Whilst calibrating my Motic Microsope's camera, I am able to calibrate the 10x object lens against the stage micrometer accurately. However when I calibrate (using the Motic 2.0 Plus software) the 40x object lens, it always sets the ratio incorrectly.

For example, in the calibration panel, I load a photo of the scale that I took and then I draw the circle so that it measures the pixels both in height and width and then I tell it that the circle is 10 µm. But it doesn't seem to get the ratio correct. When I did this for the 10x lens it worked perfectly.

So I took a photo of the scale with the 40x object lens and then went to calibrate that.

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Here you can see how the calibration is performed: you position a circle over the photograph and tell it the size in µm (it's rather large, sorry):

enter image description here

Here you can see the measuring line out of whack. It gives me 8 µm for a length that should only be 4 µm.

enter image description here

Am I missing something?


It looks like the shape is an ellipse rather than a circle. The ellipse has major and minor axes, which are described in terms of half-axes (177 and 175 pixels respectively). Is it possible the software is incorrectly using the half-axis measurements, whereas when you draw a perfect circle it will correctly use the diameter? This is consistent with the x and y scale numbers being different from each other.

So I would suggest re-calibrating with a perfect circle.

Hope this helps.

  • $\begingroup$ When I calibrate, should I calibrate to the edges of that black box? It's supposed to represent 4µm. If they were tick-marks I would think to measure to the insides... It's kind of confusing. Do I count the tick mark or not? Or do I measure to half of it? $\endgroup$ – bgmCoder Nov 2 '15 at 23:31
  • $\begingroup$ With my own calibration slide that has a linear scale (like a ruler), I measure from the left side of one tick mark to the left side of another one. I'm not familiar with your calibration slide, so I don't know what the outer lines represent. Do you have any documentation or are there other areas of the slide that are more clearly labeled? $\endgroup$ – TSwayne Nov 3 '15 at 13:33
  • $\begingroup$ My Mycologist says that it's supposed to be 10 microns from one tick to the next - whether left to left as you say (which is easy to see - thanks!) or from half to half of the tick. So I tried this by capturing the scale with the 10x lens and calibrated four ticks to be 40 microns. But when I measure from tick mark to tick mark I get 18.10 microns. I'm still missing something. $\endgroup$ – bgmCoder Nov 7 '15 at 2:09
  • $\begingroup$ My Stage Micrometer has five calibrators: a circle at 1.5mm, a circle at 0.6mm, a scale cross (1 DIV=0.01mm), a tiny circle at 0.15mm and a circle at 0.07mm. Should I be using a particular one of these? $\endgroup$ – bgmCoder Nov 7 '15 at 2:12
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    $\begingroup$ Okay, I think I've got this. Instead of calibrating by drawing a circle over the scale cross, I used a different calibration setting where I just drew a straight line vertically and a different line horizontally. with my Mycologist's directions that each length between tick marks = 10 µm I am now able to come up with correct dimensions. Thanks for your time and attention! Should I post this as it's own answer, or should you update yours to match so that I can accept a solution? $\endgroup$ – bgmCoder Nov 8 '15 at 18:32

The way I solved this was by using the scale line method. First, I understand that each division on the stage micrometer is equal to 10 microns. Then, using my photograph of my stage micrometer and the Motic software on my computer, I draw a horizontal line over oh, say, 3 divisions and tell it that this distance (measured in pixels) is equal to 30 microns.

Now, using the same method, this is also possible:

I found another Windows program called Meazure which you can use in the same fashion without any special microsoft software. For this I use XnView to open the photograph of my stage micrometer, set the zoom to 100% and then draw a straight line over oh, say, 3 divisions. Then I see how many pixels long that is. Then I tell Meazure that this many pixels is equal to 30 microns. Now I can measure, in XnView, any image taken by the microscope's camera - I just have to remember to assure the view is exactly the same.

The Meazure program is very nice and easy to use, and I actually prefer it, although the Motic software provides labels and such that are useful.


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