Spec for Objectives: Terminology

[linuxusr]

Taped to my lab table I have a chart for FOV and WD.

There's another spec I need for my objectives, but before I can search for those, I need to clarify some confusion I have around nomenclature. The spec I need is for the depth (Z direction) of the image plane. I am assuming that "image plane" and "focal plane" are synonyms. Put simply, I am referring to the vertical distance that a specimen remains in focus when the stage is moved up or down. I am confused about the relationship between the above terms and the common one, DOF, or Depth of Focus.

I'm sure you know what I'm talking about but I need to make sure that I'm using the right term when I search.

[Scarodactyl]

DoF is depth of field, which is what you want (directly related to numerical aperture). Depth of focus happens after the objective and is basically how thick the plane is where the image is in focus. Higher NA optics have a narrower depth of field, but a woder depth of focus, which is why high mag objectives care less about eyepiece positioning than low mag ones.

[Phill Brown]

WD is working distance, from the focal plane to the front lense of the objective.
LWD is long working distance, usually for inverted microscopes. eg WD 3.2 is in mm.
0.17 is corrected for a cover slip.
1.2 would be corrected for viewing from underneath/through the slide.
Always happy to be corrected if needed.

[linuxusr]

DoF is depth of field, which is what you want (directly related to numerical aperture). Depth of focus happens after the objective and is basically how thick the plane is where the image is in focus. Higher NA optics have a narrower depth of field, but a woder depth of focus, which is why high mag objectives care less about eyepiece positioning than low mag ones.

With your explanation, I thought "Bingo! This is what I need!" Then I started thinking about depth of field versus depth of focus and started to get confused. Adding to the confusion, note that both terms are abbreviated DOF!!

I then went to the Nikon knowledge base and the explanation was confounding. The glossary in the knowledge base of the Olympus website was clearer: https://www.olympus-ims.com/en/microsco ... al_depth/ What I need is the Depth of FOCUS. The focal depth or depth of focus is, quoting

the depth of the specimen layer which is in sharp focus at the same time."

. The article notes that this is also related to NA. To calculate this, you need to solve the equation for the Berek Formula. At first it looks hairy, but on second look, I realized that I have the values I need and that the article supplies the other ones, so I should be able to calculate this for each objective with a scientific calculator.

[linuxusr]

OP: I believe that image plane and focal plane are synonyms and that depth of focus refers to the depth of focus of the image/focal plane . . .

[Dubious]

Whatever Olympus says, I think it's "depth of field"--which, from my long-ago amateur photography days, I have (rightly or wrongly) always considered as depth-of-apparent-focus rather than depth-of-actual-focus. That is, with a camera lens of a certain focal length closed to a certain aperture, when the camera is focused precisely on a 2-dimensional target, depth of field is the distance from in front of the target to behind the target that appears to be in focus even though it actually is not.

[MichaelG.]

Whatever Olympus says, […]

I think Edmund Optics gives a clearer explanation

https://www.edmundoptics.co.uk/knowledg ... -of-focus/

Their page starts with:
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Due to similarity in name and nature, depth of field (DOF) and depth of focus are commonly confused concepts. To simplify the definitions, DOF concerns the image quality of a stationary lens as an object is repositioned, whereas depth of focus concerns a stationary object and a sensor’s ability to maintain focus for different sensor positions, including tilt.

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Nikon has another good page, which is more specifically about microscopy:

https://www.microscopyu.com/microscopy- ... h-of-focus
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and, for a model of written clarity, I would go to Peter Evennett

viewtopic.php?p=89233#p89233

MichaelG.

[linuxusr]

Yes, definitely, "depth of field" is a bone of contention as it relates to "depth of focus." I still need to clarify the latter and perhaps I'll do that when I read through some of your posts that I'm behind on.

Meanwhile, I calculated the Berek Formula for all of my objectives and this is what I came up with for Depth of Focus:

The order is objective, NA, D.O.F. The results are in μm's.

4x 0.10 33.75 μm
10x 0.25 14.40 μm
20x 0.45 4.13 μm
40x 0.65 1.6 μm
100x 1.25 0.19 μm

1/20 of 1 μm for oil?? Hmm. On the whole, the values makes sense. I have to wait 'til I get to my scope and play around. Unfortunately, I'm leaving the country for a few weeks, so this will be in a while.

[linuxusr]

and, for a model of written clarity, I would go to Peter Evennett

viewtopic.php?p=89233#p89233

MichaelG.

I agree that Evennett is the paragon of clarity but, I think, not so much in this case. The definitions he gives for Depth of Field and Depth of Focus are exactly the same except that, as he says, the depth of field refers to the object plane and the depth of focus refers to the image plane. But then he does a poor job distinguishing between the object and the image planes. Just from the nomenclature it seems as if the image plane is the plane that includes the object (specimen) and the object plane, well, perhaps that's the apex of the light cone. I'm wondering if depth of field isn't a term which makes more sense in photography and less in microscopy.

Call it what you will, I am interested in the distance that my specimen remains in focus when I move the stage in the Z direction. I think no one has any doubt about the meaning of that and I think that Depth of Focus, simply as a descriptor, is a better fit than Depth of Field.

[MichaelG.]

If only for my sanity, linuxusr … could you please edit your post to clarify the ‘quotations’

Thanks
MichaelG.

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Edited to correct my spelling

[MichaelG.]

Call it what you will, I am interested in the distance that my specimen remains in focus when I move the stage in the Z direction. I think no one has any doubt about the meaning of that and I think that Depth of Focus, simply as a descriptor, is a better fit than Depth of Field.

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I think we need to step back a little [you may want to read some old photographic books]

Depth of Focus relates to the captured image, and always has done
… it is based upon the concept of the ‘Circle of Confusion’

Depth of Field relates to the subject … and the physical geometry of what we are imaging

We can estimate Depth of Field by working backwards from the chosen [acceptable] Circle of Confusion

You say:

I am interested in the distance that my specimen remains in focus when I move the stage in the Z direction

But the point is … first you need to define what you mean by “in focus”

MichaelG.

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PostScript: Unfortunately, the historic explanations tend to relate to situations where the subject distance is large and the image distance is small … and microscopy is contrariwise.

[MichaelG.]

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I’ve just found this : https://www.photopills.com/calculators/coc

which [although I have not yet studied it in detail] seems to be a good modern take on the matter.

MichaelG.

[linuxusr]

If only for my sanity, linuxusr … could you please edit your post to clarify the ‘qotations’

Thanks
MichaelG.

Sorry, MichaelG. I realized that the way I responded was a little wonky. I read the paper on the interview questions with Evennett re: difference between depth of field and depth of focus. Everything I said about that article was a paraphrase. I have no quotes except for my quote of your post.

That being said, to recap, Evennett's definition of both are 100% identical except that, he says, that depth of field refers to the object plane and that depth of focus refers to the image plane. I have since reviewed various optical charts and I see that there are two image planes. The primary image plane is in the retina. The secondary image plane is in the ocular tube. The object plane proceeds horizontally through the specimen and is the apex of the cone that resolves the specimen.

Since I'm interested in using the formula for Depth of Focus, I suppose I could say that when I move the stage in the Z direction that it is moving in the vertical axis through the object plane, where the specimen is, but that I am "realizing" that image in the image plane, first, when I look down the ocular tube, second, when what I see is being resolved in my retina.

[linuxusr]

@MichaelG

I'm packing for an international flight, so I'm going to have to return to this topic in about two weeks . . .

You ask what is meant by "focus." I mean sharp and not blurry, i.e. being able to resolve discrete objects, e.g. a chloroplast within a cell or the cell wall of a chloroplast versus the color of chlorophyll within the organelle versus the transparent cytoplasm without. The example I'm thinking of is an Elodea leaf at 400x TM.

You reference an article that I will be reading. I also want to look at the definition of depth of field as it is used in photography. As I remember, "depth of field" is critical in photography. There is a precise definition for it and also methods for manipulating the depth of field. I've never heard in microscopy that "depth of field" is something that can and should be manipulated in microscopy as it is in photography. So I want to look at the formal definition in photography than compare it with the definition in microscopy and see how they are the same or different.

Also, I have purchased the hardcover textbook, "Fundamentals of Light MIcroscopy and Electronic Imaging," Second Edition, Murphy and Davidson. I also want to go to that index, when I return, and seed if I can get further clarification.

[MichaelG.]

Enjoy your reading ... we can pick this up in a couple of weeks.

MichaelG.

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Meanwhile: You may like this very brief video describing Circle of Confusion: https://youtu.be/Pdq65lEYFOM

Edit: __ I have just downloaded this [Free] from the Apple AppStore:
https://apps.apple.com/gb/app/depth-of- ... 1540947216
… If you have an iOS device, it’s worth playing with.

[MichaelG.]

If only for my sanity, linuxusr … could you please edit your post to clarify the ‘qotations’

Thanks
MichaelG.

Sorry, MichaelG. I realized that the way I responded was a little wonky. [..] I have no quotes except for my quote of your post.

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Which you somehow managed to attribute to 'Dubious'

MichaelG.

[MichaelG.]

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EUREKA !

I am posting this for linuxusr, but others might find it of interest

Please download a copy of Carpenter’s masterpiece: The Microscope and its Revelations
… available in various formats, here: https://archive.org/download/microscopeitsrev00carp_1
[ the PDF is, conveniently, searchable ]

You will note that the term ‘Depth of Field’ is completely absent [because it hadn’t been coined yet]
But he uses the terms ‘Depth of Focus’ and ‘Penetration’ to describe what we now term ‘Depth of Field’

He seems to jump directly from ‘Circle of Confusion’ to ‘Penetration’ without analysing the intervening process in any detail. But language develops … and in this case, better comprehension comes from looking separately at ‘Circle of Confusion’ ‘Depth of Focus’ and the resulting ‘Depth of Field’

Note: ‘Penetration’ also aligns with the concept of ‘Focus Depth’ used by Olympus, and mentioned earlier.

MichaelG.
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Unfortunately; although the Carpenter text is scanned very adequately, the frontispiece plate is poor … and visual comparison of [7] and [8] is futile. [if anyone has a hard copy, I would be most grateful for a high resolution scan of that frontispiece]

Edit: A better image of the page is available here: https://ia800400.us.archive.org/view_ar ... 1_0008.jp2
… But it still isn’t good.