A resolution target or standard sample for DIC microscopy

[John Oreopoulos]

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Hello confocal hive mind,

Measuring resolution or PSF for fluorescence microscopes is fairly  
straightforward through the use of fluorescent beads and image  
analysis tools such as PSFj (Knob lab), or other standard samples like  
the Argolight HM-slide (no commercial interest). I was wondering if  
the community could recommend to me a similar test sample for  
assessing the resolution and contrast of the DIC mode of a microscope.  
Does such a standard exist?

I've browsed around the web and come across a few possibilities (no  
commercial interest):

Arranged diatoms:
http://www.diatomshop.com/price-list.html

Siemens stars:
https://www.edmundoptics.com/f/high-resolution-microscopy-slide-targets/38663/

Ronchi rulings:
https://www.thorlabs.com/thorproduct.cfm?partnumber=R1L3S5P


One of Shalin Mehta's publications mentions something called the  
MBL/NNF test slide which is similar to the Siemen's star above. Does  
anyone know where I can purchase a sample like that?
https://www.osapublishing.org/boe/abstract.cfm?uri=boe-5-6-1822

Would the fluorescent beads in the standard Life Technologies  
TetraSpeck bead kit show any DIC contrast? Could they be used to  
measure a DIC PSF?

What does PSF even mean for DIC?

Sincerely

John Oreopoulos

[mmodel]

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Hi John,

I suggest you ask Michael Shribak at MBL. He knows a lot about DIC.

Mike

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of [hidden email]
Sent: Monday, January 14, 2019 2:09 PM
To: [hidden email]
Subject: A resolution target or standard sample for DIC microscopy

*****
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*****

Hello confocal hive mind,

Measuring resolution or PSF for fluorescence microscopes is fairly straightforward through the use of fluorescent beads and image analysis tools such as PSFj (Knob lab), or other standard samples like the Argolight HM-slide (no commercial interest). I was wondering if the community could recommend to me a similar test sample for assessing the resolution and contrast of the DIC mode of a microscope.  
Does such a standard exist?

I've browsed around the web and come across a few possibilities (no commercial interest):

Arranged diatoms:
http://www.diatomshop.com/price-list.html

Siemens stars:
https://www.edmundoptics.com/f/high-resolution-microscopy-slide-targets/38663/

Ronchi rulings:
https://www.thorlabs.com/thorproduct.cfm?partnumber=R1L3S5P


One of Shalin Mehta's publications mentions something called the MBL/NNF test slide which is similar to the Siemen's star above. Does anyone know where I can purchase a sample like that?
https://www.osapublishing.org/boe/abstract.cfm?uri=boe-5-6-1822

Would the fluorescent beads in the standard Life Technologies TetraSpeck bead kit show any DIC contrast? Could they be used to measure a DIC PSF?

What does PSF even mean for DIC?

Sincerely

John Oreopoulos

[Craig Brideau]

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DIC converts phase information to intensity information, so there are two
steps to determining resolution. The intensity information is just a
widefield image, so you can use the same concepts for a widefield
microscope to determine the resolution there. For the phase information,
you have to consider the wavelength you are working with, and the phase
distortion caused by your optics, coverslip, etc. A piece of glass etched
to a known depth, or perhaps the structure of a ground glass diffuser may
be useful in evaluating that aspect of the system.

Craig

On Mon, Jan 14, 2019 at 12:17 PM <[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.
> *****
>
> Hello confocal hive mind,
>
> Measuring resolution or PSF for fluorescence microscopes is fairly
> straightforward through the use of fluorescent beads and image
> analysis tools such as PSFj (Knob lab), or other standard samples like
> the Argolight HM-slide (no commercial interest). I was wondering if
> the community could recommend to me a similar test sample for
> assessing the resolution and contrast of the DIC mode of a microscope.
> Does such a standard exist?
>
> I've browsed around the web and come across a few possibilities (no
> commercial interest):
>
> Arranged diatoms:
> http://www.diatomshop.com/price-list.html
>
> Siemens stars:
>
> https://www.edmundoptics.com/f/high-resolution-microscopy-slide-targets/38663/
>
> Ronchi rulings:
> https://www.thorlabs.com/thorproduct.cfm?partnumber=R1L3S5P
>
>
> One of Shalin Mehta's publications mentions something called the
> MBL/NNF test slide which is similar to the Siemen's star above. Does
> anyone know where I can purchase a sample like that?
> https://www.osapublishing.org/boe/abstract.cfm?uri=boe-5-6-1822
>
> Would the fluorescent beads in the standard Life Technologies
> TetraSpeck bead kit show any DIC contrast? Could they be used to
> measure a DIC PSF?
>
> What does PSF even mean for DIC?
>
> Sincerely
>
> John Oreopoulos
>

[Glyn Nelson]

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Hi John,

I have tried these recently released slides: https://www.psfcheck.com .  The etched pattern is visible under brightfield (it is how I find the structures before using for widefield and confocal fluorescence), so I would expect you could use them for DIC too.  The smallest 'spots' are sub-diffraction.  The commercialisation of them was announced at the Light Microscopy Manager's meeting in the UK the other week, and Alex Corbett said they would be £365 per slide, if I remember rightly.  No affiliation with him/ them, just tested the slides during development.

Glyn.

[Talley Lambert]

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if you're specifically looking for a sample with polarization sensitivity, you might also consider a birefringent resolution target:
https://www.thorlabs.com/thorproduct.cfm?partnumber=R2L2S1B

[Kristy Wendt]

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Hi John,


My thoughts:


Bear in mind that each specimen examined in DIC has a different optimum bias retardation setting that generates a maximum level of contrast in the final image, so SOP that optimizes the microscope for a standard reference sample just prior to image collection of a biological sample is less appropriate for DIC.


Your question about psf in DIC is delightful, and I didn't know either.  There's a succinct refresher of psf theory in the context of DIC in Lola Rozo's thesis here. I am CCing her to see if she can comment on the sample she used.

https://tel.archives-ouvertes.fr/tel-01576339v2/document


In general, each point in the the specimen has a pair of overlapping point spread functions, which just like in brightfield are generated by the diffraction of light through the objective's NA, but split by the prism in the DIC. In most of the things that a cell biologist looks at in DIC -- that is to say larger than what is typically the resolution limit of the microscope -- the image is a 2D/3D array of overlapping Airy Disk pairs, where everything cancels out except the object's edge.  But if you know the NA of your objective and condenser and the wavelength of your light, and you're not at the resolution limit, you can define the psf and resolution with a reference sample.


How this practically translates depends on the make and model of your DIC but that what sample you use doesn't matter; the contrast depends on the beam shearing direction and the phase shift between the interfering beams.  You're either going to be rotating the stage to make the shear direction parallel to the sample's refractive index gradient, moving the DIC prism laterally (less likely), or rotating a compensator (more likely).


I do think the TetraSpeck bead kit would work for this purpose but I agree with Craig that an etched glass or diatom might be better. Let's see if Lola can comment on the sample she used.


Best,

Kristy




Kristy Wendt

Graduate Researcher
Biomedical Engineering<https://www.engr.wisc.edu/department/bme/>
Laboratory for Optical and Computational Instrumentation<http://loci.wisc.edu/>
<http://loci.wisc.edu/>University of Wisconsin-Madison
271 Animal Sciences
1675 Observatory Drive
Madison, WI 53706
Ph: 573-489-0552


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Craig Brideau <[hidden email]>
Sent: Monday, January 14, 2019 2:26:19 PM
To: [hidden email]
Subject: Re: A resolution target or standard sample for DIC microscopy

*****
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.
*****

DIC converts phase information to intensity information, so there are two
steps to determining resolution. The intensity information is just a
widefield image, so you can use the same concepts for a widefield
microscope to determine the resolution there. For the phase information,
you have to consider the wavelength you are working with, and the phase
distortion caused by your optics, coverslip, etc. A piece of glass etched
to a known depth, or perhaps the structure of a ground glass diffuser may
be useful in evaluating that aspect of the system.

Craig

On Mon, Jan 14, 2019 at 12:17 PM <[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.
> *****
>
> Hello confocal hive mind,
>
> Measuring resolution or PSF for fluorescence microscopes is fairly
> straightforward through the use of fluorescent beads and image
> analysis tools such as PSFj (Knob lab), or other standard samples like
> the Argolight HM-slide (no commercial interest). I was wondering if
> the community could recommend to me a similar test sample for
> assessing the resolution and contrast of the DIC mode of a microscope.
> Does such a standard exist?
>
> I've browsed around the web and come across a few possibilities (no
> commercial interest):
>
> Arranged diatoms:
> http://www.diatomshop.com/price-list.html
>
> Siemens stars:
>
> https://www.edmundoptics.com/f/high-resolution-microscopy-slide-targets/38663/
>
> Ronchi rulings:
> https://www.thorlabs.com/thorproduct.cfm?partnumber=R1L3S5P
>
>
> One of Shalin Mehta's publications mentions something called the
> MBL/NNF test slide which is similar to the Siemen's star above. Does
> anyone know where I can purchase a sample like that?
> https://www.osapublishing.org/boe/abstract.cfm?uri=boe-5-6-1822
>
> Would the fluorescent beads in the standard Life Technologies
> TetraSpeck bead kit show any DIC contrast? Could they be used to
> measure a DIC PSF?
>
> What does PSF even mean for DIC?
>
> Sincerely
>
> John Oreopoulos
>