How does microscope image measurement work?

The microscope image aquisition and measurment software give you the convenience of taking the measurement and annotation of the distance easily.  While you draw a line across an object on the digital image, the length of the line just automatically showed up on the screen.  How does it work?  Today, our microscope cameras take digital images which consist of pixels.  Each pixel can be located by its coordinates.  The distances between two points on the image can be calculated by their coordinates. Likewise, you can calculate the length of your object in pixels using the coordinates.  You need a calibration slides with know distance in micrometer (or millimeter) to provide the conversion factor from pixel to micrometer.  Do you know that you can do it with the paint program that comes with Windows operating system or any program that gives you the coordinate of the pixel on the image?  I will show you how.

1. Take a digital image of your calibration slide.

2. Open the image of the calibration slide

3. Position your mouse cursor at one end of the ruler and record the coordinate of the pixel.

 4. Position your mouse cursor at the other end of the ruler and record the coordinate of the pixel.

5. Calculate the distance between two end of the ruler in pixels using the following formula:

If you like, you can use the following formula in Excel to calculate the distance:

= SQRT(ABS(B3-B2) ^2 + ABS(C3-C2)^2) 

6. Calculate the conversion factor by dividing the distance between the two ends of the ruler and the distance in pixel calculated in step 5

7. Open the digital image containing the object that you want to measure.

8. Draw a line across the distance across the object and measure the length in pixel like the you do for the calibration slide (step 3 to 5).

9. Using the conversion factor obtained in step 6 to convert the pixel into micrometer.

10. Record the distance on the image

If you prefer, you can use the following spreadsheet (formula.xlxs) to calculate the distance.  Just enter the coordinates of two points on the calibration slide and the object that you want to measure (highlightedin blue).  The distance is highlighted in green. 

WOW! that's lots of steps.  The microscope measuring software surely make your life a lot easier.