Stupid new comer question

[MicroPunter]

Hi everyone, I’m very new to microscopy. Just got my first microscope, a Trinocular Swift 380. I am keen to do some photography and maybe videography. I ordered a camera and am awaiting it eagerly.

But there’s some thing I don’t understand. I’ve been watching lots of YouTube videos and one of my favourite channels is the Microcosmos channel. I don’t expect my photos or videos will be as good as their stuff, they have lots of experience. Watching their videos they often have the magnification in the upper left of the screen. They often use 400x, 630x, 1000x and so on.

Now, I understand how the magnification works, the magnification of the objective lens multiplied by the eyepiece lens: 40x objective and a 10x eyepiece gives 400x. But how can they get those magnification from the camera port, which doesn’t have an eyepiece lens, or at least my microscope doesn’t? Are they using digital zooming using computer software? Or do they have a lease system on the camera port? This is confusing me.

I know, I am such a ‘moon’ as my teenage son would say but we all have to start from somewhere.

Thanks for any help on this.

Kevin

[macnmotion]

Honestly, once you leave the eyepiece and start imaging for playback on various devices, magnification is essentially meaningless. You would do better getting a micrometer slide, taking photos of it with your different objectives, and calculating your field of view. Adding scale bars based on your fov to your videos is much more helpful to people who see your work.

[macnmotion]

Also, some scope setups do include optics for magnification in the photographic optical path

[mete]

It is much easier to think about this with the concept of field of view. With a micrometer slide on the stage, you can check how much of that you see on the camera (this may mean on the sensor, on the display, on the paper depending on your aim). That is basically your magnification.

I think your microscope has finite optics, that means I think it has to have an optic before the camera. I have an infinite optics microscope so it is not a must to have this but usually one is used because the field of view of microscope (lets say 23mm) is quite different than cameras. I decided to use a camera with MFT size sensor so it fits pretty good to this so I dont use any optics. My point is there can be many optics until camera, so the best thing to do is to measure it with a micrometer slide. I made a small ruler for my display connected to my camera, to somehow quickly measure something I see on the screen.

Naturally some of this magnification might not be meaningful in terms of resolution, that is the absolute limit and also probably it is further limited by the pixels in the sensor.

[MicroPunter]

Thank you for the information, I had never heard of a micrometer slide and will look at getting one. As my camera hasn’t arrived yet, the question was really just out of interest and academic. I guess it really all boils down to are you satisfied with your photographic results. But I do like the idea of a micrometer slide.

Thanks again for the replies.

[ADi]

Hi Kevin,

I guess it really all boils down to are you satisfied with your photographic results.

Yes that is the point

The term magnification is used in microscopy.
In photography, it is the scale of reproduction, the proportion of the size of the photographic object to the size of the sensor.
The maximum scale of reproduction I can achieve with a microscope lens is 100x.
The decisive factor is not the magnification but the resolution, the numerical aperture.

Some microscopists confuse the microscopic magnification with the scale of reproduction,
and write that they took a photo at 500x. But this is only possible with electron microscopes.
So, they have observed the object at 500x, but not photographed it at 500x.
Mostly the factor of the relay lens is forgotten.

Best, ADi

[MicroPunter]

Some microscopists confuse the microscopic magnification with the scale of reproduction, and write that they took a photo at 500x. But this is only possible with electron microscopes.
So, they have observed the object at 500x, but not photographed it at 500x.
Mostly the factor of the relay lens is forgotten.

Best, ADi

Thanks ADi,

Yes, that’s what I came to see after some more research and a little thinking about it. But you put it far better than I could have.

Regards,
Kevin

[Scarodactyl]

It's actually really simple: if you give the optical magnification (objective + any resizing if present) people in the comments think you have a weak microscope, and FoV is hard for randos online to interpret. So even if you're particular about optical accuracy you tack that extra 0 on the end for clarity of communication, because even if it's less technically accurate it gives people the right idea.

[SuiGenerisBrewing]

I teach microscopy to undergrad students, and your confusion as to how magnification works with digital cameras is completely normal. Through the eye pieces it makes sense - something at 100X magnification will look 100X larger than in normal life. But with digital images, you can zoom in and out, look at it on a different sized monitor, etc, meaning the "magnification" changes. So for digital images, we instead work with calibrations which can then be used to assess size. The process is fairly straight forward:

What you need:

1. A microscope with digital camera
2. A stage micrometer (essentially a microscopic ruler you can view through the microscope). If you're willing to wait, you can get one for a few bucks off of ali express, otherwise look to Amazon or similar sources
3. ImageJ - free image processing software from the NIH

How to calibrate:

1. Place the micrometer on your microscope stage, and using your lowest magnification lens, focus on the micrometer
2. Centre the micrometer in the image, and rotate the slide or camera to get the micrometer as close to horizontal as you can (as viewed through the camera)
3. Take a picture of the micrometer
4. Open the micrometer image in ImageJ
5. In ImageJ, Select "Analyze --> Set Scale", then set "Distance in Pixels" to 1, "known distance" to 1, and "pixel aspect ratio" to 1. You can also change the "unit of length to "pixels", although this is more for you to keep track of your settings than for the computer. What you've done at this point is "tell" ImageJ to measure all distances in pixels.
6. Using the line tool, draw a horizontal line between two of the micrometer markings on the image (hold down the shift key on your keyboard to force the line to remain horizontal). For the most accurate results it is best to measure from one side of the marking on the micrometer to the same side of the next marking (e.g. left-side to left-side), as trying to find the middle can be difficult. Measure across multiple lines, ideally going across ~1/3rd of the image. Once the measurement line is drawn, click "Analyze --> Measure". The "Length" that appears in the pop-up window will be the length of the line you drew, in pixels. At this time, make a note of how many markings on the stage micrometer your measurement crossed.
7. Repeat the previous step at least 2 more times, drawing each line between different markings on the stage micrometer.
8. For each measurement, divide the length of the measurement line (in pixels) by the number of micrometer markings it crossed. Average these values together - this will give you the number of pixels per division on the stage micrometer.
9. There will be a calibration value printed on the stage micrometer; this is the distance between neighbouring lines on the micrometer. It is usually 0.01 mm (10 microns), but some micrometers break with this convention, so it is always good to double check. Divide the number you calculated above by this calibration value - this will give you the scaling of images captured by that lens, with that camera, in pixels/um.
10. Repeat this process for your remaining lenses except the 100X; generally speaking, the magnification of the 100X is too much to accurately calibrate with a micrometer. For higher-mag lenses you can simple calculate the calibration from a lower-mag lens. E.G. there will be ten times as many pixels/um for a 100X lens compared to a 10X lens

**Important note: these calibration values are only valid for that specific lens, on your scope, with your camera. Change any of those and you'll need to repeat the calibration. If precision is important you may even want to do this before taking critical measurements - factors like thermal expansion can slightly alter the length of the light path, and in doing so, slightly change the area covered by a single pixel.

That's probably clear as mud, so here's the slide I use in my teaching to demonstrate this to students...hopefully it makes sense.

Micrometer.jpg (85.5 KiB) Viewed 3062 times

How to measure:
In ImageJ:

1. Open the image in which you want to measure the length of an object.
2. click "Analyze --> Measure", and enter your calibration values into the window. E.G. if you measured 10.5 pixels/micron, enter "10.5" into "Distance in pixels" and "1" into known distance. Keep the aspect ratio at 1.0, and enter "microns" in the the "Units of Length" window.
3. Using the line tool, draw a line across the object you want to measure, then click "Analyze --> Measure". The "Length" that appears in the pop-up window will be the length of your subject.

To add a scale bar to an image:
In ImageJ:

1. Set the scale for your image as per step 1&2 in "How to measure"
2. Under "Analyze" select "Tools --> Scale bar"
3. Configure the scale bar as you see fit; you can change its size, location, thickness, colour, etc, to make it informative and easy to see.
4. I would suggest turning off the "overlay" button, as this creates a new layer to draw the scale bar on, but many other programs will ignore that layer (e.g. your scale bar won't be visible outside of imagej)
5. Once you're happy with your scale bar, click "OK"

[MicroPunter]

The process is fairly straight forward:

Great answer, thank you.

[MitchW]

I had never heard of a micrometer slide and will look at getting one.

A micrometer slide really gives you a lot of information about your microscope and objectives and informs you more directly regarding size of objects in your specimen. I bought two different ones. One with just the 1mm broken into 100 divisions, and one that looks like the attached photo. Next I'm thinking of getting a measurement reticle for the eyepiece. If anyone has any information about their experience using one it would be greatly appreciated.

[Sure Squintsalot]

Honestly, once you leave the eyepiece and start imaging for playback on various devices, magnification is essentially meaningless. You would do better getting a micrometer slide, taking photos of it with your different objectives, and calculating your field of view. Adding scale bars based on your fov to your videos is much more helpful to people who see your work.

This.

[Kinetochore]

I teach microscopy to undergrad students, and your confusion as to how magnification works with digital cameras is completely normal.

What? Your answer is awesome! Thank you!

Does this mean I don't need to calibrate the ratio between the eyepiece reticle and the stage micrometer every time when I'm using the digital camera? ( As long as I'm using the same camera and microscope kit during the same session to minimise temperature differences where they're an issue ).

Endorphins are still kicking in over this. You rock

[SuiGenerisBrewing]

You shouldn't have to recalibrate every time, so long as your camera mounts in more-or-less the same place each time.

[Kinetochore]

You shouldn't have to recalibrate every time, so long as your camera mounts in more-or-less the same place each time.

That's a huge relief. Queue more endorphins, maestro