Microscope Activities, 5: Free Working Distance

In the past, Hooke College of Applied Sciences offered a microscopy workshop for middle school and high school science teachers. We thought that these basic microscope techniques would be of interest not only for science teachers, but also for homeschoolers and amateur microscopists. The activities were originally designed for a Boreal/Motic monocular microscope, but the Discussion and Task sections are transferable to most microscopes. You may complete these 36 activities in consecutive order as presented in the original classroom workshop, or skip around to those you find interesting or helpful. We hope you will find these online microscope activities valuable.

EXPERIMENT 5: Free Working Distance of Objectives

Goal

To measure the amount of space between the front of the objective and the top of the coverglass, when the specimen is in focus; this space is known as the free working distance (FWD).

Level

Basic

Materials Needed

• U.S. Lincoln pennies—preferably little used
• U.S. currency (e.g., dollar bills)
• Optional: mechanic’s micrometer

Procedure

Focus on any specimen using, successively, all three of your objectives, 4X, 10X, 40X. With each objective in focus on any specimen, fill the space between the objective and specimen coverglass with Lincoln pennies, until no more will fit. If there is leftover space, use a dollar bill, or any other denomination of U.S. currency, to fill the space between the pennies and the front of the objective; fold the dollar bill once or twice, or however many times it is necessary to completely fill the space between the pennies and the front of the objective. Figure 5-1 is what this will look like when using your 4X objective.

Record the total number of pennies and total number of layers of U.S. currency needed to fill the space between the front of the 4X objective and the top of the coverglass with the specimen in focus.

Repeat this procedure using the 10X objective and the 40X objective (Figures 5-2 and 5-3).

Discussion

The free working distance (FWD) of any objective is an extremely important characteristic of that objective. The reason for this is that professional microscopists seldom merely look at a sample. They want to manipulate the sample—move it, rotate it, apply a microchemical test to it, remove it for analysis by another analytical instrument, etc. All of these procedures require that the working microscopist know if they have enough room under the objective to handle their specimens. Some microscopists manipulate their samples under a 40X objective, and to do this, they select those 40X objectives that have the longest free working distance. Some objectives are made to be Long Working Distance, such as those made to focus through Petri dishes or tissue culture chambers, or to be used with high-temperature hot stages, where they must be kept away from excessive heat, or those that must focus through the thick glass hemispheres of the universal stage. There are 40X objectives constructed on the reflection principle that have a free working distance of 12-16 mm!

Professional machinists have quite a number of tools that can be used to measure spaces very precisely, but these highly specialized tools are very expensive. For our purposes, we can obtain results of reasonable accuracy by using readily available U.S. Lincoln pennies and U.S. currency. The reason for this is that the U.S. government controls the thickness of these coins and bills to within fairly close tolerances.

• The official thickness of a U.S. Lincoln penny is 0.061 inches.
• The official thickness of U.S. currency (any denomination) is 0.004 inches.

We noted (Figure 5-1) that it requires 14 pennies (one hidden under the two bills) and two dollar bills—one folded once, and one folded twice (i.e., 6 layers)—to fill the space between the front lens of the 4X objective and the top of the coverglass of a focused specimen.

Fourteen pennies, at 0.061 inches thickness each, equal 0.854 inches (or, converting to millimeters by multiplying 0.854 inches by 25.4 millimeters per inch, we obtain 21.69 millimeters).

Six layers of dollar bill, at an official thickness of 0.004 inches per layer, equal 0.024 inches (or, converting to millimeters by multiplying 0.024 inches by 25.4 millimeters per inch, we obtain 0.6096 millimeters).

The total thickness for 14 pennies and 6 thicknesses of dollar bill equals 0.854” + 0.024” = 0.878” (or, 21.69 mm + 0.6096 mm = 22.30 mm).

Thus, the free working distance of the 4X objective is 22.3 mm (0.878”).

To test this conclusion, we used a professional micrometer to measure the entire stack of 14 pennies and 6 layers of dollar bill, and obtained a reading of 0.803 inches (Figure 5-4)—a difference of seventy-five thousandths of an inch—which is close enough for our purposes—and it did not cost us anything.

We determined the free working distance of the 10X objective (Figure 5-2) and the 40X objective (Figure 5-3) in the same way, using pennies and dollar bills.

Be careful with the 40X objective—and all higher magnification objectives—because the front lens is spring loaded to reduce the possibility of damage when focusing down excessively; be careful that you are measuring the true free working distance, and not, unintentionally, retracting the front lens into the body of the objective.

Task

Measure the free working distance (FWD) of your 4X, 10X, and 40X objectives, using Lincoln pennies and U.S. currency.

Prepare a Table in which you record the objective magnification, the number of pennies, and number of bills required to fill the working distance spaces; the conversion of official thicknesses from inches to millimeters; and the free working distance.

Teacher’s Note: Here are the results we obtained with our microscope: