Re: [EXTERNAL] Numerical aperture and spatial resolution

Posted by Julian Smith III on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Numerical-aperture-and-spatial-resolution-tp7591293p7591294.html

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Hi, Javier
We don't teach this to first-year students, but I cover it in my microscopy course for 3&4th-year students and beginning graduate students.
The iBiology microscopy series does a pretty good job of it.
I don't know whether your course has a laboratory section or not, but in the lab portion of my course, we do several of the standard exercises:
Use a laser to generate diffractions patterns from inexpensive ruled gratings.
Set up Koehler illumination, add a green interference filter over the field iris, view an inexpensive singled ruled grating with various objectives to determine whether or not the vertical lines are resolvable.
Then, remove one eyepiece and replace it with a phase telescope, close both field and condenser diaphragms  fully.  The diffraction spots can easily be seen in the back focal plane.
We supplement this with an exercise in FIGI/ImageJ where the students start with a photo of the ruled grating, do a Fourier transform, confirm that the reverse transform restores the image, and then to the first transform, use the eraser to remove the first-order diffraction spots and all data to the outside of them, then do the reverse transform on that image, and confirm that the grating has disappeared.
Most of this, I owe to Ted Salmon from his microscopy course when I was a grad student, and to the excellent book by Douglas & Murphy (Fundamentals of light microscopy and electronic imaging, 2nd edition.
Best,
Julian
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From: Confocal Microscopy List <[hidden email]> on behalf of F Javier Diez Guerra <[hidden email]>
Sent: Saturday, September 19, 2020 5:38 AM
To: [hidden email] <[hidden email]>
Subject: [EXTERNAL] Numerical aperture and spatial resolution

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Hello,

I wonder if anybody in the list could help.

I want to convey to biology undergraduate students (very allergic to
physics and mathematics) the understanding of the relationship between
numerical aperture and spatial resolution.

I have already given them links to the different microscopy primer
sites. They find difficult to understand why the airy disk is generated
in the image plane, how the diffraction orders affect resolution and why
increasing NA reduces the image spot.

Could anybody share a basic and intuitive infographic slide, animation
or any other resource that could help to ease comprehension in this context?

Thanks

Javier


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Fco. Javier Diez-Guerra, PhD

Servicio de Microscopía Confocal
Centro de Biologia Molecular Severo Ochoa
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