1 An Introduction to Light Microscopy and the Cell
After completing this chapter, you should be able to:
- Name the parts and describe the use of the light microscope
- Describe other types of microscopy, specifically digital microscopy and electron microscopy
- Recognize major features of a tissue in light microscopy
Cell
? Nucleus
? Cytosol (cytoplasm)
Extracellular matrix
- Briefly describe the procedures used to prepare a specimen for visualization with the light microscope
Recognize the most common histologic stains, including the basic principle of the stain, the colors of the stain, the cellular components that are stained, and how variations in staining can provide clues about the structure and function of cells and tissues
? Hematoxylin and eosin (H and E)
? Periodic acid-Schiff (PAS)
Provide examples of how tissue preparation affects the appearance of a tissue and how those features may provide clues to the structure and function of the cells or tissues in that specimen
- Propose a histologic technique (type of microscopy and stain) that would be ideal for a specific study
1.1 Microscopy
1.1.1 The Light Microscope
Samples too small to see with the naked eye are visualized with the aid of a microscope. Although digital slide technology is slowly replacing the traditional microscope as the preferred method to study histologic tissues, understanding how to use a traditional microscope is still a useful skill and will help in analyzing both glass and digitized slides. Note that the following is a brief overview, and that individual microscopes may be slightly different.
A light microscope is designed to magnify the image of a tissue mounted on a glass slide (Fig. 1.1). Light generated by a light source (bulb) is focused with condensers, passes through the tissue specimen mounted on a stage, and then passes through sets of lenses (the objectives and the eyepiece), which magnify the image.
Fig. 1.1 Typical light microscope.
In Fig. 1.1, the head (blue box) is oriented away from the user for storage. Loosening the head locking screw at the base of the head allows it to rotate. It is best to rotate the head 180° from its position in this image to use the microscope, and then rotate it back for storage.
A glass slide holding the specimen is placed on the stage, where it is held in place by a specimen holder or clip. The X-Y translation mechanism moves the stage forward and back or from side to side to allow the user to view different portions of the slide.
Helpful Hint
The image seen through the microscope is upside down and reversed left to right. This makes the image seen through the lenses move in the opposite direction from the actual movement of the specimen.
A bulb in the base of the microscope is the source of light. There is an on-off switch and an illumination intensity control to adjust the brightness.
A condenser focuses the light from the light source before it passes through the specimen. There is a condenser adjustment knob that can raise or lower the condenser. For most slides, the proper position of the condenser is 1 mm below the slide.
Helpful Hint
There will be some instances in which it is helpful to lower the condenser. For example, elastic fibers are more easily visualized with the condenser lowered.
After light passes through the specimen on the slide, the image is magnified by two objectives. There are usually three or four lower objectives on a revolving nosepiece, or turret, which can be rotated to select the desired magnification. The upper objective is the eyepiece (see below). The magnification stated results from the combination of the upper and lower objectives. For convenience, and to avoid damaging the objectives and slides, always start with the objective with the lowest magnification (usually 4X), focus the image, and then progress to higher magnifications (typically 10X, 40X, and 100X oil).
Helpful Hint
Most microscopes are parfocal, which means that, as objectives are changed, the image remains in focus (or at least close to it). This is quite helpful because the user can easily focus on a structure at 4X and then increase magnification, adjusting the focal plane as necessary. Higher-magnification lenses have a narrow focal plane; jumping straight to higher-magnification lenses without first progressing from lower-magnification lenses will make it difficult to find the focal plane.
The specimen is focused using knobs that raise or lower the stage. The coarse focus knob moves the stage more quickly and should be used only under low magnification. After an image is focused under low power, switch to higher magnifications and then use the fine focus knob.
Helpful Hint
The higher-magnification lenses have longer tubes on the turret and, therefore, sit closer to the glass slides. Using the coarse focus knob with high-magnification lenses has the risk of driving the lens through and breaking the slide or, worse, damaging the objective lens.
Finally, the eyepieces, or oculars, also magnify the image (usually 10X). It is beneficial to use both eyes when looking at a specimen; this will save a lot of headaches. One important adjustment to the eyepieces is the interpupillary distance, which can be adjusted on the microscope to match the user's eyes. Note that some microscopes have an interpupillary adjustment roller, while others allow free movement of the eyepieces.
Another important adjustment of the eyepieces ensures that both eyes see the image in focus. One of the eyepieces has a diopter adjustment ring. This changes the focal plane for that eyepiece. To make this adjustment, start by closing the eye corresponding to the adjustable eyepiece (the left eye on the microscope in Fig. 1.1) and adjust the focus for the other eye (the "fixed" eyepiece) using the coarse and fine focus knobs until the image is sharp through that eyepiece. Then close that eye and adjust the focus on the other eyepiece with the diopter adjustment ring until the image is sharp through that eye as well.
1.1.2 Digital Microscopy
Traditional microscopes have been used for centuries to look at samples on glass slides and are still used today by many practitioners and researchers. However, digital slide technology is becoming more common and is the format that many schools utilize to study histologic specimens.
To create digital slides, glass slides are scanned in a slide scanner. There are several formats for the resulting data, but ultimately digital slides can be viewed using any computer (Fig. 1.2). Mouse controls are fairly intuitive. For example, dragging and dropping is used to move around the slide. The mouse may have a scroll wheel that can be used (sometimes with the Ctrl key) to change magnification, or magnification can be selected from a menu (Fig. 1.2, circle). The inset in the upper right shows a scanning view of the slide. The box in the inset shows the user where the view is on the slide, and it changes size depending on magnification. As mentioned, each digital slide platform is different, and functionality varies based on operating system, type of mouse, and other features of the computer system. The best way to learn is by experimentation.
Fig. 1.2 Digital slide.
1.1.3 Traditional versus Digital Microscopy
When using a traditional light microscope, magnification is determined by multiplying the magnifications of the objective and ocular lenses. The magnification of the objective lens is selected by the user, while the eyepiece magnification is typically 10X. So, with the nosepiece set to the 40X objective, then the total magnification of the image is 400X (40 × 10).
When creating digital slides, only the objective lenses equivalent to the ones on a traditional microscope are used. Depending on the system, the digital slide view may include a zoom bar that shows the magnification of the image. The magnification can be changed by adjusting the cursor on the magnification bar or by zooming in and out using the mouse scroll wheel. Typically, the magnification bar goes only to 20X or 40X, so one would naturally think that the magnification of digital slides pales in comparison to traditional microscopes. However, remember that the traditional microscope image is projected through tiny eyepieces, while digital slides are projected on large computer screens at high resolution.
Helpful Hint
To approximate magnification on digital slides, users typically multiply the magnification in the zoom bar by 10. This isn't exact but close enough, and it compares quite well to what is seen in the traditional light microscope. In this publication, scale bars will be included in images as often as...
