116 Church St. SE
> >>> Minneapolis, MN 55414
> >>
>

[berkserk]

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First of all thanks a lot for the responses. I will try the suggestions.
 
I tried placing the sample in different orientations to see if the coverslip
is tilted but i see no change. Also playing with the set screws on the
corners of the stage holder didn't make any difference.

Thanks
Serkan

[berkserk]

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I realized that the uploaded image in my first message was taken with 100x
oil objective not with 63x water obj.

Image taken with 63x water is here,
https://www.dropbox.com/s/9g0swtwgueorere/63x%20water%20obj.%205s%2025%25%20110nm.tif

I also uploaded z-stack with water objective,
https://www.dropbox.com/s/49xl9csoxxzijov/110nm%2063x%20water%20obj.%200.173%20axiocam%202x%201s%2025nm%20step%20-5um%20to%20%2B5um%20z-stack_MMStack.ome.tif



Changing the collar setting to a greater value makes nice looking airy disk
when the focal plane is above the particle, while setting collar to a lower
value makes nice airy disk when focal plane is set below the particle. But
in both conditions PSF in the axial plane becomes more asymmetric than the
PSF at correct collar setting. Also intensity is highest at correct collar
position.

Thanks
Serkan

[cail]

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Dear all,

Due to the fund limitation, we cannot afford a commercial system. We would like to setup one by purchasing lasers, lens, mirrors and holders. Is there any instruction available? Anyone has a successful experience to share?
Thank you so much for your time.

Sincerely,
Liang

[Craig Brideau]

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Big difference. The oil negates any coverslip tilt. This brings us back to
the possibility that there is something misaligned in your microscope.
Jiggle anything you have access to, like filters, and if you can, check the
position of your tube lens. Also see if/how much the problem changes with
different objectives.

Craig
On 2013-09-13 5:47 PM, "Serkan Berk" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I realized that the uploaded image in my first message was taken with 100x
> oil objective not with 63x water obj.
>
> Image taken with 63x water is here,
>
> https://www.dropbox.com/s/9g0swtwgueorere/63x%20water%20obj.%205s%2025%25%20110nm.tif
>
> I also uploaded z-stack with water objective,
>
> https://www.dropbox.com/s/49xl9csoxxzijov/110nm%2063x%20water%20obj.%200.173%20axiocam%202x%201s%2025nm%20step%20-5um%20to%20%2B5um%20z-stack_MMStack.ome.tif
>
>
>
> Changing the collar setting to a greater value makes nice looking airy disk
> when the focal plane is above the particle, while setting collar to a lower
> value makes nice airy disk when focal plane is set below the particle. But
> in both conditions PSF in the axial plane becomes more asymmetric than the
> PSF at correct collar setting. Also intensity is highest at correct collar
> position.
>
> Thanks
> Serkan
>

[Craig Brideau]

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This looks like a good place to start:

http://www.tirf-labs.com

Craig
On 2013-09-14 6:48 AM, "cail" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Due to the fund limitation, we cannot afford a commercial system. We would
> like to setup one by purchasing lasers, lens, mirrors and holders. Is there
> any instruction available? Anyone has a successful experience to share?
> Thank you so much for your time.
>
> Sincerely,
> Liang

[David Baddeley]

having been involved in the setup of a number of systems, I
*****
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Hi Liang,

having been involved in the setup of a number of systems, I can confirm that it's fairly easy, although there are a few questions you need to ask before you begin. The most important of these are:

- What are you going to be using the system for? If you are going to try and measure depth using the evanescent decay, you need a well controlled angle and good beam quality. In practice this tends to mean spatial filtering (e.g. by using a single mode fibre) and illuminating a large field of view so as to get a reasonably uniform illumination in the bit you're interested in. If you're doing PALM/STORM or related work, you want to maximise the intensity you get, which means illuminating only the area you need too and probably skipping the spatial filter (the method is surprisingly robust against beam quality). Because you're only illuminating a small area, however, you will need reasonably easy to use controls to centre it on your field of view. Single particle tracking will be somewhere between these two extremes.

- Do you need computer control of the TIRF angle? This will influence how you couple the light in - the easiest approach to computer control is probably to put either a mirror, or the end of a fibre on a motorised stage at one of the systems fourier planes.

In general I like to build a folded 4-f system with mirrors at both the conjugate image and fourier planes. This allows you to set the illumination position and angle (mostly) independently. The other trick I use is to use a polarising beam splitter to combine the laser based TIRF illumination with the arc lamp. You lose 50% on the arc lamp, but the lasers pass without loss and the alignment is much more stable than if using a flip mirror.

cheers,
David


________________________________
 From: cail <[hidden email]>
To: [hidden email]
Sent: Saturday, 14 September 2013 8:37 AM
Subject: Anyone has suggestion on setting up an objective based TIRF system
 

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

Due to the fund limitation, we cannot afford a commercial system. We would like to setup one by purchasing lasers, lens, mirrors and holders. Is there any instruction available? Anyone has a successful experience to share?
Thank you so much for your time.

Sincerely,
Liang

[John Oreopoulos]

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

Dear Liang,

Many of the review papers written by Dan Axelrod cover the basics of how to setup a TIRF microscope (prism- and objective-based). For example, see here:

Axelrod, D., Total internal reflection fluorescence microscopy in cell biology, in Biophotonics, pt B. 2003. p. 1-33.

Axelrod, D., Total internal reflection fluorescence microscopy in cell biology. Traffic, 2001. 2(11): p. 764-774.

Axelrod, D., Total internal-reflection fluorescence microscopy, in Methods in cell biology, T. Langsing and Y. Wang, Editors. 1989, Academic Press: San Diego. p. 245-270.

But actually, the best step-by-step instruction on building an objective-based TIRF microscope I ever came across was in this book chapter by Ian Parker:

Parker, I., Photonics for biologists, in Biophotonics, Pt A. 2003. p. 345-382

If using the objective-based approach, at a bare minimum you will need an oil immersion objective of at least 1.4, but greater than 1.4 will make your life easier in terms of laser alignment. If you're really strapped for cash, try building a "white-light" TIRF system as described in the review papers by Axelrod. Good luck.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-09-15, at 11:33 AM, David Baddeley wrote:

> having been involved in the setup of a number of systems, I
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Liang,
>
> having been involved in the setup of a number of systems, I can confirm that it's fairly easy, although there are a few questions you need to ask before you begin. The most important of these are:
>
> - What are you going to be using the system for? If you are going to try and measure depth using the evanescent decay, you need a well controlled angle and good beam quality. In practice this tends to mean spatial filtering (e.g. by using a single mode fibre) and illuminating a large field of view so as to get a reasonably uniform illumination in the bit you're interested in. If you're doing PALM/STORM or related work, you want to maximise the intensity you get, which means illuminating only the area you need too and probably skipping the spatial filter (the method is surprisingly robust against beam quality). Because you're only illuminating a small area, however, you will need reasonably easy to use controls to centre it on your field of view. Single particle tracking will be somewhere between these two extremes.
>
> - Do you need computer control of the TIRF angle? This will influence how you couple the light in - the easiest approach to computer control is probably to put either a mirror, or the end of a fibre on a motorised stage at one of the systems fourier planes.
>
> In general I like to build a folded 4-f system with mirrors at both the conjugate image and fourier planes. This allows you to set the illumination position and angle (mostly) independently. The other trick I use is to use a polarising beam splitter to combine the laser based TIRF illumination with the arc lamp. You lose 50% on the arc lamp, but the lasers pass without loss and the alignment is much more stable than if using a flip mirror.
>
> cheers,
> David
>
>
> ________________________________
> From: cail <[hidden email]>
> To: [hidden email]
> Sent: Saturday, 14 September 2013 8:37 AM
> Subject: Anyone has suggestion on setting up an objective based TIRF system
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Due to the fund limitation, we cannot afford a commercial system. We would like to setup one by purchasing lasers, lens, mirrors and holders. Is there any instruction available? Anyone has a successful experience to share?
> Thank you so much for your time.
>
> Sincerely,
> Liang

[Andreas Bruckbauer]

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Dear Liang,

one concern is laser safety, when the angle is below the critical angle, the laser exits the objective as a straight beam and not divergent as in a confocal microscope where the intensity quickly decays with distance. It will generally not be parallel to the laser table as in a normal optical setup, so could be potentially dangerous. Commercial systems have controls to prevent any eye injuries, you would need to discuss your setup with your local laser safety officer.

best wishes

Andreas

 

 

 

-----Original Message-----
From: cail <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sat, 14 Sep 2013 13:48
Subject: Anyone has suggestion on setting up an objective based TIRF system


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Dear all,

Due to the fund limitation, we cannot afford a commercial system. We would like
to setup one by purchasing lasers, lens, mirrors and holders. Is there any
instruction available? Anyone has a successful experience to share?
Thank you so much for your time.

Sincerely,
Liang

 

[Siddharth Sivankutty]

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Dear Serkan,
You might need to check if your dichroic mirror is deformed (bent) or too
tightly screwed in its holder. This could introduce a fair bit of
astigmatism, and I've observed a similar psf when that has been the case,
albeit in a confocal microscope.

Best regards,
Sid