Microscope Activities, 24: Coverglass Thickness
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 24: Coverglass Thickness
To measure the actual thickness of all of the coverglasses in a ½ ounce or 1 ounce box, to prepare a histogram or table from the results, and to understand the significance of coverglass thickness as it relates to the numerical aperture of objectives.
Boxes of coverglasses, preferably representing #1, #1½, and #2; a machinist’s micrometer—regular or pocket; metric or English measure.
Using a machinist’s micrometer, measure the thickness (in millimeters) of every coverglass in a ½ ounce or 1 ounce box, and make a record of the results in the form of a table or histogram. Record the total number of coverglasses, the name of the manufacturer of the coverglasses, the indicated thickness class (#1, #1½, #2, etc.), the dimensions, and, if available, the Lot Number and/or date of manufacture.
Figure 24-1 illustrates two different styles of machinist’s micrometer and three boxes of coverglasses. The three boxes of coverglasses represent 1 ounce each of #1, #1½, and #2 thickness coverglasses; these all happen to be round coverglasses, but for this experiment the shape and outside dimensions do not matter. The object will be to measure the thickness of each coverglass in a box, using a machinist’s micrometer.
The micrometer being held in the hand in the figure is a pocket dial-type micrometer; it is metric, and reads to 0.01 mm, for a maximum opening of 9 mm; each one of the smallest divisions on the dial face represents 0.01 mm (or 10 μm); every 10 such divisions is numbered (10, 20, 30….representing 0.10, 0.20, 0.30 mm); when the long needle goes completely all around once for 100 divisions, that represents 100 x 0.01 mm = 1.00 mm, and the needle on the small dial moves to “1” representing 1 mm. In the figure, the dial reads exactly 17 divisions, or 17 x 0.01 mm = 0.17 mm (or 170 μm). This kind of pocket dial-type metric micrometer is the fastest and handiest type to use for measuring coverglass thickness, but it is expensive.
The micrometer shown at the lower left in the figure is a conventional 0-1” Outside Micrometer reading to one ten-thousandth of an inch (0.0001”). Although domestic micrometers like this of professional grade cost $150-$200, the imported one illustrated here is a remarkable value selling for $14-$15, and, when on sale, about $12. It has a satin-chrome finish, carbide faces, ratchet stop, lock, plastic faces on the “C” portion to prevent heat transfer from the hand that would change the reading and it reads to 0.0001”. With this type of outside micrometer reading in inches, you will have to convert your readings to metric.
The moving cylinder (spindle) that bears against the anvil is moved via an internal screw that has a pitch of 40 threads per inch—it takes 40 full turns of the knurled handle to move the cylinder through 1 full inch. Therefore, 1 full turn of the handle moves the cylinder 1/40 of an inch, or 1” ÷ 40 = 0.025”. The body (sleeve) of the “mike” has graduations at each 0.025” interval, but there is a Vernier inscribed on the rotating handle (thimble) that is divided into 25 divisions so as to read each 0.025” interval to 0.001” (one thousandth of an inch). This particular micrometer has a second Vernier of 10 parts, which divides each 0.001” into ten further divisions, giving final readings to 0.0001” (one ten-thousandth of an inch)—not bad for 12 bucks!
In Experiment #2 you learned that the objectives on your microscopes were designed to be used with coverglasses that are 0.17mm (170 μm) thick; this specification is engraved on each objective. The thickness of the coverglasses in a box are not indicated; they are sold by number: #0, #1, #1½, #2, #3, etc—#1½ was only added recently. It is interesting to hear microscope users justify their choice of number: “Buy #0 or #1 because you get more of them in an ounce,” “Buy #3’s because they are thicker, and students don’t break them as easily,” “Buy #1’s because then you can use them for oil immersion.” All of these are incorrect.
For objectives with numerical apertures of 0.70 to 0.95—the “high drys”—the acceptable tolerance for thickness departure from design value is 0.17 ± 0.01 (that is, 170 μm ± 10 μm). This is the really critical range where coverglass thickness is vital. Remember, the higher the numerical aperture of an objective, the higher is the resolving power.
The mechanical drawtube mentioned in Experiment 4 can be used to correct problems of spherical aberration introduced by too thick or too thin coverglasses.
To complicate matters even more, one has to take into account the thickness of the mounting medium if it is on top of the specimen, as it is when tissue sections are attached to the slide, the mountant is placed on top, and then the coverglass is added. An interesting side experiment in this regard, as long as you have a micrometer handy, is to measure the thickness of your slide (that’s a whole other story!); then measure the thickness of a coverglass. Now, make a blank preparation; that is, pretend like you have a specimen on the slide, and add your mountant and coverglass.
When solid, measure the entire thickness and substract the sum of the slide and coverglass to determine your mountant thickness (our blank averaged 0.0077 mm, or about 8 μm mountant thickness)—probably only the geeks and the obsessive compulsives will actually do this!
Spherical aberration in the image due to too thick or too thin coverglass and/or mountant may also be corrected by the use of objectives equipped with so-called “correction collars”; rotating a knurled ring on these objectives actually moves the upper set of lenses up or down inside of the objective! Changing the position of these lenses introduces spherical aberration, and the idea is to introduce an aberration which is equal in magnitude, but opposite in sign, to that introduced by the incorrect coverglass, thus cancelling it out. This is accomplished by critically evaluating some fine structure in a sample while making small changes in the correction collar; if turning it in one direction makes the image worse, it is turned the other way, and so forth, until the best quality image is obtained. Objectives of high numerical aperture, equipped with correction collars, cost several thousand dollars each.
The reason that Table 1 has two yellow-color highlighted lines is that most manufacturers today specify the use of coverglasses that are 0.17 mm thick; in the past, Bausch & Lomb specified 0.18 mm thick coverglasses for use with their objectives. Note also that these thickness distributions depend of each manufacturer’s lot number for that particular run!
Measure the thickness of the coverglasses in a ½ ounce or 1 ounce box; working with a lab partner, or making this a class project will make the task go faster. Tabulate the results. Consider the results in terms of the requirements for your objectives. Now what thickness coverglasses are you going to buy?