The Proper Way to Examine Tissues

Courtesy: Wikipedia

Most beginners approach slide examination in a somewhat haphazard, nonsystematic way. In the process, they get frustrated with a poor image or by losing their orientation, by losing the feature they are examining, or by losing the specimen's image entirely.  The reality is that the microscopic examination of tissues is quite straight-forward, as long as it is done in a systematic way.   A standard, systematic approach to tissue examination can make the work less frustrating and the examination more thorough. The following stepwise approach to tissue examination with a compound microscope is recommended for the beginner.  With experience, the user will learn how to adapt the appraoch to meet their needs.

The correct approach to the microscopic examination of tissue is really based on common sense and a simple appreciation for how a modern compound optical microscope works.  More specifically, it is based on the understanding that 1) the user can see more of the tissue with a lower-power objective than with a higher-power objective (i.e., the field of view of a lower-power objective covers more tissue area than a higher-power objective), and 2) that microscopes of reasonable quality are reasonably parcentric and parfocal.  That is, an object that is in the center of the field and in focus will remain in the center of the field (parcentric) and in focus (parfocal) when the next objective is rotated into position.  So the approach is very simple, start out at the lowest possible magnification, bring the specimen to focus, find a feature of interest, center it, and move to the next objective to study the feature in more detail.

To introduce this approach, you are going to examine the hair follicles of thin skin. 

Place a Tissue Slide on the Stage of your Microscope

As was mentioned earlier, there is a right way and wrong way to place a slide on the satge of your microscope.  Follow the steps below every time you want to place a slide on the stage of your microscope.

1.  Prepare the microscope to receive a new tissue slide. 

a.  Lower the stage as far as it will go by turning the coarse focusing knob.  This will give you plenty of room to work on the stage.

b.  Remove any tissue slide that you might have on the mechanical stage and return it to its slide box.

c.  Rotate the scanning objective into position.

2.  Obtain a slide of skin containing hair follicles and follow along these instructions to prepare for your examination of this slide.

a. Clean the slide. Use a common lint-free lab wipe wetted with common glass cleaner to clean the slide. Wipe gently; be careful not to dislodge the coverslip.

b. Read the slide’s label. Important information about the specimen can be found on its label. Often, the plane of section, e.g. crossection (CS) or longitudinal section (LS) and the type of histological stain are indicated on the label.  Knowing this information can be very helpful in understanding the specimen.

The most commonly employed histological stain is a double stain consisting of hematoxylin and eosin, usually called an H & E stain. In an H& E stain, nuclei are stained a dark purple and the cytoplasm is usually stained a light pink.  If your silde label does not mention the stain, an H & E stain was likely used. 

c. Examine the slide with the unaided eye.  Hold the slide up to the light and examine it with the unaided eye. Mentally note any visible macroscopic features, such as the number of tissue slices (there may be more than one), their approximate position on the slide, their shape, orientation, and the intensity of the stain. This information will help you know what to expect as you begin your microscopic examination.

3.  Place the slide on the microscope stage.

a.  Carefully place the slide on the stage and secure it with the slide holder. Using the stage translation mechanism, and with the scanning objective in position, position the tissue section directly under the objective lens.

b.  While observing your set-up from the side, adjust the course focusing knob to bring the objective lens to within a few millimeters of the surface of the slide. Then, while viewing through the eyepiece, slowly adjust the course focusing knob upward, i.e., away from the specimen. By focusing upward rather than downward, you reduce the risk of accidently driving the objective lens into the slide and breaking the slide.

c. Once you have brought the specimen in focus with the coarse adjustment knob, refine your focus if necessary with the fine focusing knob.

 

Getting a Satisfactory Image

Look through the eyepiece to view the image.  This image might be OK, it might not.  Whatever the case, the user should check certain basic adjustments to make sure the image is a good as one could reasonably expect.  In optical microscopy, there are four basic adjustments one can make to an image: eyepiece, brightness, focus and contrast (with resolution).

1. Adjust the Image Brightness. Next, you might need to adjust the image brightness.  Several controls affect iamage brightness.  However, to adjust the brightness of the image without affecting other image characteristics, simply adjust the intensity control on the light source.

2. Adjust the Interocular Distance.   If you have a binocular scope,  you might need to adjust the interocular distance  to achieve a suitable image.  Binocular compound microscopes are equipped with two eyepiece adjustments. The interocular distance can be adjusted by gently moving the eyepieces toward or away from each other. The distance should be adjusted to the point where a single comfortable image is viewed.

3. Aperture Diaphragm Adjustment. The final adjustment is usually an adjustment of the aperture diaphragm.  As discussed earlier, the aperture diaphragm controls the beam of light entering the condenser, thus controlling the effective numerical aperture (N.A.) of the condenser and thereby controlling image contrast and resolution.

For an optimum image, the N.A. of the condenser should match that of the objective lens in use. If the aperture diaphragm control has a numerical scale, it is easy to match the condenser aperture to that of the obective lens.  However, if your aperture diaphrgam control is not graduated, adjusting the aperture diaphragm to match the condenser and objective lens N.A.s is easier said than done.  Microscope manufacturers recommend setting the diaphragm to about 70% - 80% of the exit pupil of the objective. This can easily be done by removing the eyepiece, then adjusting the aperture iris diaphragm control while looking into the eyepiece sleeve.

  Short of this, the user must understand that the aperture diaphragm should be adjusted every time the objective lens is changed.  At lower magnifications, the aperture diaphragm should be slightly closed.  At higher magnifications, the aperture diaphragm should be more opened. So, at the very least one should adjust the aperture diaphragm every time the objective lens is changed until a visually comfortable balance of resolution and contrast are achieved.

Now that you have a reasonable image, you are ready to examine the tissue.

 

Examine the Slide

Step 1.  Examine the slide with the scanning objective. 

The proper approach to tissue examination begins with a scan of the tissue using the scanning objective, which is usually a 4X objective.  The total magnification with a 4X objective and 10x eyepiece is 40X.   Under this very low magnification it is relatively easy to survey the entire slide.  The purpose of a scanning examination of a specimen is to note the general structure and orientation of the tissue. Famialiarize yourself with the major structural features of the tissue, then focus your attention on the features of interest.

In the specific case of our examination of thin skin (containing hair follicles), slowly scan the specimen by moving the X-Y stage control knobs.  Note the presence of hair follicles, their appearance, relative abundance, and relative position.

Now you are ready to begin examining the base of the hair follicle in more detail.  Locate the base of a representative hair follicle, center it, refine your focus, then rotate the low-power objective (10X or 20X objective) into position.


Step 2.  Examine the slide with the low-power objective. 

The low-power objective is usually a 10X objective.  The total magnification with a 10X objective and 10x eyepiece is 100X. When you move the 10X objective into position, the base of the hair follicle should be very close to the center of the field, a characteristic of the scope called parcentric.  You might need to slightly adjust the stage to bring the base of the hair follicle into the center.  The base of the follicle should also be in focus or very close to focus, a characteristic of the scope called parfocal.  Refine the focus if necessary.

In the specific case of our examination of the base of a hair follicles, note the various layers of cells and the bulb-like feature at the root of the follicle.

Now you are ready to examining the root of the follicle in greater detail.  Center the root of the follicle, refine your focus, then rotate the high-power objective (40X or 60X objective) into position.


Step 3.  Examine the slide with the high-power objective. 

Examine the slide with the high-power objective. Now you are ready to examine your specimen at an even greater magnification. Use the same basic stepwise process described in step 7 to examine the base of the hair follicle with the high power objective in position. That is, center the feature, refine the focus if needed, then rotate the high-power objective into position. Because you took the time to locate the feature with lower power objectives, finding the feature with the high-power objective is easy!

During your examination of detail under dry conditions, you may find it necessary to re-examine the specimen with an objective of lesser magnifying power. If so, feel free to drop down to the lower power. If you need to refine your focus, be careful. You will likely be focusing downward, which carries a risk of pushing your objective into the slide. So focus carefully!


Step 4.  Examine the slide with the oil immersion objective. 

Microscopic examination with the oil objective is not always necessary, but often important when studying fine details of tissue structure. When advancing to the 100X objective, use the same systematic approach used above (Center, focus, the rotate to the next objective) with one additinal step. When advancing the turret to the oil objective, stop your rotation at a point halfway between the high-power and oil objective, then place a drop of immersion oil directly on the beam of light passing through the slide. Once done, complete the rotation of the turret to place the oil objective into position. Then proceed with your examination. Follow this procedure now to study the base of the hair follicle with you oil objective.

 

The Examination is Complete:  Removing and Returning the Slide

When you are finished with any examination, remove the slide and and the microscope are properly cleaned and returned to where they belong.

1.  Lower the stage.

2.  Rotate the scanning objective into position

3. Remove the slide, clean it, and return it.

If you examined the slide with oil, be sure to gently, but thoroughly, clean the slide with glass cleaner. Be gentle, though, you do not want to loosen the coverslip.

4. Clean the oil objective. Be sure to clean the oil-immesion lens with lens paper immediately after examination with the oil objective.

5. Shut down the microscope.

Turn off the power to the lamp.  Unplug and wrap the cord neatly. Cover the scope with its dust cover. Then, using both hands, grasp the microscope by its base and arm and gently return it to its cabinet.

 

Summary

This systematic approach to the microscopic study of tissue is based on the understanding that the field of view offered by lower-power objectives reveals more tissue area than that of higher-power objectives. Therefore, it is easier to find a feature of interest with an objective of lower-power.

Before examining the slide microscopically, examine the slide with the unaided eye. The slide itself offers valuable information that can help you know what to expect as you begin the microscopic examination of the tissue, such as the number of tissue slices (there may be more than one), their approximate position on the slide, their shape, orientation, and the intensity of the stain.

The initial examination of a slide should always begin with the scanning objective, which is usually the 4X objective. Relying on the fact that microscopes of reasonable quality are reasonably parcentric, one can then study a feature of interest at progressively higher magnifications by centering the feature, refine the focus if needed, then rotate to the next objective.

During your examination you may find it necessary to re-examine the specimen with an objective of lesser magnifying power. If so, feel free to drop down to the lower power. If you need to refine your focus, be careful. You will likely be focusing downward, which carries a risk of pushing your objective into the slide. So focus carefully!

By using a systematic approach to the examination of slides of tissue, your examination should be less frustrating, more efficient, and more valuable.