What is Unit Distance Per Fine Focus Knob Increment??

[linuxusr]

Greetings All:

Help! As I’m generally looking down my eyetube, I just noticed that my fine focus has increments and realized that I can calculate DOF from these increments. But what is the unit distance (Z axis) for each increment?

Numerically, each increment represents 2 and the range is 0-200.
Further, there are two arrows and the distance between them is 50.

a. Is the unit distance per increment (numerical value 2 on fine focus knob) standard for all microscopes?
b. The range 0-200 seems arbitrary. It certainly is not degrees. Maybe it’s fine if it is arbitrary. This would mean you could take the same measurement regardless of the position of the knob.


I’ll have to play with it to see how it works out but it seems to me that one might place one of the knob’s arrows at 0, then the range between the two arrow points would be 0-50, and it would be from this range that you would calculate the depth of the focal plane, two points on the Z axis. But whether you can actually place the knob here and focus at the same time, I don’t know.

[MichaelG.]

(a) No

But my guess would be that yours is graduated at 2 microns per division.

MichaelG.

[microcosmos]

You may also be able to find the information in the microscope’s user manual.

If you need to be sure of accuracy, I recommend calibrating it using a broken coverslip to see how many graduations of the fine focus knob it takes to get from the lower surface to the upper surface of the coverslip using a high-powered objective of working distance more than the coverslip thickness. The broken edge of the coverslip is vertically slanted so it is easier to see the edges of the two surfaces. Determine the actual thickness of the coverslip using a micrometer screw gauge. Take multiple measurements of everything.

But I believe most good microscope makers are quite accurate with their engineering so you can probably just take their word for it if they specify a particular z distance, unless accuracy is critical, such as for measuring birefringence the calculation of which depends on the thickness of the sample. In fact, the calibration process described above itself has several sources of potential error if not careful.

[PeteM]

The graduations vary from maker (and model) to maker. Easy enough to check on the calibrations for whatever you have.

The range of the fine focus also varies. Many earlier models (Olympus, Leitz, Zeiss etc.) had beautifully made fine focus mechanisms -- but with a limited range. This limited range was sometimes indicated by a couple small marks to indicate upper and lower limits. More modern scopes may have fine focus systems that can cover the entire focusing range, should someone be that patient to crank and crank and crank . . . Sometimes in the better models with the same attention to build quality as earlier microscopes.

An accurate and finely-divided fine focus system with a large-enough range to cover subjects of interest is a big plus for focus stacking.

[75RR]

.
Looked it up out of interest. This is from the Zeiss Primostar 3, 25 page brochure

500/200 = 2.5µm by my calculation, so each division would be 5µm
.

[MichaelG.]

.
Looked it up out of interest. This is from the Zeiss Primostar 3, 25 page brochure

500/200 = 2.5µm by my calculation, so each division would be 5µm
.

.
Slightly confused here

I can see no graduations on the illustrated knobs.
… is this just ‘artistic license’ ?

MichaelG.
.

EE119E58-58DE-4EFA-ACAE-A49C576432F4.jpeg (148.82 KiB) Viewed 7433 times

[BramHuntingNematodes]

well, 200 graduations was specified and this table shows the total distance traveled with a full revolution.

[MichaelG.]

well, 200 graduations was specified and this table shows the total distance traveled with a full revolution.

.

Forgive me asking, Bram … where, in the opening post, was the microscope specified ?

MichaelG.

[apochronaut]

The markings on the fine focus are not that relevant : as long as the focusing mechanism has a smooth and fluid movement, for most microscopy that is all that is required. Most of the time, microscopists do not normally use the graduations on the focusing knob for anything.
However, 1 micron used to be a standard for a good microscope , where locating an aspect of your sample in depth or measuring in depth based on the fine focus graduations was used.
Now we have focal distance used as a tool for stacking as well. Probably the precision of some systems exceeds the reference graduations , so one could use the interspaces as 1/2 graduations.

[BramHuntingNematodes]

There is a Zeiss Primostar 3 in the signature line. I suppose there may only be 100 increments if each increment is 2 and the range is between 0-200.

[MichaelG.]

There is a Zeiss Primostar 3 in the signature line.

There is also a Nikon AlphaPhot 2

MichaelG.

[BramHuntingNematodes]

well heck what do the graduations on an alphaphot look like? Does it have any?

My only Nikon microscope says "Nippon Kogaku" on it

[75RR]

.
The Zeiss Primostar is linuxusr's go to microscope. All his posts on microscopes since he got it are about it.
.

[MichaelG.]

well heck what do the graduations on an alphaphot look like? Does it have any?

This one appears to have graduations : https://www.ebay.co.uk/itm/154564379201

MichaelG.

[MichaelG.]

.
The Zeiss Primostar is linuxusr's go to microscope. All his posts on microscopes since he got it are about it.
.

Thanks for the photo … which shows graduations that do not feature in the drawings

That’s all I was asking, when I wrote:

I can see no graduations on the illustrated knobs.
… is this just ‘artistic license’ ?

MichaelG.

[MicroBob]

A simple check is to take a cover slip and make a line on the top an bottom with a permanent marker. Only accurate enough as he cover slip thickness but useful to determine whether one has calulated right. In engineering literature one has to be careful to avoid mixing up dimensions, are we talking about 1/1000 mm or inch?

[linuxusr]

.
Looked it up out of interest. This is from the Zeiss Primostar 3, 25 page brochure

500/200 = 2.5µm by my calculation, so each division would be 5µm
.

.
Slightly confused here

I can see no graduations on the illustrated knobs.
… is this just ‘artistic license’ ?

MichaelG.
.
EE119E58-58DE-4EFA-ACAE-A49C576432F4.jpeg

MichaelG, you are right. It was artistic license. It seems that the purpose of these figures was to show principal controls and functions, so no details.

Here is a shot I just took of the fine focus knob: https://imgur.com/9KJsoYO

[linuxusr]

.
The Zeiss Primostar is linuxusr's go to microscope. All his posts on microscopes since he got it are about it.
.

That is correct 75RR. The Nikon in my signature line was my first microscope 25 years ago.

[linuxusr]

.
Looked it up out of interest. This is from the Zeiss Primostar 3, 25 page brochure

500/200 = 2.5µm by my calculation, so each division would be 5µm
.

75RR, you are on the money! Thanks. As many times as I read the manual, I never saw this. I repeated your calculations and arrived at the same value, 2.5 μm per increment where the value of each increment is 2, so 2(2.5) = 5 μm. I think what I'm going to do now to confirm and also for fun, is to use microcosmos' suggestion and to take the measure of a coverglass with a caliper, calculate the depth in μm, figure out the equivalency in increments on the focus knob, and see if I can use these increments to calculate the DOF of the coverglass. It I can do that, then using the final focus as a measurement tool could be useful. I'll try that now and see what happens and get back.

[linuxusr]

You may also be able to find the information in the microscope’s user manual.

If you need to be sure of accuracy, I recommend calibrating it using a broken coverslip to see how many graduations of the fine focus knob it takes to get from the lower surface to the upper surface of the coverslip using a high-powered objective of working distance more than the coverslip thickness. The broken edge of the coverslip is vertically slanted so it is easier to see the edges of the two surfaces. Determine the actual thickness of the coverslip using a micrometer screw gauge. Take multiple measurements of everything.

But I believe most good microscope makers are quite accurate with their engineering so you can probably just take their word for it if they specify a particular z distance, unless accuracy is critical, such as for measuring birefringence the calculation of which depends on the thickness of the sample. In fact, the calibration process described above itself has several sources of potential error if not careful.

Good idea to break it and get an angle. 75RR found the spec in the manual, which I had overlooked, and I confirm his calculation at 5 μm per increment. Now I'm going to apply your test and compare. I'll post a result. To me, the only interesting question is to find out if the fine focus knob can be used as a useful measurement tool for the Z axis. I think it's the only tool, right? Reticles are for the X and Y axes.

[linuxusr]

There is a Zeiss Primostar 3 in the signature line. I suppose there may only be 100 increments if each increment is 2 and the range is between 0-200.

There's 200 numbered increments but each as the value of 2: 2, 4, 6, 8, etc.

[linuxusr]

A simple check is to take a cover slip and make a line on the top an bottom with a permanent marker. Only accurate enough as he cover slip thickness but useful to determine whether one has calulated right. In engineering literature one has to be careful to avoid mixing up dimensions, are we talking about 1/1000 mm or inch?

Marking a line on each side is a good idea. The distance between the top line, when it is in focus, and the bottom line when it is in focus, will be the focal plane depth, Z axis.

[MichaelG.]

Good idea to break it and get an angle. 75RR found the spec in the manual, which I had overlooked, and I confirm his calculation at 5 μm per increment. Now I'm going to apply your test and compare. I'll post a result. To me, the only interesting question is to find out if the fine focus knob can be used as a useful measurement tool for the Z axis. I think it's the only tool, right? Reticles are for the X and Y axes.

.

Yes, as you correctly deduce, it should effectively be a ‘depth micrometer’
… Glad to see that the collective discussion got you to where you want to be.

What astonished me was that I could find no illustration of the graduations in any of the PrimoStar 3 ‘handbooks’ and no explanation of how and why one might use them.

If it was not for the photographic evidence, I would have not known they existed !

MichaelG.

[linuxusr]

@ et al

Hello All:

OP Summation:

I did not have success calibrating my FF knob such that the distance I turned it agreed with the distance of the Z axis between the top and the bottom of a coverglass. More on that later.

The data:

(a) The Zeiss spec for the FF knob is 0.5 mm rev. or 500 μm/rev.
Earlier I made an arithmetic mistake, confused by the fact that each increment on the FF knob equals two digits. Imagining the knob as a circle equally divided by 200 units, in this case a range of numbers from 0-200, then you have to divide 500/200 = 2.5 μm every time you change from one digit to the next on the knob.

(b) The coverglass is one by EMS (Electron Microscopy Sciences), Micro Cover Glass #1. All are < 0.17 mm or 170 μm. I measured 5 using my digital caliper and each had a depth of 0.15 mm or 150 μm. This is the distance of the Z axis from the top to the bottom of the coverglass.


The test purpose is to determine if this value corresponds to the value calculated from the FF knob when both the top and the bottom of the coverglass’ specimens are in focus.

The procedure:

I made three tests. The first used a coverglass that was marked by a dot from a permanent marker, one dot on the top, the other on the bottom in a different location (easier to find when they are not superimposed). The second used a slide with cheek cells (a dry mount), with the second set of cells on the top of the cover glass, so, essentially, cheek cells on the top and the bottom of a cover glass.

The procedure is to focus on the top of the specimen, then to note the digit on the FF knob (there is an arrow.) Then focus on the specimen that is on the bottom and note that digit.

I’m calling Zt the top of the coverglass’ Z axis and Zb the bottom of the coverglass’ Z axis. Having written both values, one for the top and one for the bottom, then you calculate the difference between Zt and Zb and multiply that by 2.5 μm. That value, which is taken from the Zeiss spec, should equal the depth of the coverglass.

All tests at 100x TM.

Test 1:

The marker dot result:

Zt = 130 (FF knob value)
Zb = 79
130 – 79 = 51(2.5) = 127.5 μm.

But the depth of the coverglass is 150 μm.
127.5 μm does not equal 150 μm. Error!


Test 2:

Broken coverglass:

This test failed because I could not focus Zb.

Test 3:

Cheek cells.

Zt = 106
Zb = 7

Zt – Zb = 99(2.5) = 247.5 μm

I think it’s interesting to compare the result of 127.5 μm from the marker spots with 247.5 μm with the cheek cell result. Except for slight differences in focusing the numbers should match but they don’t. I think that this can be accounted for by the depth of the cheek cells versus the depth of the spots. The cheek cells clearly add distance x 2 (top and bottom of coverglass) to the Z axis that corresponds only to the coverglass.

The difference between 247.5 and 127.5 is 120. 120/2 = 60, indicating, possibly, that the depth of each cheek cell is 60 μm. That’s a big “if” because it seems that that value should be less.

And, again, 247.5 μm does not equal 150 μm. Error!

So I cannot at this time use my FF knob to measure the depth of a specimen. I think it’d be great if one of you could try and get an accurate result. There’s an assortment of variables here that could explain the discrepancy.

[PeteM]

While backlash and wear in the gears can be a slight factor -- as another check -- you might try fixing a dial indicator to the stage and actually measuring how much movement you get in a complete rotation of the fine focus knob.

Lacking an indicator, just come to a firm landing on something like a slide or flat piece of metal, then remove the slide/metal and see how many graduations are needed to get the same firm landing on the stage. Measure the slide/metal thickness with your caliper and divide by the number of graduations traversed.

You could use an old/bad objective for the touch-off point and a thin bit of paper in between. Once you get to a point where you can remove the paper, but with just a bit of drag, the measurement should repeat within a couple thousandths.

[MichaelG.]

@ et al

Hello All:

OP Summation:

I did not have success calibrating my FF knob such that the distance I turned it agreed with the distance of the Z axis between the top and the bottom of a coverglass. More on that later.

The data:

(a) The Zeiss spec for the FF knob is 0.5 mm rev. or 500 μm/rev.

< etc. >

.
Please forgive me if I have misinterpreted your description, but : It appears that you are trying to measure through glass.
… if so, your results will inevitably be distorted by the Refractive Index of that medium.

My approach would be to first check that the Zeiss claim of O.5 mm per rev. is accurate.
In the absence of a set of ‘Jo Blocks’ I would start with a 0.5 mm Feeler Gauge.
Check the 0.5 mm claim [in Air], and then proceed to ‘calibrate’ linearity.

MichaelG.
.
Ref. __ https://en.wikipedia.org/wiki/Gauge_block
Ref. __ https://en.wikipedia.org/wiki/Feeler_gauge

.

P.S. __ If you are doing the test mechanically, rather than optically, it is worth noting that cigarette papers are approximately one thousandth of an inch thick … and provide a convenient way of checking ‘touch-down’

[75RR]

.
Have a look at this article: https://www.microscopy-uk.org.uk/mag/ar ... rslip.html

[microcosmos]

Please forgive me if I have misinterpreted your description, but : It appears that you are trying to measure through glass.
… if so, your results will inevitably be distorted by the Refractive Index of that medium.

Thanks for mentioning this. It prompted me to take another look at the reference I was using (Guide to Thin Section Microscopy by Raith et al.).

Apparently this is why it is preferable/easier to use the slanted broken edge of a coverslip, rather than using a marker to mark the two surfaces. If the broken edge is slanted with a positive slope, you can observe and focus on both surfaces through air. Then the distance traveled by the stage per graduation on the fine focus knob is simply the measured thickness of the coverslip divided by the number of graduation marks traversed while focusing between the two surfaces, as Pete explained.

If the broken edge has a negative slope (i.e. overhanging), you will have to focus on the lower surface through the glass of the coverslip. In this case you can still get an accurate calibration by further dividing the above result by the refractive index of the glass (assuming you know it accurately - it's given in my book as 1.5). You can do the same if you're using the marker method.

I was also wondering if the marker ink could introduce another (minor) potential source of error as it has some thickness itself. You have to be sure to focus on the part of the ink that's right next to the surface of the coverslip, or perhaps you could include the ink marks in the overall micrometer screw gauge thickness measurement.

[Hobbyst46]

Please forgive me if I have misinterpreted your description, but : It appears that you are trying to measure through glass.
… if so, your results will inevitably be distorted by the Refractive Index of that medium.

Thanks for mentioning this. It prompted me to take another look at the reference I was using (Guide to Thin Section Microscopy by Raith et al.).

Apparently this is why it is preferable/easier to use the slanted broken edge of a coverslip, rather than using a marker to mark the two surfaces. If the broken edge is slanted with a positive slope, you can observe and focus on both surfaces through air. Then the distance traveled by the stage per graduation on the fine focus knob is simply the measured thickness of the coverslip divided by the number of graduation marks traversed while focusing between the two surfaces, as Pete explained.

If the broken edge has a negative slope (i.e. overhanging), you will have to focus on the lower surface through the glass of the coverslip. In this case you can still get an accurate calibration by further dividing the above result by the refractive index of the glass (assuming you know it accurately - it's given in my book as 1.5). You can do the same if you're using the marker method.

I was also wondering if the marker ink could introduce another (minor) potential source of error as it has some thickness itself. You have to be sure to focus on the part of the ink that's right next to the surface of the coverslip, or perhaps you could include the ink marks in the overall micrometer screw gauge thickness measurement.

Moreover, running these calibrations on a slide instead of a coverslip will be inherently more accurate. The slide thickness of ~1mm is fairly accurately measurable with a micrometer or even calipers. A slanted edge would indeed enable focusing through air. Notice - start with the fine focus control midway to allow several full rotations while the coarse focusing stays untouched.

[linuxusr]

OP: I read with interest your various methods for measuring this value. Some of those methods were too sophisticated for this amateur!

-- The Zeiss spec indicates a 15 mm stage lift. I set my caliper to 15 mm, held it against a cutting board perpendicular to the stage, with the bottom jaw flush with the stage. As I raised the stage it exceeded 15 mm. The stage lift is more like 22 mm. So I'm a little uncertain about those specs.

-- I tried measuring the depth of a slide, using top and bottom markings, without a coverglass. The slide was 1,130 μm. I had to rotate the FF knob so many times that it was hard to count, so I abandoned that and went back to the coverslip marked on top and bottom with a marker. There are tiny specs within the marker liquid from which you can get a sharp focus.
Also, I measured two marker strips with no marker strips and the caliper readout was the same. Probably that depth is in nm and can be ingnored.

From the top to the bottom of the coverglass I passed through the range 77-18 = 59. 150 μm for the depth of the coverglass /59 gives me 2.54 μm which is the distance between two integers on the FF knob on the Z axis. This is the same value I got the first time except for 4/100 or 1/25 of a μm which is negligible.

Unless you think this value is really, really suspect, I think that in the future, if I want to know the depth of a specimen, I'll focus on the top and bottom, take the difference between the two numbers and divide that into 150 and that should give me Z axis μm through the specimen.