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Scale Bars, Part II

 
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Charles Krebs



Joined: 01 Aug 2006
Posts: 5762
Location: Issaquah, WA USA

PostPosted: Sun Nov 05, 2006 5:35 pm    Post subject: Scale Bars, Part II Reply with quote

There are a few methods that can be used to arrive at the proper dimensions for a scale-bar to be placed in a photomicrograph. Photographing a “stage micrometer” (a slide that contains, in essence, an extremely small, calibrated ruler) with your various optics is the best way. (See Grahams message: http://www.photomacrography.net/forum/viewtopic.php?t=848).

But many of us, myself included, do not have a proper stage micrometer. So for non-critical work this is the procedure I use...

First I get the actual physical dimensions (in mm) and the pixel dimensions of the camera sensor I will be using. (If you don’t have this already, a little exploration on a site like www.dpreview.com should turn up this information). My Canon 350D has a sensor size of 22.2mm (in the longer, or “x” dimension), with pixel dimensions of 3456 pixels along that dimension.

Using my two lowest power objectives, a 2X and a 4X, I photograph a rule that is marked in millimeters. (This becomes my crude “stage micrometer”. It would be far better to have a calibrated rule with much smaller units, but this will work fairly well if you use low power objectives). What we want to do is determine the magnification that is provided by the optics between the objective and the camera sensor. These “relay optics” include any eyepieces as well as any camera lens attached to the camera. This need be calculated one time, as long as the relay optics are not changed.

Examine the resulting picture and see how many millimeters are included in the long dimension of the frame. For example…when using my 2X, I record about 5.25mm on the long dimension, and with the 4X it is just a bit more than 2.5mm.

The magnification at which the picture was taken can be determined with the following relationship:

Total Recorded Magnification = sensor dimension (mm)/# of mm recorded from scale

So with my example the recorded magnifications would be as follows:

With 2X objective Total Recorded Magnification = 22.2mm/5.25 = 4.23
With 4X objective Total Recorded Magnification = 22.2mm/2.6 = 8.54

The recorded magnification is the product of the objective magnification and the “relay” magnification. So to determine the magnification that is being provided by all relay optics you simply divide the recorded magnification by the objective power. In my example:

4.23/2 = 2.11
8.54/4 = 2.13

So in my example it appears that my relay optics are providing a magnification of about 2.12X. Now I can determine what the recorded magnification will be with any of my other objectives:

With 10X… (10)(2.12) = 21.2X
With 20X… (20)(2.12) = 41.4X
With 40X… (40)(2.12) = 82.8X
With 100X… (100)(2.12)= 212X

Now… on to the scale bars

Once we know the recorded magnification, the sensor size, and the number of pixels along the width of the sensor, we can determine the length (in pixels) of the bar that will be used to represent any given length in our photo. To make it simple, I have created a spreadsheet that does all the necessary calculation. It can be opened or downloaded here:
http://www.krebsmicro.com/scale_bars.xls

All you need to do is enter three values:
-recorded magnification
-x sensor dimension (mm)
-number of pixels in x dimension of picture

The spreadsheet will display a chart that shows you the length (in pixels) of the bar needed to represent a chosen measurement. Most photo-editing software has the ability to show the length of a line being drawn.
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