How is magnification in a microscope defined?

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Stuart4444
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How is magnification in a microscope defined?

#1 Post by Stuart4444 » Thu Jun 17, 2021 11:08 pm

Hi,

I'm new to this forum, and am trying to solve a puzzle. I hope a microscopist might help!

I'm a macro photographer, and might get into microscopes in the future, but presently own Olympus and Canon cameras to take photos up to 5x magnification. My knowledge of microscopes is small.

In photography, we define magnification as the ratio of the size of the object being photographed to the size of its image on the camera sensor.

When looking at the site https://www.nikonsmallworld.com/, I found an image of a varroah mite on a honey bee (see https://www.nikonsmallworld.com/galleri ... a-honeybee). I was interested as the mite in life is about 1mm long and it occupies about 40% of the width of this image, so if this were a full frame sensor (36mm long) the image of the mite would have been about 40% of 36 = 14mm, so the magnification would be 14x by the photographer's definition. If it were a smaller sensor, eg micro 4/3 whose sensor is 18mm long, the magnification would have been 7x.

However, the site says it is 1x magnification. Which make me wonder, how did they work that out? Is the microscopy definition of magnification different to the photography one? I guess it must be! Any explanation on how to relate microscope magnification to camera magnification would be very helpful.

Thanks,

Stuart

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Re: How is magnification in a microscope defined?

#2 Post by MichaelG. » Fri Jun 18, 2021 8:06 am

Welcome Stuart

I will start the ball rolling, but you should get a multitude of answers.

Your photographer’s interpretation of magnification is perfectly logical; as far as it goes … But, of course, you could also state magnification in terms of a poster-sized print, or a projected image.

Without much more information, the mention of a 1x Objective being used for that Nikon image is trivial in regards to magnification. The safest approach with photomicrography is either to include a scale-bar in the image, or to declare the size of some ‘Region of Interest’ [which could just be the total width represented by the image, or the length of that mite].

For visual microscopy, the notional magnification is generally expressed by comparison with ‘naked eye at 250mm’
and in a compound microscope is the product of Objective and Eyepiece magnifications.

for example … 40x multiplied by 10x equals 400x

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Re: How is magnification in a microscope defined?

#3 Post by Phill Brown » Fri Jun 18, 2021 8:51 am

From everything I have seen so far I would conclude 1x is a misprint.
10x plan 160 is a useful lens,easy enough to make up 160mm of tube,
RMS to 42mm is cheap adapter.
For image stacking an adjustable stage would be ideal.
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Re: How is magnification in a microscope defined?

#4 Post by Phill Brown » Fri Jun 18, 2021 8:52 am

Should add, only one axis adjustable is needed but the extra help with centering the image.

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Re: How is magnification in a microscope defined?

#5 Post by patta » Fri Jun 18, 2021 9:09 am

If there are no misprints, my guess is that the photo has been taken with a zoom stereomicroscope.
True reproduction ratio (image on sensor /object size) can then be a bit whatever, because of magnifications from the zoom and from the camera adapter lenses. For a full-format sensor, yes it should be like 14:1 or 14x.

The "1x objective" for a stereo microscope is a special lens screwed on its nose, like a close-up filter screw macro lens. This "objective" is not truly an objective and is not necessarily working at 1:1 reproduction ratio, but everybody calls it the 1x objective. You can swap it with other "objectives" like 0,5x and 2x.
(For CMO stereomicroscope, this 1x may actually be a true objective made for 1:1; but again, there are the zoom and the camera adapter that change the reproduction ratio away from 1:1)

The photo could also come out from a "true" 1x, 1:1 Mitutoyo microscope objective, heavily enlarged by a tube or relay lens and/or cropped. Again, not 1:1 reproduction ratio.

I fear that there are 3 or more definitions of "magnification" in microscopy; different and incompatible. Continuing from post #2, will take several pages! On NikonSmallWorld they usually specify only the "Objective magnification", which is more like a ballpark of what type of lens has been used, rather than the actual Sensor/Object reproduction ratio.

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Re: How is magnification in a microscope defined?

#6 Post by Phill Brown » Fri Jun 18, 2021 11:10 am

Pixel density and MOA gives the resolution?
Higher optical magnification gives lower depth of field.
Reflected light in the blue wavelength scatters more than red.
Better quality coated for reflected light helps.
LED tend to be more blue.
Only observations and serving suggestions.
Good luck, can't beat having a go with a budget in mind.

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Re: How is magnification in a microscope defined?

#7 Post by lagoonatic » Fri Jun 18, 2021 2:46 pm

Hi Stuart -
The magnification ratios in photography always bugged me. Even back in the days of film, a "life-size" (1:1) ratio meant the image was the same size on the film as in real life. So when I shot an image of that mite on 35mm film, it would be, as you pointed out, 1mm across on the negative. Nobody is looking at a contact print of that negative (except the photographer, maybe). It will be enlarged. So why do we call it 1x? The same with images from a microscope. And when those images are viewed on an electronic device, could be a smart phone or a projection screen or a laptop or tablet etc, without a reference, who knows what the magnification ratio is.
All that said, sometimes it is nice to know what combination of optical equipment was used to obtain the image - be it that amazing Canon MP-E 65mm f/2.8 or a Nikon CFI Plan Fluor 60XC or a homemade Leeuwenhoek scope.
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Re: How is magnification in a microscope defined?

#8 Post by patta » Fri Jun 18, 2021 4:00 pm

A short answer:

Yes, the number on the objective is true magnification as in macro.
A 10x objective takes a 1mm bug and produces an image of 10mm.
We call this "primary magnification"

But, usually we don't put the sensor directly there: for convenience we use some more lenses, like a relay, a projection eyepiece, camera adapter or zoom that project to the sensor.
Those extra lenses add their own magnification. We call it "secondary magnification".
If I have a secondary magnification 2x, and primary magnification 10x, the total true magnification ( bug size on sensor/ life size) is 10*2=20x

But usually we don't bother much about the secondary nor about the true magnification.
Why?
The primary magnification is the one that matters most, because it is the "heavy lifting" from the micro to some more manageable size.
The secondary magnification is more a matter of convenience, depends on what sensor size you have. Microscope cameras have sensors of all sizes from puny 1/4" to 35mm and more; most often sold together with their lens adapter.
You can think that the microscope objective is projecting the image on a wall; then you take a photo of it with a full format camera, or with a Micro4/3. The photos will look more or less the same, even if the sensors have different size; you're using different "secondary magnification"; and it doesn't affect much the image quality.
Also, almost all normal microscope objectives are designed to project the "primary image" over a size of about 20mm (illuminated disk); it is like an unwritten standard. You then magnify or reduce those 20mm to fit your sensor size with the "secondary magnification".

So normally we care only about the "primary magnification" of the objective, 10x, and this is the one that gets written on the photo description. The secondary magnification, it is important only in the system setup phase, to make sure we are not vignetting or over-cropping. The total, true magnification, may be useful for measurements, estimate of exposure, etc. but it is not so fundamental as with macro, where you have only one lens and play with it to change the magnification on the sensor.

The image you've linked is weird because it is clear that they've used a very large secondary magnification - or a very small sensor. Or, a zoom stereomicroscope, where primary and secondary magnifications are a bit mixed up.

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Re: How is magnification in a microscope defined?

#9 Post by MichaelG. » Fri Jun 18, 2021 5:11 pm

If it’s any help … Antoine Franck is pictured here, at his microscope:
http://www.agriculture-biodiversite-oi. ... tomologist

He may, of course, have others !

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Re: How is magnification in a microscope defined?

#10 Post by patta » Fri Jun 18, 2021 5:47 pm

MichaelG. wrote:
Fri Jun 18, 2021 5:11 pm
If it’s any help … Antoine Franck is pictured here, at his microscope:
http://www.agriculture-biodiversite-oi. ... tomologist

He may, of course, have others !

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the microscope in the linked page may be actually the one of the NinkonSmallWorld stack photo, with the mystery 1x objective on a body with zoom and other gizmos.

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Re: How is magnification in a microscope defined?

#11 Post by 75RR » Fri Jun 18, 2021 8:24 pm

.

Agree that the magnification of the Objective and the Illumination Technique used is the most useful information.

The variations in the resolution of the monitors that the image is seen on effectively makes the stated magnification nonsensical, or at the very least unreliable.

Scale bars are one way to get around this problem - as in never mind the magnification, the subject is 'x' size.

Only problem with that is that the scale bars (however accurate they might individually be) are inevitably smaller than the subject,

so that one ends up measuring the size of the scale bar with one's thumb and then pacing off the length of the subject on the screen!

Much better in my opinion to measure the length or width of the subject and then simply state it.
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Re: How is magnification in a microscope defined?

#12 Post by MichaelG. » Sat Jun 19, 2021 6:11 am

75RR wrote:
Fri Jun 18, 2021 8:24 pm
Scale bars are one way to get around this problem - as in never mind the magnification, the subject is x size.

Only problem with that is that the scale bars (however accurate they might individually be) are inevitably smaller than the subject,
.

Not quite ‘inevitably‘

Adapting slightly what I wrote yesterday :
… include a scale-bar or declare the size of some ‘Region of Interest’ [either of which could be any length up to the ‘diagonal’ of the image].

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Re: How is magnification in a microscope defined?

#13 Post by Stuart4444 » Mon Jun 21, 2021 11:44 am

Wow, I'm impressed by the help being offered here! And I'm not even (yet) a microscopist. Thanks to you all.

The photography definition of magnification works fine for me, partly because it's what I've used for over 50 years since doing my first 1:1 macro, and partly because magnification of the image and then cropping it is (in my opinion) cheating! It leads to poor quality anyway, and photographically the primary aim is clear, sharp, artistic pictures, else photographic judges will find fault. But I can see now how that definition doesn't translate into microscopy. Having one lens in the light path to the user's eye and a different one in the path to camera sensor to suit different sensor sizes complicates it, too! I agree, a statement of the subject size would make microphotographs more understandable to all.

I like the stage as a means to move the subject when focus stacking. I use a "macro rail" on a carbon fibre tripod with a "3-way" head to aim the camera, but with a big lens like the Canon MP-E dangling on it, it's quite wobbly. I use radio shutter releases and an electronic shutter on mirrorless cameras to help here. It's not a problem at 1x, but obviously will get more so at higher magnification. Some computer controlled stacking kit looks very rigid, but it's not something I own at the moment.

My wife is a document examiner and uses a £40 USB microscope to examin signatures etc. I bought her a Bresser LCD microscope some years ago thinking it would be better for her, but she found the USB one more convenient to use and never used it. I tried it for my photographs but found the 3MPx camera and possible inadequate optics gave photos unacceptable for photographic competitions.

I'm thinking of trying a microscope objective and a prime lens on my camera as I've seen that advocated, to get beyond the 5x limit of my MP-E. Or perhaps a recommended stereo microscope. I have a graticule of 1cm x 0.1mm divisions, so I can figure out the magnification if I need to!

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Re: How is magnification in a microscope defined?

#14 Post by 75RR » Mon Jun 21, 2021 12:47 pm

Stuart4444 wrote:
Mon Jun 21, 2021 11:44 am
My wife is a document examiner and uses a £40 USB microscope to examin signatures etc. I bought her a Bresser LCD microscope some years ago thinking it would be better for her, but she found the USB one more convenient to use and never used it. I tried it for my photographs but found the 3MPx camera and possible inadequate optics gave photos unacceptable for photographic competitions.
Do note that in microscopy, you are photographing what you are seeing through the microscope, and what you are seeing through the microscope depends greatly on your objectives, the Illumination Technique you are using and your microscope skills.
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Re: How is magnification in a microscope defined?

#15 Post by 75RR » Mon Jun 21, 2021 1:49 pm

Stuart4444 wrote:
Mon Jun 21, 2021 11:44 am
My wife is a document examiner and uses a £40 USB microscope to examin signatures etc. I bought her a Bresser LCD microscope some years ago thinking it would be better for her, but she found the USB one more convenient to use and never used it. I tried it for my photographs but found the 3MPx camera and possible inadequate optics gave photos unacceptable for photographic competitions.
Do note that in microscopy, you photograph what you are seeing through the microscope, and what you see through the microscope depends greatly on your objectives, the Illumination Technique you are using and your microscope skills.
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Re: How is magnification in a microscope defined?

#16 Post by 75RR » Mon Jun 21, 2021 1:50 pm

.
You should find this link very useful: https://krebsmicro.com/
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Re: How is magnification in a microscope defined?

#17 Post by apochronaut » Mon Jun 21, 2021 2:05 pm

The missing link in interpreting microscope magnification in print or on a screen is knowing the size of the real field of view the image has captured as well as the objective used. Knowing that is similar to a scale bar comparison only a little easier to accomplish and relay to the viewer.
Every microscopist should know what the real field of view is for their various objectives. A simple 4 objective microscope with 10, 20, 40 and 100X objectives and 20mm field of view eyepiece will provide 2000 um, 1000 um, 500um and 200um f's.o.v. but for publishing photos of what you are viewing you need to know how your photo system copies that f.o.v.
When establishing your photo system your photo relay lens becomes the eyepiece and projects x amount of the real field to the sensor. You need to know how much of that is being captured on the sensor, because if it is not exactly the same, it is a crop: either + or - and the magnification will be either greater or less than what it is being viewed through the eyepieces. Knowing the percentage of the real field that is getting captured on the sensor irregardless of sensor size, will allow you to state the size of the real field in any copied or electronically relayed image.
As Michael G pointed out, a standard viewing distance (250mm) needs to be applied to get an impression of the magnification but knowing the diameter of the real field plus the crop factor is necessary in order to know how much over or possibly under magnification has been applied during the reproduction of the image but doesn't help in providing the actual magnification, just a guess at it.

In the case of the image of the varroah mite, using a 1X objective with 10 X 20mm eyepieces in a microscope would project a real field of 20,000 um or 20mm. A 1mm varroah mite would take up 1/20 or 5% linear of a 20mm f.o.v. Since in the image it apparently takes up 40% of the field, one would assume a crop factor or a photo system of higher amplification and small field capture. In the absence of knowing the field size in comparison to the objective used, there are few clues as to actual magnification.

For those interested , a 20mm f.o.v. is the same as 20mm across at your cornea, so an image on a screen at 250mm distance that is 20mm across at your cornea represents pretty closely that image in a microscope with a 20mm eyepiece but without knowing what real field it represents, you can't make an estimate of magnification. Something like stating :
40X objective, image covering a 500 um field would be very helpful.

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Re: How is magnification in a microscope defined?

#18 Post by Stuart4444 » Mon Jun 21, 2021 2:54 pm

Yes, I'm realising that lighting is very important. When I bought a ring flash I found a big jump in quality as I could stop down more (better depth of field without stacking) and short exposure. However it's only a step down the road I realise. Even at 1x, I can be disappointed at my result and since the equipment in use is good quality, it's the operator at fault. I have a lot more to learn.

Thanks for your link 75RR, it looks really relevant to me, lots there to read also.

Field of view is something that photographers know about, but only in a round-about way really. We think more in terms of focal length for framing and for perspective control. We think of FOV in terms of the angle that the lens sees. I can see that FOV as a linear dimension such as width is a good measure here I think, a perfect statistic really to help the viewer understand the picture. It would be easy to make a FOV vs magnification graph or table for photographers -

Sensor......Dimension........Magnification.......FOV (long dimension)
Full frame.....36x24mm.......1x................. 36mm
....................................2x..................18mm
...................................10x..................3.6mm
Micro 4/3.....18x12mm...... 1x..................18mm
...................................2x....................9mm
..................................10x...................1.8mm

And so on. APS-C is in between, closer to micro 4/3
Last edited by Stuart4444 on Mon Jun 21, 2021 2:58 pm, edited 1 time in total.

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Re: How is magnification in a microscope defined?

#19 Post by MichaelG. » Mon Jun 21, 2021 3:14 pm

Stuart4444 wrote:
Mon Jun 21, 2021 2:54 pm
[…] I found a big jump in quality as I could stop down more (better depth of field without stacking) and short exposure. However it's only a step down the road I realise. Even at 1x, I can be disappointed at my result and since the equipment in use is good quality, it's the operator at fault. I have a lot more to learn.
You will quickly come to notice that stopping-down is ‘a two-edged sword’
… with increased depth-of-field comes reduced resolution !!

This is very significant in microscopy

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Re: How is magnification in a microscope defined?

#20 Post by Stuart4444 » Mon Jun 21, 2021 7:37 pm

Thanks, Michael. Yes, indeed, we usually consider the "sweet spot" of lenses about 2-4 stops down from wide open as the two opposite effects (edge of lens quality and diffraction) add up to total image degradation which is at a minimum in this region. Having said that, I've seen macros taken at wide open f2.8 and at minimum aperture as low as f32 (nominal) which were quite sharp.

I thought most microscopes or their lenses don't have diaphragms?

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Re: How is magnification in a microscope defined?

#21 Post by MichaelG. » Mon Jun 21, 2021 7:43 pm

Stuart4444 wrote:
Mon Jun 21, 2021 7:37 pm
I thought most microscopes or their lenses don't have diaphragms?
For the very reason I mentioned
… generally speaking ‘resolution is king’ so the objectives are designed to be used wide open.

[ there are, of course, “exceptions which prove the rule” ]

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.

P.S. __ These are worth a look:
https://www.leica-microsystems.com/scie ... lculation/
https://www.microscopyu.com/microscopy- ... resolution
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Re: How is magnification in a microscope defined?

#22 Post by apochronaut » Mon Jun 21, 2021 9:48 pm

Iris diaphragms are fitted into specific models of objective lenses for specific purposes where the benefit outweighs the small reduction in resolution that occurs. In all cases except perhaps in the case of a dedicated photo microscope lens the purpose has to do with an improvement in contrast or special types of contrast. In the case of objectives manufactured as microscope photo lenses made in the past, contrast does play a role but also does depth of field.
Diaphragms are installed that can usually be adjusted to reduce the N.A. to about 1/2 . Objectives are almost never used that way. The reduction in resolution is severe. Nor are microscope objectives traditionally used wide open due to the reduction in contrast that occurs due to diffraction and scatter. The aperture is controlled by means of one or two iris diaphragms that restrict the N.A. of the illumination beam, therefore lowering the functional N.A. of the objective. Thus, when an integrated objective iris is closed slightly in order to increase contrast, it doesn't have as much effect on resolution as one might think, until it is closed too far.

Ray tracing, coatings and improved optical glasses have allowed many objectives to be made that can be used at fuller apertures for the past 15 or maybe 20 years. At wide apertures though, there is always a duel berween resolution and contrast. Although technically contrast has nothing to do with resolution, higher contrast imaging has the effect of highlighting detail and therefore assists resolution.

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Re: How is magnification in a microscope defined?

#23 Post by patta » Tue Jun 22, 2021 5:58 am

to add some - all known things, but clears my mind at least

Angular FOV and perspective control basically disappear in high magnification; the depth of focus is so thin, that there is no much perspective to play with. Perspective can instead be a nuisance for focus stacking. So a "telecentric" lens is more welcome here. High magnification objectives are usually almost telecentric. ("zero FOV angle")
Unless somebody start fiddling with micro-fisheyes ;) ...

Also, microscope objectives are designed very differently than photographic ones:
A 10x microscope objective is, in practice, a 20mm f/2 lens. But won't absolutely work as a 20mm wide angle! It is optimized only for 10:1 reproduction ratio; maybe it will be ok also at 9:1 or 11:1, but not much farther.
As from post #22, the 10x objective has no aperture diaphragm, so it usually works at f/2 (object-side) and will show horrible aberrations if refocused at distances different than what's intended for. Like if you want to use a standard 50mm f/1.8, straight, with macro spacers to get 1:1, fully open at f/1.8... it is not its job, the image ends up in a sea of aberrations and haze.
This is very different from standard macro lenses, which (money allowing) provide good performance from 1:1 to infinity, and we can close the aperture to get more depth of field and sharpness.
Microscope objectives are actually more similar to photographic lenses mounted inverted; at least the 5x and 10x. An old 55mm macro lens, that was designed to give optimum at 1:10, works almost just as well, inverted, for 10:1 magnification. The microscope 10x objectives have design quite similar to those 55mm macros, inverted ( or a much simplified version of it).

From 5:1 and beyond, the aperture (object-side) needs to be high (low f/number) otherwise diffraction comes in. That's why objectives with large magnifications have very large apertures ( 10x has NA 0.25 = f/2; the 40x has NA 0.65 = f/0.6!); as sad consequence, we need a different objectives for each magnification: a lens with f/0.6 won't collaborate unless used at the (only) distance and reproduction ratio it was designed for. If I try push the 40x to give me a 100x primary magnification, it will answer with confused blobs of light. To get a decent 100:1, I need to buy another lens, the 100x objective. With even larger aperture.

This "diffraction softening" issue can be translated also sensor-side, more understandable for the pixel peeper photographer:
I put my camera directly on the image of the 10x objective ("primary magnification" only). The 10x has NA=0,25 =f/2, object-side. I use it at magnification 10:1, so sensor-side it is working at aperture 0,25 /10 = 0,025 = f/20. This f/20 is what the camera see. It get even worse for higher magnification, with the 40x the camera will see 0.65 /40 = 0,016 = f/32.
Those apertures are puny when used on modern cameras with small pixels! That is why the sensor choice, and the addition of more lenses for "secondary magnification", are not so critical; they work at very narrow aperture and are not bothersome (apart from high ISO issues...). Many troubles come instead from the large-aperture objective and its illumination.

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Re: How is magnification in a microscope defined?

#24 Post by Stuart4444 » Wed Jun 23, 2021 4:26 pm

This thread is going a bit away from my original question, but it's really helpful by answering questions I never thought to ask!

I think I've got an understanding of magnification as used in microscopy now - thanks for that.

It seems my school level physics understanding of optics and ray diagrams is woefully inadequate. I gather that f = 1/2NA approximately, which is amazing given that f is defined as iris diameter / focal length whereas NA is defined in terms of the objective light cone angle. I'd also seen, but paid little attention to, the effective f stop decreasing below the one set on the lens. I see it is an important factor as magnification increases.

I'm struggling to understand why resolution increases as the aperture increases. Is this just referring to resolution loss due to diffraction?

Yes, I can see that perspective is more or less meaningless in microphotography. Indeed it is a nuisance in focus stacking even at my normal magnification level. Generating the set of photos by moving the camera with the focus fixed, and alternetively by moving the focus distance whilst keeping the camera still, can give quite different results. Which is used more with microscopes? Or are they these same thing here? I'm thinking they must be the same as a microscope is focussed by moving the subject rather than be altering the setup in the lens. Or is that a simple microscope?? Very puzzling for an outsider jumping in at the deep end!

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Re: How is magnification in a microscope defined?

#25 Post by MichaelG. » Thu Jun 24, 2021 9:06 pm

Stuart4444 wrote:
Wed Jun 23, 2021 4:26 pm
I'm struggling to understand why resolution increases as the aperture increases. Is this just referring to resolution loss due to diffraction?
.
If you want to understand, Stuart … I can do no better than refer you to Peter Evennett’s video, which we have been discussing here: https://www.microbehunter.com/microscop ... =5&t=13120

You will probably need to watch it more than once … but it is an excellent demonstration.
Direct link: https://youtu.be/60_jgZtyR6U

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Re: How is magnification in a microscope defined?

#26 Post by Stuart4444 » Sat Jul 03, 2021 6:51 am

Many thanks, Michael. He's a very clear speaker and demonstrator, taking it slowly, just what I need.

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Re: How is magnification in a microscope defined?

#27 Post by Stuart4444 » Fri Aug 06, 2021 8:33 am

In the last month I've bought a bellows, a 10x microscope objective, and adapters to suit, the last of which arrived just yesterday. The bellows are at full extension to give the required 160mm distance from the sensor plane to the lens mount.

It seems to work fine, and my first pictures of a small graticule that I have (0-10mm x 0.1mm) amazed me. 2.1mm fills the horizontal distance of my Canon M50ii (field of view), so as its sensor is 22.3mm wide, this is a magnification (by the photographer's definition) of 22.3/2.1=10.6:1. Which it what it says on the lens, more or less.

Seems I've come full circle. :D

And the information on the Nikon World which set me off must be an error, as Phill surmised.

viktor j nilsson
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Re: How is magnification in a microscope defined?

#28 Post by viktor j nilsson » Sat Aug 07, 2021 5:31 am

Stuart4444 wrote: The bellows are at full extension to give the required 160mm distance from the sensor plane to the lens mount.
Glad it's working for you. Minor comment: the distance should be 150mm from sensor to the objective shoulder. This is because the image is designed to be placed 10mm down the tube in a 160mm tube length microscope.

Stuart4444
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Re: How is magnification in a microscope defined?

#29 Post by Stuart4444 » Tue Aug 31, 2021 6:25 pm

Thanks, viktor. I had it set a little more than that, have adjusted it now.

mete
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Re: How is magnification in a microscope defined?

#30 Post by mete » Fri Sep 10, 2021 6:42 pm

I also have more experience with photography and some with macro/stereomicroscope, and very little with microscopy. Like it is mentioned in a few posts above, I find it much easier to think about magnification using FoV. Also I am using a stage micrometer whenever I am confused. There are too many moving parts comparing to traditional photography (objective mag, extra front objective in stereo, camera adapter, sensor size, the image size/crop you select in the camera etc). Also, if not using a bellow/view camera, vignetting is normally not an issue in photography, but it might be here, and again thinking about FoV helps for that.

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