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How to Measure Microscopic Objects

by Jim Ekstrom

It is often important for a biologist to know the dimensions of the object being observed under the microscope. A simple method is described below to obtain estimates of the size of microscopic objects. This method enables you to obtain an approximate estimate of the size of an object by comparing it with the diameter of the field of view. To do this it will first be necessary to measure the size of the field of view using the metric system.

The unit of length that is commonly used for microscopic objects is the micrometer. The prefix micro- is given the symbol of the Greek letter mµ (µ). A micrometer (µm) is equal to one-millionth of a meter. More appropriately, for microscopic objects a micrometer is equal to one-thousandth of a millimeter (0.001 mm.), or 1000 µm = 1 mm.


Procedure:
  1. With the lowest power objective (4X) in position, place the millimeter ruler on the center of the stage so that the scale is visible through the microscope.
  2. Line up the 0 mm. vertical line with the left side of the circular field of view. (A total magnification of 40X will give you a view similar to Figure 2.)
  3. Count the number of millimeters included from one side of the field to the opposite side. If the right side of the field of view does not coincide with one of the lines, you will have to estimate to a fraction of a millimeter. (Be particularly careful that the edge of the ruler is across the middle of the field of view.)

    40X field of view  ________ mm. _________ µm

    100X field of view ___________ mm. ___________ µm

    400X field of view ____________ mm. ___________ µm

  4. Turn the 10X objective into place and fine adjust. Estimate this new field of view of 100X.

  5. Now turn the 40X objective into place. You will observe the field of view is less than 1 mm. Instead of measuring the field directly it is more accurate to calculate the diameter by the following method:

              (Field of View 1) X (Magnification 1) = (F of View 2) X (Mag. 2)

  6. The electron micrograph below shows a Red Blood Cell (RBC) in cross-section. Using your millimeter ruler and the scale that is included with the photograph, calculate the greatest diameter of this cell.(Show calculations)

  7. What is the number of RBC's that would fit across the following fields? (show calculations)

    1. across diameter of 40X field?


    2. across diameter of 100X field?


    3. across diameter of 400X field


  8. After you have determined the number of RBCs that will fit across the diameter of the high power field of view, do the following:

    1. Obtain a prepared slide of a human blood smear.
    2. Focus the slide under low power and find an area of the slide with a dense collection of cells which span the diameter of the field of view.
    3. Center the dense region you have located and switch to high power.
    4. After focusing with the fine adjustment, carefully adjust the slide and count the number of cells that fit across the diameter of the high power field of view.

  9. How does your actual count compare to the calculated number of cells expected to fit across the diameter of the high power (400X) field?


  10. What is the percent difference between the two values? (Percent difference is determined by dividing the difference between the actual and calculated values by the average of the two values and then multiplying by 100.) (show calculations)

  11. Identify a number of possible sources of error in this method of measuring microscopic objects.



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