magnifications on photomicrography

soniclink · Post by **soniclink** » Sun Oct 30, 2011 6:30 pm

hi, i would like to know if there's a way to estimate the magnification or the objective in which the photography was taken just by using the image information from camera (aperture value, zoom, shutter speed, focal length)

i think there could be a way to know the magnification through these info due to similarities on the info in photos from the same magnification

ChrisR · Post by **ChrisR** » Sun Oct 30, 2011 8:17 pm

Not from those alone. You 'd need distance(s) as well, and when very close that wouldn't be very accurate.

ChrisR · Post by **ChrisR** » Mon Oct 31, 2011 4:32 am

Edit - I hadn't taken note that this is a microscopy section, where I normally keep quiet and try to learn from others' answers! Did you mean to ask in a general Macro context? That was what I addressed.
I'm sure the answer's still a "no", with the rider that it's relatively easy to put a scale in place of the subject and take an extra photo, just of that. Divisions go down to 10 microns, maybe finer.
"Magnification" is a slippery customer - it depends how big you make the picture. "Image width", or a scale included in the image, tell everyone more.

If you're using a microscope objective on a microscope, it's usual to quote the number on that, and any other lenses in the optical train. If you chop the top off a scope and put a camera there, the size of the sensor may or may not become a factor.

Mitch640 · Post by **Mitch640** » Mon Oct 31, 2011 4:56 am

Since there is no lens on the camera in most photomicrography, aperture value, zoom and focal length will be blank in the EXIF anyway.

Charles Krebs · Post by **Charles Krebs** » Mon Oct 31, 2011 11:21 am

Generally like Mitch said (at least with a lens-less DSLR):

Since there is no lens on the camera in most photomicrography, aperture value, zoom and focal length will be blank in the EXIF anyway.

But if you use the "afocal" method, where basically a camera with an attached lens is held up to an eyepiece, you will get these values in the EXIF data. But they can't help you determine magnification/objective values unless you have more info about the microscope hardware you are looking through, or the real-life subject size.

soniclink · Post by **soniclink** » Wed Nov 02, 2011 9:06 pm

sorry if i didn't explain clearly before, the fact is that i'm using an inverted microscope with 2 principals objectives 10x and 20x with 10x and 15x eyepieces and also it has additional magnifications on trinocular head

i do know which objectives and magnification i had used on each photograph, i'm just trying to make it easier to identify some magnifications from the photos i've taken through the microscope, and Charles you are right the EXIF info i use to categorize images comes from a camera that i attach to the eyepiece manually

now, by looking at this info i can organize some photos but it seems some info looks similar: photos from 10x obj with additional magnification of 1,1x has similarities with EXIF info from photos from 20x objectives 1,1x whereas the same happens on greater magnification (i.e: lower focal length on each greater mag from any of the two objectives)

i had the chance to measure a micrometer through an eyepiece with yet another micrometer. Problem is that i don't have the micrometer "objective" or "ruler" anymore, only the 15x eyepiece with the micrometer, and i only took photos through the eyepiece WITHOUT the intern micrometer, only the one from the stage (the "ruler") those photos are attached to this reply

the problem now is that, all photos that i took have the same configuration (to ease work) but happens to make measuring more difficult

first line: 10x objective ,10x eyepiece
second line: 20x objective,10x eyepiece

rjlittlefield · Post by **rjlittlefield** » Wed Nov 02, 2011 9:39 pm

When images are shot using the afocal method, then the final magnification is pretty close to:

objectiveMagnification * eyepieceMagnification * cameraLensFocalLength * constantScalingFactor

You have to determine the constantScalingFactor from test images. The images you've shown here should be enough to do that.

Be aware that the EXIF values are not completely accurate or repeatable. If your workflow involves changing the zoom (cameraLensFocalLength), there'll be some uncertainty in the numbers you get.

--Rik

Starshade · Post by **Starshade** » Thu Nov 03, 2011 11:33 pm

Can I say something like "optical magnification is 50x" when I have a 5x objective lens + 10x lens in the camera connector? I also have lens on a camera but never counted it since I though that magnification is a term for people who are not good at optics (like me).

Or the word "optical" does not make it different?

Charles Krebs · Post by **Charles Krebs** » Fri Nov 04, 2011 12:18 am

Starshade

Can I say something like "optical magnification is 50x" when I have a 5x objective lens + 10x lens in the camera connector? I also have lens on a camera but never counted it since I though that magnification is a term for people who are not good at optics (like me).

No not really. If you are looking through 10X eyepieces and using a 5X objective it is fair to say that you are viewing the subject at a magnification of 50X. But the magnification recorded on the camera sensor/film will be significantly less (if, as I interpreted from the info you gave, this is using "afocal" method, lens on camera, and a 10X eyepiece in the trinocular tube). The actual "recorded" magnification will depend on the focal length of the camera lens.

rjlittlefield · Post by **rjlittlefield** » Fri Nov 04, 2011 8:36 am

You could, however, say "This is what you'd see through a 50X microscope." That's the way I usually explain my photos informally.

--Rik

Starshade · Post by **Starshade** » Fri Nov 04, 2011 8:59 am

Thank you guys for replies, sometimes there's a need to indicate something like a magnification and at the same time not to make a technical mistake...

Charles Krebs · Post by **Charles Krebs** » Fri Nov 04, 2011 12:16 pm

Scale bars are always informative, but depending on the circumstances and image display, can sometimes look intrusive when placed in a picture.

If there is any descriptive info supplied with a picture it can be quite effective to provide the field size covered in the image.... something like:

"Photograph covers an area of:"
446 x 297 microns (or)
0.45 x 0.30 mm (or)
0.018 x 0.012 inches (notice how when you use inches it really seems smaller

. But here in the "States" there are plenty of people that have no notion of "microns" or even "millimeters".)

soniclink · Post by **soniclink** » Mon Nov 07, 2011 10:19 am

rjlittlefield wrote:When images are shot using the afocal method, then the final magnification is pretty close to:

objectiveMagnification * eyepieceMagnification * cameraLensFocalLength * constantScalingFactor

You have to determine the constantScalingFactor from test images. The images you've shown here should be enough to do that.

Be aware that the EXIF values are not completely accurate or repeatable. If your workflow involves changing the zoom (cameraLensFocalLength), there'll be some uncertainty in the numbers you get.

--Rik

How do i know the constantScalingFactor? and also as you can see in the last image that i previously posted (10X eyepiece, 20X objective, 2,5X) there's almost 0.1 mm in the center of the field of view. If i overlay this division "scales" with a photo from the same magnification/objective/zoom/camerafocallength, can i say that said scale can be used to get a accurate measure of any organism in the field of view?

the next image shows measurements on excel from the observations with the micrometer from the same time i had taken the pictures previously posted

sorry if measurements are in mm, i had no time to convert them to inches, but the microns scale is in the microphotography language don't you think?

all measurements made with the 15X eyepiece (micrometer eyepiece)

rjlittlefield · Post by **rjlittlefield** » Mon Nov 07, 2011 5:50 pm

Well, I have just re-read all your posts four more times, and I'm gradually realizing how confused this situation is -- or at least how confused I am. I don't think there's any useful information in the EXIF of your images. The six images posted above all say the same thing: Canon PowerShot G7 at 7.4mm focal length. I was definitely confused about how your images were shot. I thought you were adjusting the camera's zoom; now I think you were adjusting zoom at the microscope head. But I'm not sure.

If you're as confused as I am, then the best path forward is to purchase a "stage micrometer", photograph that using the same optical setups as for your real subjects, and figure out for yourself what the correspondences are.

A stage micrometer is just a tiny ruler printed on a microscope slide, typically 1 mm divided into 100 ticks of 10 microns each. They sell regularly on eBay as new parts for $25-30, like HERE and HERE.

When you have an eyepiece reticle, the standard procedure is to use a stage micrometer to calibrate the reticle given the other optics, then use the reticle to measure subjects. In that case camera settings don't matter, as long as you can see the subject and the reticle at the same time.

The other standard procedure is to photograph the stage micrometer and subjects separately but using the same optics and camera settings, then use the image of the stage micrometer to measure the corresponding images of the subjects. If that's what you mean when you write

If i overlay this division "scales" with a photo from the same magnification/objective/zoom/camerafocallength, can i say that said scale can be used to get a accurate measure of any organism in the field of view?

then the answer to your question is "Yes".

--Rik