BIOL 1406

PreLab 4.1

How do I calibrate a reticle?

A reticle is used to measure objects viewed with the microscope. When you look through the ocular of your microscope, the reticle looks like a small ruler superimposed on the specimen you are viewing. As you increase the magnification of your objective, the image of your specimen increases in size, but the size of the reticle does not change.

The cross-lines on a reticle do not represent standard units of measurement, such as millimeters or micrometers. They are simply arbitrary markings superimposed on the specimen. In order to use these “reticle units” to measure a specimen, you must calibrate the reticle. This means you must determine the relationship between a “reticle unit” and a standard unit of measurement. As an analogy, suppose you asked a carpet installer to give you an estimate for carpeting your living room based on the following information: “My living room is 10 tiles wide by 15 tiles long”. It would be impossible for the installer to provide an estimate based on this information because a “tile” is not a standard unit of measurement. Are these large Saltillo tiles, medium vinyl tiles, or small ceramic tiles? In other words, the installer doesn’t know what the size of a “tile” is, although he does know the size of standard units such as inches, feet, or meters. In order for the installer to know how large your room is, you could use a ruler to “calibrate” your tiles, i.e. convert the non-standard unit called a “tile” into standard units such as inches. If the installer knows that each tile is 8 inches long by 8 inches wide, then he can give you an accurate estimate.

To further complicate matters when measuring objects with a reticle, if you increase the magnification of your objective, the size of the image you are viewing gets larger but size of the reticle units remains unchanged. This means your reticle must be calibrated for each objective that you use


To calibrate a reticle, switch to the scanning or low-power objective, place a clear plastic millimeter ruler on the stage, and then focus on the mm lines. What you see will resemble the illustrations below. Notice that microscope A is using an objective with a higher magnification than microscope B. Therefore, the mm lines will look farther apart on microscope A compared to microscope B, even though the size of the reticle units on both microscopes looks the same.

Figure 4.1 Calibrating a reticle on 2 different microscopes using a ruler with lines 1 mm apart

Note: Different types of reticles are available, and individual microscopes may vary slightly in their optics and powers of magnification. Therefore, you must calibrate the reticle for each objective and for each particular microscope that you use.

Careful! Do not be distracted by numbers on the reticle, if present. A reticle unit is the smallest unit visible on the reticle. Notice that the reticles of both microscopes shown above have a total of 50 reticle units, not 10.

 

Your Turn
Observe and record the number of reticle units (r.u.’s) which correspond to 1mm as seen through each microscope in the sample illustrations above.

IMPORTANT: You must count the number of reticle units starting from the MIDDLE of the first mm line and ending at the MIDDLE of the second mm line.
r.u.’s per millimeter for microscope A when using the 5X objective r.u./mm Hint Check your answer.
r.u.’s per millimeter for microscope B when using the 4X objective r.u./mm Hint Check your answer.
From this information, calculate the number of µm’s per r.u. for microscope A when using the 5X objective and for microscope B when using the 4X objective. To do this, first convert one mm to 1,000 µm, and then calculate the number of µm’s equal to one reticle unit.

IMPORTANT: Make sure you calculate µm / r.u., not r.u./ µm!!!
µm’s per reticle unit when using microscope A with the 5X objective µm/r.u. Hint Check your answer.
µm’s per reticle unit when using microscope B with the 4X objective µm/r.u. Hint Check your answer.


Assuming you have correctly determined the number of µm’s per reticle unit when using one objective on your microscope, you can calculate the number of µm’s per reticle unit for any other objective on the same microscope using the following formula:
 

µm / r.u. (using original objective) x (Magnification of original objective / Magnification of the new objective) = µm / r.u. ( using new objective)

 

 

Example calculation:

Using the 4X objective, let’s say you determined that for a particular microscope one reticle unit represents 50 µm. If you want to know the number of µm’s per r.u. when using the 40X objective on the same microscope, use the formula above:

50 µm / r.u. (using 4X obj.) x (4X / 40X)  = 5 µm / r.u. (using 40X obj.)

or

50 µm / r.u. x (0.1) = 5 µm / r.u.

In other words, if one r.u. equals 50 µm when using the 4X objective, then one r.u. equals 5 µm when using the 40X objective.

 

Your Turn
Using the formula above and the measurements you made for microscopes A and B, calculate
1. µm’s per r.u. when using a 10X objective with Microscope A. Hint Check your answer.
2. µm’s per r.u. when using a 40X objective with Microscope B. Hint Check your answer.


 


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