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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.

enter image description here enter image description here

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?

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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.

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  • $\begingroup$ 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
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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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