Max resolution in extreme macro. Is lighting a factor?

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Beatsy
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Location: Malvern, UK

Max resolution in extreme macro. Is lighting a factor?

Post by Beatsy »

Hi,

I am familiar with the concepts and formulae regarding resolution of microscope-based optics and illumination. There's handy summary here...

https://www.leica-microsystems.com/scie ... lculation/

Darkfield and fluorescence seem pretty straightforward too as the NA of the illumination is simply not included in the calculation. Presumably because the subject is considered self-illuminating (it *is* the light source) from the lens's perspective.

I've been working with shorter wavelength lighting to increase resolution in microscopy. It works great! It crossed my mind this might be useful in certain (very) extreme macro situations too.

Then I thought that there could be an "N.A." component to EPI lighting in this situation after all. If you shine a pencil beam of light on the subject you get bright specular highlights for sure, but I wondered if the subject then becomes a low-NA source which reduces the max resolution achievable. Further "thinking" just confused me...

I assume shorter wavelength WILL yield better resolution in a macro, EPI illuminated situation but is the light "structure" a critical factor too? What is the NA of diffuse illumination hitting a subject from all directions? Presumably it's high and equivalent to darkfield illumination - the subject is effectively the light source. But what if that illumination comes from an un-diffused point source? Is maximum resolution compromised?

I haven't experimented yet as the answer is likely very simple and staring me in the face. I'm hoping the collective PMN brain can shed some light (haha) and spare me the effort.

Thanks in advance...

enricosavazzi
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Re: Max resolution in extreme macro. Is lighting a factor?

Post by enricosavazzi »

Beatsy wrote:
Mon Feb 20, 2023 2:59 am
[...]What is the NA of diffuse illumination hitting a subject from all directions? Presumably it's high and equivalent to darkfield illumination - the subject is effectively the light source.
It depends on whether the subject is a specular reflector or a diffuse reflector. A specular reflector may change the NA of the illumination source by a little, but often not by much. A diffuse reflector can give a much higher NA than the illumination source. What you need to strive for is achieving a high enough NA of the reflected light from the subject that the objective's NA becomes the limiting factor.

Another way of seeing this is by considering the reflection of a point light source on a specular subject. The cone of light reflected by the subject and entering the lens will be much narrower than the cone of light that could enter the lens if the field of view was uniformly illuminated. In these conditions, the utilized aperture of the objective will only be a fraction of the actual objective's NA.

Starting with a diffuse illumination, in practice, has better chances of producing reflection with a high NA from the subject, and is the best bet at high-magnification photomacrography. There isn't much you can do about the type of reflection from the subject, unless you coat it with a diffusing layer. Often there isn't much you can do about the NA of the objective, either.
--ES

rjlittlefield
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Re: Max resolution in extreme macro. Is lighting a factor?

Post by rjlittlefield »

I agree completely with Enrico's assessment.

There is a long thread at viewtopic.php?t=19582 ("False color and detail/DOF from shiny metal") that touches on this issue.

The discussion of "partial coherence" is particularly relevant. That is on page 6. viewtopic.php?p=164914#p164914 is a good place to start.

I see at viewtopic.php?p=164939#p164939 that there are several papers by H.H. Hopkins, the fellow who wrote my standard reference about MTF of a defocused lens. They are described as behind paywall but at least today the first two are accessible as free download of PDF. I have collected copies of those two but have not yet started to read them. If Hopkins' work on defocused optical systems is any guide, it will take me several years to digest most of these two.

--Rik

Beatsy
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Location: Malvern, UK

Re: Max resolution in extreme macro. Is lighting a factor?

Post by Beatsy »

Thank you Enrico. I had fumbled my way to that conclusion (that NA is derived from the subject and how it reflects incident light), but had nagging doubts I was missing something. Confirmation is comforting...

And thanks for your confirmation of that confirmation Rik, and the links. I'll look through those this evening.

Cheers

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