Resoloution and Light Wavelengths

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75RR
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Re: Resoloution and Light Wavelengths

#31 Post by 75RR » Wed Jul 21, 2021 2:35 pm

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This is my latest effort with a Kemp slide:

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Stonius
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Re: Resoloution and Light Wavelengths

#32 Post by Stonius » Wed Jul 21, 2021 2:37 pm

Wow - that's amazing!
His wife thinks he does protist too much.
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Re: Resoloution and Light Wavelengths

#33 Post by Hobbyst46 » Wed Jul 21, 2021 3:35 pm

However resolution is not everything. Contrast is as important. Illumination is important. I would assume that there is nothing wrong with the resolution of your microscope and start looking at interesting specimens instead. After some specimen-related adventures, one forgets about theoretical vs practical resolutions. There are modern light microscopes that give much better resolution than implied by diffraction, but they are very very expensive, and most of them are dedicated to fluorescence rather than broad band illumination.

crb5
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Re: Resoloution and Light Wavelengths

#34 Post by crb5 » Wed Jul 21, 2021 4:43 pm

Whether you go with graticules or diatoms, the numerical value assigned to the resolution depends on the criterion used to define the separation required to distinguish two objects. The Rayleigh limit is rather generous in this respect and boosting contrast may resolve (i.e. distinguish) features which are slightly smaller than this limit.

Thanks, Hans for the https://www.dspguide.com/ch25/1.htm reference which I found very useful. Figure 25.2 shows how as the graticule lines get closer together, there is a region where the intensity becomes modulated, before finally merging into one featureless line. It is in this modulated region that contrast enhancement can help to resolve the lines. Modern camera and digital technology gives a distinct advantage over the eyes of Victorian diatomists.

Thanks, Marcus for the oil immersion image, which is more contrasty and sharper than the original image. From an intensity profile plot using ImageJ, the slope at the edges of the black lines, as defined by the 10% to 90% intensity criterion, comes out at 0.3 um which is pretty close to the expected Abbe limit.

Hans – you were right to question my numerical aperture equation – still thinking about that but it seems to depend on the depth of the air layer and NA approaches 1 as the air layer -> 0.
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Phill Brown
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Re: Resoloution and Light Wavelengths

#35 Post by Phill Brown » Wed Jul 21, 2021 5:24 pm

Definitely should have diatom slides, and dark field.

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Re: Resoloution and Light Wavelengths

#36 Post by hans » Wed Jul 21, 2021 8:38 pm

Stonius wrote:
Wed Jul 21, 2021 8:58 am
The insert at 100% shows a line separated from the edge of the black division marker by about 50 pixels, or 0.0005mm. I take it this line is an effect of diffraction and represents the point beyond which no further detail can be resolved, even if all else were perfect?
Seems more common to measure the apparent distance it takes for the intensity to go from 10% to 90% relative to the values far from the edge, which is also an effect of diffraction as crb5 mentioned earlier. Measuring the distance from the center of the transition to that dark band is conceptually similar but more sensitive to the exact shape of the response, so not as robust and more difficult to interpret without prior knowledge of what the edge response should look like for the specific optical system in question. Figure 25-4 in that dspguide.com link is illustrating how 10% to 90% transition width is not strongly dependent on the exact shape of the PSF/MTF.
Hobbyst46 wrote:
Wed Jul 21, 2021 7:50 am
There is nothing wrong with the plot, except that it does not indicate resolution in the classic sense (defined according to the Abbe or Raleigh criteria).
Hobbyst46 wrote:
Wed Jul 21, 2021 10:03 am
The thin line around the scale mark, which is probably an artifact (besides diffraction, there is chromatic aberrations), is nevertheless a distinguishable object in the image, So I treat it as if it were a real object.
Its distance from the scale mark visible border on my monitor is ~2mm. Hence, its true distance in the image created by the microscope is 5.6*2/32 = 0.35 um.
If you will infer resolution from this dark band diffraction artifact then why not from the apparent transition width, which is also a diffraction artifact? (Assuming the physical transition on the slide is sharper than the objective can resolve.) Transition width 10% to 90% (as opposed to other details/features of the edge response) appears to be widely used and has some practical benefits discussed on the two pages I linked earlier.
crb5 wrote:
Wed Jul 21, 2021 4:43 pm
... numerical aperture ... seems to depend on the depth of the air layer and NA approaches 1 as the air layer -> 0.
Yeah as the air gap approaches zero, it is simpler, rays corresponding to NA >= 1 are just lost due internal reflection going glass-to-air but there are no other effects on imaging. With significant air gap it gets more confusing to think about because of spherical aberration. In the extreme case with only air like Markus's first photo (guessing no cover glass either in that one) I believe the paraxial focus is shifted closer to the front element by a factor ~1/1.5, similar to the focus shift caused by plane parallel plate elements. But marginal focus approaches the surface of the front element as NA approaches 1 indicating extreme spherical aberration. So I guess the real question is, at what air gap thickness does resolution become limited by spherical aberration, so that NA is no longer a useful metric?

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Re: Resoloution and Light Wavelengths

#37 Post by Hobbyst46 » Wed Jul 21, 2021 9:35 pm

hans wrote:...
crb5 wrote:...
Thank you both for the link and comments. Admittedly my previous claims are only relevant within the two popular (at least in microscopy texts) definitions of resolution. I agree that the width of the 90% to 10% decay is a reliable indicator of resolution for the image obtained under oil immersion. It is far less reliable, IMHO, for the image of the scale bars under 100x objective, dry.

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Re: Resoloution and Light Wavelengths

#38 Post by 75RR » Wed Jul 21, 2021 10:00 pm

Hobbyst46 wrote:
Wed Jul 21, 2021 3:35 pm
However resolution is not everything. Contrast is as important. Illumination is important. I would assume that there is nothing wrong with the resolution of your microscope and start looking at interesting specimens instead. After some specimen-related adventures, one forgets about theoretical vs practical resolutions.
There is a great deal of skill in light microscopy, and as such it can be considered a craft as well as a science.

At the end of the day however, pursuing resolution for its own sake is more about seeing if one can get close to what one’s microscope can do –

of proving that you deserve such a microscope - of matching skills if you like.

That is well and good and should not be discouraged, however, the parameters needed to achieve such resolution are not the ones one normally encounters

or can arrange in day-to-day viewing of say a wet slide.

As such, one has to accept that the maximum resolution attainable at the moment is the one that counts.

In a nutshell, better a slightly less sharp Loch Ness Monster (or its animalcule equivalent) than no Nessy at all!
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
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Sure Squintsalot
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Re: Resoloution and Light Wavelengths

#39 Post by Sure Squintsalot » Mon Jun 20, 2022 5:31 pm

Bumping this discussion.

I'd like to know, in microns, what the resolution is of this setup. I'm looking at a 90s era Pentium chip using a fiber optic halogen light, reflected at a low angle. Within that pink bar, parallel to it's long dimension, I can see distinct lines:
Screenshot 2022-06-20 112521.jpg
Screenshot 2022-06-20 112521.jpg (60.32 KiB) Viewed 1536 times
Based on the information solely in this image, can I determine the separation between the pink lines? And what is that separation?

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