Multiphoton microscope - Field of View

[Heping Yuan]

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Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?

Thanks,
Tim

[Marc Reinig]

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If you have a stage that is calibrated, find a target, move it to the
edge of your FOV, note the position, move it to the opposite edge of
your FOV, note the position, the difference is your linear FOV.

Marco
Marco

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



On Wed, Nov 30, 2016 at 11:01 AM, Tim <[hidden email]> wrote:

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> *****
>
> Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?
>
> Thanks,
> Tim

[Gary G. Li-3]

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Look at something with a known size, e.g., a fluorescent slide with known grid spacing.  

On Wed, Nov 30, 2016 at 2:01 PM, Tim <[hidden email]> wrote:

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Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?

Thanks,
Tim

[Heping Yuan]

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Thanks Gary. Would such a slide be commercially available? I did not see any slides with fluorescent grids after doing a search.

[George McNamara]

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Hi Tim,

If you are attending ASCB, you can pick up a box with several different
color fluorescence slides from the Chroma Technology booth (if not, you
could email them and very politley ask if they would mail a box to you
... might help to offer to pay shipping).

For a grid - just stick a hemacytometer on the microscope. If you want
to get fancy, put a (dilute) solution of fluorescein or a dilute
solution (to not waste money) of fluorescent antibody on it. You can
focus in brightfield.

enjoy,

George


On 11/30/2016 2:51 PM, Tim wrote:
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> Thanks Gary. Would such a slide be commercially available? I did not see any slides with fluorescent grids after doing a search.

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Gary G. Li-2]

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There are some commercial fluorescent targets with known grid patterns, for example,  

http://www.maxlevy.com/category/fluorescent-tragets.html

Please make sure you select one that suits for your purpose.

On Wed, Nov 30, 2016 at 3:51 PM, Tim <[hidden email]> wrote:

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Thanks Gary. Would such a slide be commercially available? I did not see any slides with fluorescent grids after doing a search.

[Hongtao Chen]

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http://www.edmundoptics.com/test-targets/resolution-test-targets/

On Wed, Nov 30, 2016 at 1:31 PM, George McNamara <[hidden email]> wrote:

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Hi Tim,

If you are attending ASCB, you can pick up a box with several different color fluorescence slides from the Chroma Technology booth (if not, you could email them and very politley ask if they would mail a box to you ... might help to offer to pay shipping).

For a grid - just stick a hemacytometer on the microscope. If you want to get fancy, put a (dilute) solution of fluorescein or a dilute solution (to not waste money) of fluorescent antibody on it. You can focus in brightfield.

enjoy,

George

On 11/30/2016 2:51 PM, Tim wrote:

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Thanks Gary. Would such a slide be commercially available? I did not see any slides with fluorescent grids after doing a search.

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

--

[Reece, Jeff (NIH/NIDDK) [E]]

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Hi Tim,

 

If you are happy with precision of a few percent, you could use Powerpoint or similar software, and a decent printer, to print out your own crude sample on regular paper, and image it in transmitted or reflected light.  The spacing on my sample goes down to 500 microns, but your printer might do much better.

 

Cheers,

Jeff

 

From: Gary G. Li [mailto:[hidden email]]
Sent: Wednesday, November 30, 2016 3:00 PM
To: [hidden email]
Subject: Re: Multiphoton microscope - Field of View

 

***** 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. *****

 

Look at something with a known size, e.g., a fluorescent slide with known grid spacing.  

 

On Wed, Nov 30, 2016 at 2:01 PM, Tim <[hidden email]> wrote:

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*****

Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?

Thanks,
Tim

 

[Gary G. Li-2]

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You can also paint the printed grid with a fluorescent marker, to make it a crude fluorescent target.   

On Wed, Nov 30, 2016 at 5:52 PM, Reece, Jeff (NIH/NIDDK) [E] <[hidden email]> wrote:

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Hi Tim,

 

If you are happy with precision of a few percent, you could use Powerpoint or similar software, and a decent printer, to print out your own crude sample on regular paper, and image it in transmitted or reflected light.  The spacing on my sample goes down to 500 microns, but your printer might do much better.

 

Cheers,

Jeff

 

From: Gary G. Li [mailto:[hidden email]]
Sent: Wednesday, November 30, 2016 3:00 PM
To: [hidden email]
Subject: Re: Multiphoton microscope - Field of View

 

***** 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. *****

 

Look at something with a known size, e.g., a fluorescent slide with known grid spacing.  

 

On Wed, Nov 30, 2016 at 2:01 PM, Tim <[hidden email]> wrote:

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Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?

Thanks,
Tim

 

[Tamara Howard]

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If you have a spare etched micrometer slide, you could "paint" the etched lines with the fluorescent marker. We have a very old, etched line slide that someone probably modified to be fluorescent at some time. Swipe the marker (or make a dye paint) over the etched area, let it dry, then carefully wipe the surface clean & at least some of the fluor should stay in the etched lines.

Tamara

...................................................
Tamara Howard
Dept. of Cell Biology & Physiology
University of New Mexico
Albuquerque, NM

---

From: Confocal Microscopy List <[hidden email]> on behalf of Gary G. Li <[hidden email]>
Sent: Wednesday, November 30, 2016 4:09 PM
To: [hidden email]
Subject: Re: Multiphoton microscope - Field of View

 

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You can also paint the printed grid with a fluorescent marker, to make it a crude fluorescent target.   

On Wed, Nov 30, 2016 at 5:52 PM, Reece, Jeff (NIH/NIDDK) [E] <[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 Tim,

 

If you are happy with precision of a few percent, you could use Powerpoint or similar software, and a decent printer, to print out your own crude sample on regular paper, and image it in transmitted or reflected light.  The spacing on my sample goes down to 500 microns, but your printer might do much better.

 

Cheers,

Jeff

 

From: Gary G. Li [mailto:[hidden email]]
Sent: Wednesday, November 30, 2016 3:00 PM
To: [hidden email]
Subject: Re: Multiphoton microscope - Field of View

 

***** 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. *****

 

Look at something with a known size, e.g., a fluorescent slide with known grid spacing.  

 

On Wed, Nov 30, 2016 at 2:01 PM, Tim <[hidden email]> wrote:

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*****

Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?

Thanks,
Tim

 

[Benjamin Smith]

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We use the following grid micrometer with a layer of fluorescein and a coverslip: http://www.edmundoptics.com/test-targets/image-analysis-test-targets/multi-grid-standard-stage-micrometer/58607/

The fluorescence reflects off of the mirrored grid nicely, and allows for the width and height to be measured simultaneously.  It is also really helpful for aligning a brightfield system to an MP scan field, including any transformations.

Cheers,

   Ben Smith

On Wed, Nov 30, 2016 at 4:16 PM, Tamara Howard <[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. *****

If you have a spare etched micrometer slide, you could "paint" the etched lines with the fluorescent marker. We have a very old, etched line slide that someone probably modified to be fluorescent at some time. Swipe the marker (or make a dye paint) over the etched area, let it dry, then carefully wipe the surface clean & at least some of the fluor should stay in the etched lines.

Tamara

...................................................
Tamara Howard
Dept. of Cell Biology & Physiology
University of New Mexico
Albuquerque, NM

---

From: Confocal Microscopy List <[hidden email]> on behalf of Gary G. Li <[hidden email]>
Sent: Wednesday, November 30, 2016 4:09 PM
To: [hidden email]
Subject: Re: Multiphoton microscope - Field of View

 

***** 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. *****

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Imgur: The most awesome images on the Internet www.imgur.com Imgur is the best place to share and enjoy the most awesome images on the Internet. Every day, millions of people use Imgur to be entertained and inspired by funny ...

You can also paint the printed grid with a fluorescent marker, to make it a crude fluorescent target.   

On Wed, Nov 30, 2016 at 5:52 PM, Reece, Jeff (NIH/NIDDK) [E] <[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 Tim,

 

If you are happy with precision of a few percent, you could use Powerpoint or similar software, and a decent printer, to print out your own crude sample on regular paper, and image it in transmitted or reflected light.  The spacing on my sample goes down to 500 microns, but your printer might do much better.

 

Cheers,

Jeff

 

From: Gary G. Li [mailto:[hidden email]]
Sent: Wednesday, November 30, 2016 3:00 PM
To: [hidden email]
Subject: Re: Multiphoton microscope - Field of View

 

***** 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. *****

 

Look at something with a known size, e.g., a fluorescent slide with known grid spacing.  

 

On Wed, Nov 30, 2016 at 2:01 PM, Tim <[hidden email]> wrote:

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Hi all, sorry if this has been discussed before (which I'm sure it has) but I couldn't find any relevant discussions through search. I was wondering how does one go about measuring field of view on a custom designed multiphoton microscope?

Thanks,
Tim

 

--

[Bill Bennett]

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We use the Convollaria (lily of the valley) root cross-section slide that came with our old Zeiss LSM500.   It gives a beautiful bright-field image, and fluoresces intensely right across most of our Mai Tai wavelengths.   So you can use it to align BF and 2P captures and transformations, as previously mentioned.  But we also find it very useful as a startup check for our 2P system to check detectors,  beam alignment etc.

We got ours with the Zeiss, but I believe you can buy these slides quite cheaply e.g.
http://www.msmedia.com.au/convallaria-rhizome-training-slide-stained-for-fluorescence.php

Bill Bennett