Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Magnification of a photomicrograph
I'm trying to find the calculation used to determine the magnification of a photomicrograph. I know you have to take into consideration several things besides the objective. Can someone help?
There are a couple of "gotchas" in figuring magnification. You need the magnification of the objective multiplied by the magnification of the ocular. However, and here is where you need to do some double checking, be sure the ocular in the path to the camera is the magnification you use. On some microscope/camera combinations, a different magnification is used for the camera ocular.
Then there is the matter of whether the microscope has a "tube lens." If the microscope you used is not one of the newest infinity corrected types, then there is most likely a magnification lens BETWEEN the objective and the ocular. These generally fall into the magnification range of 1.5 x, which again would have to be multiplied with the other two magnifications. On some microscopes, the tube lens magnification is marked on a surface betwen the objectives and the oculars, but on others, theres is no external marking. In that case, you will need an original manual for the scope. To complicate matters even further, many camera connect to the microscope trinocular tube with a reduction tube. So the magnification the camera sees is the combination of the various lenses used, divided by the reduction tube. The reduction tubes commonly fall into the range of 0.25 to 0.75 x. The reduction factor is generally printed on the outside of the tube that connects the camera to the microscope.
As a general procedure, for any microscope used to take photomicrographs, one should take a picture of a stage micrometer with each objective on the scope, and keep these pictures in a "calibration" file for that camera/microscope combination. The stage micrometer will be a true "ruler" with divisions of 0.1 and 0.01 mm, so it is easy to check the true magnification of prints or slides. If you don't have a stage micrometer, then use the built in standard: the average diameter of red blood cells after most processing procedures is approximately 7 microns. That is not exact, but is a good way to check that your magnification calculations are in the right ballpark
Alton D. Floyd, Ph.D.
The best way is to photograph a calibrated slide using the same objective and other variable things as for the section. Print the photos at the same enlargement, and measure with a ruler. If a 100 micrometre distance is 32 mm on the print, the magnification is 32000/100 = 320.
Calculations based on the optics commonly lead to ridiculous mistakes. As a rough check, measure something in the photo and see if it's a sensible size. If there are cell nuclei 50 micrometres across, somebody has made an arithmetic error.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1