BASIC LIGHT MICROSCOPY
The following exercises are provided for guided study and self-evaluation in the histology laboratory. Your laboratory goal should be to develop a comfortable familiarity with the human body as a structure built from cells organized into four basic tissues.
Effective "seeing" often requires knowledge beyond that directly available to your eyes. Any attempt to look at human histology slides with the microscope should be accompanied by textbook reading and by examination of labelled micrographs (including electron micrographs) in various histology atlases.
The electron micrographs, especially, will extend your vision beyond what your own instrument can resolve. This additional resolution can often show why, in terms of particular functional specialization, a cell has the appearance it does. The atlas by Rhodin is recommended as an excellent source for numerous electron micrographs of various tissues and organs, with each illustrated at a wide range of magnifications.
If you are unfamiliar with cells and tissues, you should try to answer all the questions posed in the exercises below. In completing the exercises, you are expected to use effectively all the resources available to you, including not only your microscope and reference slide set, but also textbooks and atlases, your fellow students, your faculty, and this website. The multiviewer microscopes available in Room 206 are especially valuable for group examination and study. Remember, look beyond the obvious. Question yourself. Why do tissues look and act the way they do? What invisible structures and processes are implied by those features which you do see? How does the microscopic view fit into the economy of the whole body?
Be aware that tissue sections (slices) are quite thin, commonly less than 10µm (micrometers, microns). Since the diameter of many cells is greater than this, cells often appear in partial section (i.e., cytoplasm without a nucleus, or only a small portion of the nucleus).
Any three dimensional object, such as a cell or a blood vessel, may display many different appearances when sliced, depending on where and at what angle it is cut. Think carefully (and use diagrammatic sketches) to interpret such variations.
Most structures seen under the microscope fit into one of several simple categories (surfaces, tubes, strings, lumps, or extensive solid masses). Surfaces, in turn, may have wrinkles, evaginations (protrusions, bumps, fingers) or invaginations (dents, pits, crypts). Tubes or strings may be straight or curved or tangled, simple or branched. Solid masses may be similar in any direction (isotropic) or their appearance may vary depending on the angle at which they are cut (anisotropic).
The shape-category of a structure is a significant clue to the structure's identity. For example, most surfaces are epithelial (unless they are artifacts of cutting). Most tubes are either blood vessels or exocrine ducts.
Be alert to the ways in which one shape-category may masquerade as another. For example, a tangential slice across the edge of a curved tube or surface can resemble a solid lump.
Here is a list of some commonly encountered patterns, with examples.
- a smooth surface (example -- the surface of the epidermis)
- a surface with wrinkles, indentations or invaginations (example -- the interface between epidermis and dermis, or the surface of the tongue)
- a straight, curved or branching tubule (example -- a duct or blood vessel)
- a twisted or coiled tubule (example -- a sweat gland)
- a solid lump (example -- a sebaceous gland)
- a cord or string (example -- a nerve)
- fibers arranged in parallel (example -- tendons, some muscles)
- fibers arranged in a meshwork (example -- collagen in dermis)
But note that many shapes can mimic one another if sectioned appropriately, so judicious interpretation is necessary for effective "seeing".
Your own slides will hand you many puzzles. Always be aware that apparently simple structures can assume several very different appearances in different planes of section.
- Islands of connective tissue can appear isolated "within" an epithelium if an irregular interface with finger-like evaginations is cut at an angle.
- Any elongated structure (nerve, muscle fiber, vessel) will appear very different in cross section and longitudinal section.
- When the epithelium of a hollow structure is cut obliquely or tangentially, it can look like a thick mass of cells or solid lump.
Most illustrations in textbooks and lectures are chosen to minimize the difficulty of interpretation of three-dimensional structure. But when "odd" planes of section are correctly interpreted, they often reveal details of cell shape or orientation that are not readily seen in "ideal" cross sections. With some careful effort you can see fascinating features on your own slides that are not illustrated in even the best atlases.
BASIC LIGHT MICROSCOPY
1. Plug in and turn on your microscope. Place a slide on the stage. Focus. Adjust eyepieces for comfortable binocular vision. Adjust the substage condenser for Kohler illumination, for optimal contrast and resolution of the image. [More]
2. Choose a slide from your reference slide box, such as skin or trachea.
3. Identify the following features: cells, cytoplasm, nuclei.
4. For one particular cell:
a. Estimate the cell's size (diameter) in micrometers. (How do I do this?)
b. Describe the cell's shape, its staining properties, and the texture of its cytoplasm. Try to use standard histological terminology (e.g., squamous, cuboidal, acidophilic, basophilic).
c. Estimate the size of the cell's nucleus, and describe the nucleus' shape, its position within the cell, and the texture of the chromatin in the nucleus. Can you find nucleoli?
5. Repeat for a cell of a different type. How many different types of cells can you distinguish on this slide? In what ways do they differ?
6. Try to find extracellular material. The most common extracellular fiber is collagen. What color is collagen on your slide? What is the natural (unstained) color of collagen? Ground substance and extracellular fluid is usually washed away during slide preparation. How do you recognize where this material used to be?
7. Identify basic tissue types. Can you distinguish epithelium, connective tissue, nerve and muscle? In what ways do these tissues differ in the appearance of their cells?
8. Find blood vessels. Blood vessels are tubular structures. On slides they often (but not always) contain red blood cells. How big is a red blood cell? Can you recognize different cell types (endothelium and smooth muscle) in the wall of a blood vessel? How big is a terminal arteriole (the smallest arterial vessel which includes smooth muscle in its wall)?
WebPath provides a number of annotated electron micrographs for examination.
Choose an electron micrograph for examination. You should be able to find many examples in any cell biology/histology text or atlas of your choice. Especially recommended is the atlas by Rhodin (out-of-print, multiple copies available in the MRC), which includes very many quality EMs at many different magnifications from many different tissues and organs.
Checklist for review of cells.
1. What is the magnification, at least approximately? If this is a textbook picture, try to deduce the magnification without reference to the caption. Your best clues for determining magnification are familiar structures like cell nuclei, mitochondria, collagen filaments, ribosomes. What are the normal size ranges for each of these structures?
2. Distinguish cells from extracellular space. What do cell membranes look like? Locate and follow a cell membrane. How do you distinguish inside (cytoplasm) from outside (tissue fluid and extracellular fibers)? Certain easily recognized structures can facilitate this task. Nuclei, mitochondria, rER, etc., are found only within cells. Collagen is a good marker for extracellular space, as is any clear area, devoid of texture. What is the EM appearance of collagen?
3. How many cells, or parts of cells are visible in the micrograph? (Hint: How many distinct nuclei are visible? Confirm by finding membranes between cells.)
4. What evidence can you see for cellular specialization? Are any organelles present in above-average concentration? Are any organelles not visible at all? Are any organelles localized in particular regions of the cell? Are there any specializations of cell surface? If your answer to any of these questions was yes, what cell functions appear to be enhanced?
5. Repeat with another electron micrograph. Repeat again. Try to become comfortable seeing all that these images reveal. Practice this exercise with micrographs at both low and high magnifications. Find the same cell types in your reference slides. As you look at these cells with your light microscope, try to "see" in your mind the cellular specialization illustrated in the electron micrographs.
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Last updated: 14 November 2003 / dgk