Magnification of oil immersion

[FungusMan]

I want to be sure that what I am doing/assuming is correct.

I need to measure some spores and assume I am using the x100 objective (oil) with the eyepiece of x10 and a reticle with 10 um (0.01mm) per division. The theoretic distance between each division is 1um. However when use a stage micrometre with oil immersion to calibrate, the length of one div of the reticle is not 1um but 1.17um, that is the reticle under oil is 1.17um per div.

1. When measuring under oil with the same reticle, I have to use the factor that 1 div is 1.17um (not 1um) and nothing else. A spore long 10 divisions is hence 11.7um (not 10um)

2. The actual magnification under oil is then not x100 but x117 (1170 with the x10 eyepiece). Is this reasoning correct (feels not but...)

Thanks

[Hobbyst46]

The stage micrometer gives the true (not theoretical) scale on the specimen slide. Prior to calibration, the exact value of the distance between reticle marks is not important (aside from giving sufficient spatial resolution).
The size of objects in the specimen might be important (depends on the microscopist); magnification is usually not important.
To measure an object in a specimen, you rely on the scale of the stage micrometer and calibrate the reticle appropriately. Like this:
Focus on the stage micrometer (typically the marks are spaced at 10um). The reticle should be simultaneously sharply visible.
Find out to which distance (from the stage micrometer scale !) corresponds a space between two scale marks on the reticle. Now the reticle is calibrated.
Repeat the procedure for every objective you use (BTW, the width of the FOV, in mm, is proportional to the magnification; i.e. the FOV through the 100X objective is 1/2.5 as wide as the FOV through the 40X; given that the same eyepiece is used).
From then on, to measure spores for example, use the reticle as ruler.

[EYE C U]

does it make sense to look through an eyepiece that has a measuring field then put a calibration slide and note real measurements per each objective power compared to the eyepiece grid

[apochronaut]

You initially use both an eyepiece reticle and a stage micrometer. The stage micrometer is a real measurement and the reticle is compared to it and thus is calibrated to it. In your case that would be done with the 100X objective.From thence forward, the calibrated reticle is used to measure. The reticle should be calibrated for each objective used, unless you use just one for measuring or you can easily transpose the measurement from that of a known f.o.v. to any other.

[MichaelG.]

This is a very good tutorial video ... “straight from the horse’s mouth”

https://youtu.be/c2KeZur8D_E

MichaelG.

[EYE C U]

YEA..JUST LIKE THAT

haven't owned an eyepiece with a scale till last week. no better time to play with the slide that came with my camera

[FungusMan]

I did all this already and I knew all the theory, but my question was that when I calibrated the oil immersion x100, the distance between the 10um grades of the reticle where 11.74um and hence asking what is the actually magnification x100 or x 85 or x117 under oil immersion ?

[FungusMan]

in the x63 obj. 95 reticle units measured 150um, hence 1 unit - 150/95 = 1.58um/div
actual magnification: 950um (reticle units) / 15um (actual) = x 63.3

in the x100 obj., 95 reticle units measured 110um, hence 1 unit - 110/95 = 1.17um/div magn 1000/1.17 = x854 (not x 1000)
actual magnification: 950um (reticle units) / 11um (actual) = x 86.6 (not x 1000 as expected)

It looks a bit screwed up but the magnification in oil immersion is not actually x1000 (with x10 eyepiece) but considerably different!

[Hobbyst46]

I approach it differently. Irrespective of the scale of the reticle.
On my PC screen, with the 63X objective, 150um = 19.5cm. And with the 100X objective, 120um = 19.5cm.
Hence, the apparent ratio of FOVs between them is 150/120=1.25.
If the images were obtained under identical conditions, the ratio would have been 100/63 = 1.5.
If either objective has a different magnification from the marked figures, of course it would not be 1.5 (unless they both differ by the same factor... ).

My question is : do these two images represent identical condition ? I mean, are they cropped ? is the camera setting the same ? any zooming was done ? from the photos and vignetting, I suspect that they were taken under different condition.
Hence cannot conclude for sure about the magnification. The camera settings, distance from eyepiece, focusing (neglecting the few micrometers focus difference between the 63X and 100X) must be constant for a reliable comparison. Just switch objectives and record.

[MichaelG.]

I did all this already and I knew all the theory

Sorry !

MichaelG.

[FungusMan]

I did all this already and I knew all the theory

Sorry !

MichaelG.

You dont need to apologize, it is ok and thanks for your time

[FungusMan]

I approach it differently. Irrespective of the scale of the reticle.
On my PC screen, with the 63X objective, 150um = 19.5cm. And with the 100X objective, 120um = 19.5cm.
Hence, the apparent ratio of FOVs between them is 150/120=1.25.
If the images were obtained under identical conditions, the ratio would have been 100/63 = 1.5.
If either objective has a different magnification from the marked figures, of course it would not be 1.5 (unless they both differ by the same factor... ).

My question is : do these two images represent identical condition ? I mean, are they cropped ? is the camera setting the same ? any zooming was done ? from the photos and vignetting, I suspect that they were taken under different condition.
Hence cannot conclude for sure about the magnification. The camera settings, distance from eyepiece, focusing (neglecting the few micrometers focus difference between the 63X and 100X) must be constant for a reliable comparison. Just switch objectives and record.

Yes the images are cropped differently and few varying factors to use the screen as a model to calculate/compare the magnification. I was simply thinking that in air, the objective is x100 magnification but in oil immersion it is less (at first I assumed it is more). So when in science there is a x100 image and captioned as x1000, in reality, it is not! Also I think measuring the reticle as an indication is better than that of the screen, but I can see how your method is applied. The 63x objective x40 and x10 objective gave the right magnification (reticle method), only the oil objective with oil was different. What I can do is try and measure without oil.

[Hobbyst46]

I approach it differently. Irrespective of the scale of the reticle.
On my PC screen, with the 63X objective, 150um = 19.5cm. And with the 100X objective, 120um = 19.5cm.
Hence, the apparent ratio of FOVs between them is 150/120=1.25.
If the images were obtained under identical conditions, the ratio would have been 100/63 = 1.5.
If either objective has a different magnification from the marked figures, of course it would not be 1.5 (unless they both differ by the same factor... ).

My question is : do these two images represent identical condition ? I mean, are they cropped ? is the camera setting the same ? any zooming was done ? from the photos and vignetting, I suspect that they were taken under different condition.
Hence cannot conclude for sure about the magnification. The camera settings, distance from eyepiece, focusing (neglecting the few micrometers focus difference between the 63X and 100X) must be constant for a reliable comparison. Just switch objectives and record.

Yes the images are cropped differently and few varying factors to use the screen as a model to calculate/compare the magnification. I was simply thinking that in air, the objective is x100 magnification but in oil immersion it is less (at first I assumed it is more). So when in science there is a x100 image and captioned as x1000, in reality, it is not! Also I think measuring the reticle as an indication is better than that of the screen, but I can see how your method is applied. The 63x objective x40 and x10 objective gave the right magnification (reticle method), only the oil objective with oil was different. What I can do is try and measure without oil.

Sorry, magnification is (at least in practice) the same with / without oil.

[BramHuntingNematodes]

So when in science there is a x100 image and captioned as x1000, in reality, it is not!

well it depends on how big they print the image

[Hobbyst46]

Please note, that of the various features of the optical microscope (and objective), magnification is always relative (depends on other variables) and is much less important than other features. The main purpose of a microscope is resolution.

[apochronaut]

Hobbyist46. In mycology, accurate measurements of spores and features are very important to correct species i.d.
It is important to calibrate high magñification systems accurately.

I assume that you bought the Zeiss planfluar and your objectives are from the same series? Objectives from different manufacturers can have surprisingly different magnifications with the same eyepiece because they were originally designed to form an intermediate image at a different point due to differences in the eyepieces employed.

I have done extensive magnification measurements and they can vary from that marked quite a bit. Older objectives are more prone to those variances .

Oil only affects the magnification a tiny amount my increasing the w.d. slightly. One would assume that manufacturers based their objective measurements on the correct usage

[FungusMan]

[/quote]Sorry, magnification is (at least in practice) the same with / without oil.[/quote]

Hobbyst, I confirm your statement, i checked without oil. The Carl Zeiss x100 code 4860960 (1.30 na) objective is actually x850 (kinda a false marketing lol!). I bought this Zeiss second hand but after I cleaned it it has a fairly good and acceptable resolution and clarity

I assume that you bought the Zeiss planfluar

Correct, coming Plan Neofluar all way from Australia

I have done extensive magnification measurements and they can vary from that marked quite a bit. Older objectives are more prone to those variances .

Very interesting apochronaut !!! So than that is the reason, somewhat bewildering but acceptable

The conclusion of this, esp. for the beginner, is to calibrate the microscope with a stage micrometer before taking critical measurements such as in mycology.

[apochronaut]

It isn't normally that much, though. 5 X would be a lot. I'm still thinking through another answer.

[Hobbyst46]

The conclusion of this, esp. for the beginner, is to calibrate the microscope with a stage micrometer before taking critical measurements such as in mycology.

This is true for beginners and experts alike, in mycology as in any other quantitative microscopy.

The Axio sports relatively modern infinity corrected objectives and Zeiss produces quality reliable optics.
Would help to see photos of the set of objectives that you compare. Are they all infinity corrected and compatible with the Axio system ?

[Hobbyst46]

Hobbyst, I confirm your statement, i checked without oil. The Carl Zeiss x100 code 4860960 (1.30 na) objective is actually x850 (kinda a false marketing lol!). I bought this Zeiss second hand but after I cleaned it it has a fairly good and acceptable resolution and clarity

I do not have infinity corrected optics, so I just double checked with the 160mm TL 100X1.3 objective. It yielded the same FOV (maybe to within a deviation of <3%) with or without immersion oil (aside from the blur, of course, without oil). Of course the objective is designed for immersion oil, otherwise the view is not sharp, yet the magnification is very nearly the same - and I accept that for Mycology higher accuracy is required.

[75RR]

in the x63 obj. 95 reticle units measured 150um, hence 1 unit - 150/95 = 1.58um/div
actual magnification: 950um (reticle units) / 15um (actual) = x 63.3

in the x100 obj., 95 reticle units measured 110um, hence 1 unit - 110/95 = 1.17um/div magn 1000/1.17 = x854 (not x 1000)
actual magnification: 950um (reticle units) / 11um (actual) = x 86.6 (not x 1000 as expected)

It looks a bit screwed up but the magnification in oil immersion is not actually x1000 (with x10 eyepiece) but considerably different!

Magnification marked on objectives is in many cases nominal - marked so for convenience - this is from the Zeiss Optical Systems Catalogue
.

[apochronaut]

Agreed , but in his case, the difference seems extreme at 86X. In actually testing about 50 older higher magnification objectives from 9 manufacturers, the biggest variance I found was in the range of 5X.