Measuring and Counting with a Light Microscope
Estimating Field Diameter of a Microscope
To determine field diameter using a stage micrometer, one places the stage micrometer on the microscope stage. Next, looking through the eyepiece (using the lowest magnification), one uses the mechanical stage controls to line up a major division of the scale with one edge of a microscope field so that the finely etched portion of the scale overlaps the other edge. By counting divisions, one can estimate true field diameter to the nearest 0.01 or 0.02 mm. Once a microscope is calibrated at one magnification, it should not be necessary to repeat calibration for other objective lenses. The scale is inversely proportional to the magnification itself. For example, if the field diameter at 40x total magnification is measured to be 5.2 mm, then the diameter at 400x must be 0.52 mm.
(field of view 1) X (magnification 1) = (field of view 2) X (magnification 2)
Knowing field diameter, one can determine the actual surface area of the specimen in view, count objects in the field, and determine the number of objects per unit area. For accuracy, one may count multiple randomly-selected fields. Often an object will overlap the edge of a field, and counting all overlapping objects results in overestimating the density. A good practice is to set criteria in advance for accepting or rejecting an object. For example, you can divide the field into four imaginary quadrants. If an object overlaps the top or right edge of the field it is accepted, but it is rejected if it overlaps the bottom or left of the field.
- Alberts, B., et al. (2002). Molecular Biology of the Cell (4th ed.). New York: Garland Science.
- Caprette, D. (1995). Light Microscopy. Retrieved 8-22-2006 from http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html
- Lodish, H., et al. (2000). Molecular Cell Biology (4th ed.). New York: W.H. Freeman and Co.
- Wolfe, S.L. (1993). Molecular and Cellular Biology. Belmont, CA: Wadsworth Publishing Company.
- Caprette, D. (2006). Field diameter.
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