Microscope Activities, 28: The Graduated Drawtube

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 28: The Graduated Drawtube

Goal

To be able to recognize a graduated drawtube, and understand how and why it is used.

Level

Intermediate – Advanced

Materials Needed

A microscope equipped with an adjustable graduated drawtube.

Procedure

• Review the Discussion of fixed tube length in Experiment 4.
• Read the Discussion below and practice introducing and eliminating spherical aberration from the microscope optical system.
• Prepare a chart of Mechanical Tube Length (mm) vs. Eyepiece Micrometer Calibration (µm/division).

Discussion

Almost all student-grade microscopes today come equipped with a fixed mechanical tube length. In the past, the most common tube lengths were 160 mm (most manufacturers) and 170 mm (Leitz). Metallurgical-type microscopes required a longer tube length so as to accommodate a light source reflecting system above the objective; mechanical tube lengths for these systems included, for example, 185 mm and 215 mm. More recent microscopes, including metallographs (those with built-in epi-brightfield illumination made for looking at opaque specimens, such as polished metal and ore samples) have tended to be corrected for an infinitely long tube length, indicated with an infinity symbol, ∞.

The reason for the move to “infinity corrected” systems is because of the trend today to use multiple modules stacked for polarized light and various contrast enhancement systems, and also for epi-fluorescence (see Experiment 23). Figure 28-1 illustrates four objectives, each requiring a different mechanical tube length; from left to right, 160 mm, 170 mm, 215 mm, ∞. If these objectives are used at mechanical tube lengths other than that for which they were designed, the resulting images will be severely degraded.

This much is taken for granted: that the user of any microscope will make sure that the objectives being used correspond to the microscope’s mechanical tube length. Now, if you will review the Discussion in Experiment 24 on Coverglass Thickness, you will recall that use of incorrect coverglass thickness will introduce spherical aberration into the final image; the image will be “soft.” Notice that the required coverglass thickness for any particular objective is always engraved on it. In Figure 28-1, the two objectives on the left require a coverglass that is 0.17 mm thick—this is indicated after the slash following the mechanical tube length; the two objectives on the right require no coverglass, indicated by the “0”, because they are intended for viewing polished metal and ore specimens by reflected light; a coverglass would reflect the incident light right back up, causing nothing but glare.

Objectives of high numerical aperture are particularly sensitive to changes in coverglass thickness, including thickness of mounting medium. In Experiment 24 it was mentioned that spherical aberration due to coverglass/mountant thickness variation could be corrected by using the so-called “correction collar” of specially-constructed objectives in which a knurled ring surrounding these objectives is used to alter the position of key lens elements within the objective itself. Altering the lens position introduces spherical aberration that is of equal magnitude, but opposite sign, so as to cancel the aberration from the specimen slide. Objectives with correction collars are, however, very expensive, and here is where the adjustable drawtube comes in!

An adjustable drawtube allows the user to change the overall length of the body tube so as to correct spherical aberration due to use of too-thick or too-thin coverglasses. In the past, adjustable graduated drawtubes were offered as an option with every monocular microscope. Figure 28-2 is a composite photograph of a Bausch & Lomb Microscope fixed-length eyepiece tube (right) having been replaced with an optional adjustable graduated drawtube screwed into the main body tube.

In the fully-closed position, this drawtube gives a total tube length of 140 mm; when fully extended, the total tube length can be increased to 174 mm. There is a reference line at 160 mm that goes all the way around the drawtube to indicate that this is the correct starting position setting for the vast majority of observations made with objectives corrected for 160 mm tube length. (Note that you could mix a Leitz objective on the nosepiece, but you would have to remember to re-set the tube length to 170 mm when that objective is being used).

Figure 28-3 shows the adjustable graduated drawtube removed from the main body tube of a Zeiss microscope; its range of tube length is 154 mm to 200 mm; it just slides into the main body tube.

How to Use an Adjustable Graduated Drawtube

There are several uses for a drawtube, including:

• Correction of spherical aberration due to use of a too-thick or too-thin coverglass—its most important use.
• Continuous change of magnification (the microscope’s total magnification can be increased by lengthing the tube length, or decreased by shortening the tube length. This feature is often helpful in photomicrography when filling a frame.
• Variable eyepiece micrometer calibration (since the total magnification changes continuously with tube length change, the micrometer value of each eyepiece micrometer division varies continuously).

The most important use of the adjustable drawtube is to correct for spherical aberration in the image that is due to incorrect coverglass thickness. You will know how to choose correct coverglass thickness to begin with, after performing Experiment 24, Coverglass Thickness. But if you are looking at a commercially prepared specimen, or any specimen prepared by someone else, you do not know if the correct thickness coverglass has been used. You will also recall from Experiment 24 that lower magnification objectives with their lower numerical apertures are not particularly affected by incorrect coverglass thickness; it is the high numerical aperture objectives that are particularly affected.

So, say you are looking for Barr bodies in the leucocytes in a stained blood film, or any fine structure within any tissue section, or live microorganisms using a 40X/0.95 NA objective. Here is how you proceed: with one hand on the fine focus knob, and the other hand on the drawtube, concentrate your view on some tiny, tiny structural feature, and adjust the fine focus carefully for best critical focus. Now change the tube length slightly—it does not matter if you lengthen it or shorten it a little—and you will discover that you have to re-adjust the fine focus for sharp focus. Evaluate the tiny fine feature you were concentrating your view on, and ask yourself if the change in mechanical tube length results in a better, sharper image, or has the image become “softer.” If the resulting image was improved, continue to lengthen or shorten the tube length as you did before. Again refocus and evaluate. Continue the procedure as long as image quality improves. When the image starts to degrade, reverse direction of the drawtube. The object is to find that tube length that results in the very best quality image. When you have done so, you will have corrected the system for spherical aberration; it does not matter what the actual tube length reads, as long as the image quality is perfect. This use of the drawtube is particularly necessary for those microscopists who desire to resolve the fine lines and “puncta” of diatoms. As for the actual, final tube length reading, it is immaterial, but you will find that tube length must be shortened for too-thick coverglass, and lengthened for too-thin coverglass.

Using tube length adjustment to change magnification is self-explanatory. When viewing your image on a screen or within framing lines, if you would like the image a bit bigger, but the next objective up in magnification is too much, you may increase the tube length to increase the image size.

There is an important use of the graduated drawtube in micrometry. The eyepiece micrometer is normally calibrated with a fixed tube length body. With an adjustable tube length, the calibration of each eyepiece micrometer division will continuously change with tube length because of the continuous magnification change. So, what you do is prepare a graph in which you plot mechanical tube length against eyepiece micrometer calibration. For example, the set-up in Figure 28-2 was used; the tube was fully closed to 140 mm, and the eyepiece micrometer was calibrated using a stage micrometer, and found to be 16.0 µm per division. Next, the drawtube was successively set to 150 mm, 160 mm, 170 mm, and 174 mm (the maximum extension), and the value of each eyepiece micrometer division was determined for each tube length setting.

Figure 28-4 shows the resulting graph; note that there is approximately a 1 µm difference in calibration for each 10 mm change in tube length. What is commonly done is that a tube length is selected that results in some even number of µm/div to facilitate measurements of structures, or for particle size analysis.

Task

Obtain a microscope that has an adjustable graduated drawtube, install a high numerical aperture objective, and practice finding the optimum tube length for some very fine structure in any specimen. Change the tube length, and repeat the operation to get an idea of the reproducibility of your findings. These are advanced tasks that require some practice, but they will result in making you a critical observer.

Not all drawtubes are graduated; ungraduated drawtubes are used in the same way described for neutralizing image error due to incorrect coverglass thickness; they just cannot be used for micrometry. Ungraduated drawtubes still have a simple line engraved all around them at e.g., 160 mm.

Suggested Further Reading

This is an advanced topic, but for those interested in pursuing further details, the following is highly recommended:

Spinell, B. M. and Loveland, R. P. (1960). Optics of the Object Space in Microscopy. Journal of the Royal Microscopical Society 79 Pt 1, 59-80.