Mayukh Goswami Presents Human Physiology identifications in Histology.
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The objectives of this lab include:
1. Familiarization with the structure, function and use of the compound light microscope.
2. Familiarization with different images of tissues, and identification of key structural elements (anatomy) that determine their function (physiology).
3. Instructions on how to complete your lab report.
Part I. The Microscope
In your previous courses you have had some experience with gross anatomy, the study of the structure of the body at the macroscopic level. Today, you will explore histology, the microscopic study of tissues, by viewing non-living, chemically treated specimens with a light microscope. It is my hope that through the study of the structure of the various tissues you will gain a better understanding of their function(s). Please consult this web page and the textbooks available to you in order to identify the key characteristics of each tissue type.
Although there are at least 200 distinct cell types, cells may be grouped into one of four major categories (tissues): nerve cells, muscle cells, epithelial cells, and connective tissue cells. The same classification system is followed here. The slides included in this website and in your tray represent a variety of techniques used to study tissue structure and morphology. These techniques include light microscopy (most slides), electron microscopy, and a variety of staining techniques. These staining techniques are necessary in order to make the important structures of the cell stand out, since they are almost invisible when untreated.
Your instructions will be given to you during lab. When using the microscope please proceed very carefully, as microscopes are costly! Carry the microscope using both hands (one on the neck and one under the base), and take special care when positioning or removing slides, and when adjusting the focus knob. It would be a great idea for you to spend some time at the beginning of the lab familiarizing yourselves with the functions of the various knobs (coarse focus, fine focus, condenser lens adjustment, mechanical stage movement), the iris diaphragm, and the lenses (ocular, condenser).
Take the time to learn how to use the appropriate light intensity to maximize clarity of image. Make sure you always start by using the low-power objectives, and work your way up. Most microscopes are parfocal, which means that once you focus a slide with a low-powered objective, it will remain focused at higher magnifications also. Please handle the slides very gently, and lower and raise the lenses slowly, to minimize the chance of breaking a slide or damaging the lens. Also, keep the slides in the box you are sharing in order, to make it easier for everyone to keep track of them. If you have doubts about what you are doing, please do not hesitate to ask questions. I ask you to refer to the various textbooks available to you in the lab for more information on the structure and function of the microscopes.
The images below should help you understand and appreciate the various adjustments necessary to see an image as clearly as possible.
Effect of brightness
Effect of contrast
Effect of focus
Effect of resolution
The ocular lens typically provides a magnification of 10X. The condenser lens can be one of three types: low-power lens, high-power lens, and oil-immersion lens, each with different magnifying and resolution capabilities. The ability to resolve images (or tell the structures apart) is determined by the amount of light that enters the microscope. You will probably find that the higher the magnification the lower light enters through the iris, thus you may need to increase the intensity of the light beam when using high-power lenses. Furthermore, you will find that your ability to determine relative depth is greater at lower magnifications. If the images are not clear, the lens(es) may need to be cleaned. Please consult with your instructor for instructions.
It is also possible to estimate the dimension of a particular cell or structure within a cell by understanding the concept behind microscopy. It is easy to understand that if the ocular lens has a magnification of 10X and the objective lens has a magnification factor of 10X, then the total magnification is 100X. Assuming the iris diaphragm is completely open, the diameter of the visual field should be approximately 1600 micrometers (mm). On the other hand, the diameter of the visual field under 450X magnification is approximately 356 micrometers (mm). This should help you estimate the size of objects as they appear in your field of vision.
The first part of this lab has to do with familiarizing yourselves with the operation of the microscope. In your tray you should find some slides with printed letters on them. Place a slide on the stage, and secure with the mechanical lever or spring clip. The slide should be oriented so that you can read the letters. Make sure one of the letters is in the path of the light beam as it crosses the stage. Adjust the various settings so the image comes to focus using the lowest power objective. Then perform the following activities, and try to answer the questions below:
1. What is the orientation (left/right, top/bottom) of the letter as it appears through the eyepiece? What is the magnification?
2. Move the stage slowly towards you. Does the image also move towards you? Repeat by moving the image to the left or right. What happened?
3. Increase the magnification, and make certain the image is still in focus. Does the appearance of the letter change? Is the image more clear or more difficult to read? Is the visual field larger or smaller?
4. Repeat the above procedures with at least one more letter. Does the size of the letter alter its appearance under the microscope?
In the second part of this lab we will examine depth of a specimen while using the microscope. Please select the slide with the two threads from your slide tray. Focusing at low magnification locate the point where the two threads cross each other. Then change the iris diaphragm in order to maximize contrast and manipulate the coarse focus so that the images are out of focus. Then use the fine focus to slowly bring the images into focus again. Notice which thread comes into clear focus first. In your preparation, which thread was at the bottom?
After you examine the prepared slide you may wish to create a simple experiment of your own: pull a hair from your scalp, cut a small (less that one cm in length) piece and place it on a slide with a coverslip. Examine the hair under the microscope.
In the third part of the lab we will examine the appearance of the various tissues under the light microscope. It is hoped that by understanding the structural components of the cells, you will be better prepared to understand the physiological processes involved. The resources available to you include the labels of the slides themselves, the textbooks located at the instructor's desk, the images in the PhysioEx 4.0 CD-ROM, and the images on this web site. I fully expect you to be familiar with these structures as we discuss material in subsequent chapters of the book.
1. Cardiac Muscle (longitudinal view)
Cardiac muscle has a striated appearance, just like skeletal muscle. More on the origin and importance of the striations will be discussed in chapters 8 and 9. The main characteristic of cardiac muscle is the appearance of the intercalated disks (dark lines between fibers). They allow for the end-to-end connection between cardiac muscle fibers, and signal communication between these fibers (I will ask you to remember this when we discuss cell-to-cell communication in chapter 3).
2. Skeletal Muscle (E.M. - longitudinal view)
This is a longitudinal view (along the length of the muscle) of skeletal muscle using electron microscopy (E.M.), a technique very different from the compound light microscope we use in our class. The E.M. uses a beam of electrons as the illumination source. The degree of detail is usually much greater, and the magnifications are significantly higher. The dark stripes define the ends of each sarcomere (the smallest functional unit of the muscle - see chapter 8 for more details). One can also identify transverse tubules and mitochondria.
3. Skeletal Muscle (E.M. - longitudinal section)
These are higher magnifications of skeletal muscle, again using E.M. . You can clearly identify the myofilaments (parallel lines within each sarcomere - see chapter 8 for more details). The dark dots (granules) are glycogen, used for energy production within the cell. You can also see some mitochondria and connective tissue surrounding each muscle fiber.
4. Skeletal Muscle (longitudinal section)
This is yet another longitudinal view of skeletal muscle, this time using light microscopy. This sample had been prepared with a different stain that only points out the striations and the nuclei. You can see the parallel arrangement of the muscle fibers.
5. Muscle-tendon junction (longitudinal view)
This image contains a very complex site in the body: the connection between two different tissues, muscular and connective. This connection is essential so that the muscular system can carry out its function (movement). Every such juncture is always the weakest link in a system. In other words, healthy muscle and healthy tendon are always stronger that the muscle-tendon junction. Most muscle tears occur in the regions closest to a tendon. As you can see, the region contains several nuclei due to the constant need for repairs and tissue regeneration. Is this electron microscopy or light microscopy?
6. Muscle-tendon Junction (cross section)
A cross-section of the muscle-tendon junction. You can see that the connective tissue does not have obvious striations. A different preparation however, would reveal that the connective tissue is also fibrous, and its main component is collagen.
7. Connective Tissue
Elastin is a component of connective tissue, along with collagen, adipose cells, blood, cartilage, and bone. It is prevalent in large arteries and the lower respiratory system. Its presence allows for tendons, ligaments, and vessels to stretch and recover their original shape, much like a rubber band. The wavy appearance of elastin disappears when the connective tissue is stretched, similar to what happens with the collagen "crimp" seen below.
The images above illustrate the structure of collagen. The two images use different staining techniques to identify different aspects of the structure of collagen. The image on the left allows the identification of fibroblasts (dark spots) and the clear separation between collagen fibers. The image on the right emphasizes the collagen crimp (wavy appearance). Some fibroblasts can also bee seen (red spots). Note that it is not possible to distinguish the individual collagen fibers.
8. Categories of Epithelial Tissue (I)
The simple squamous epithelium is mainly used for diffusion, absorption, filtration and secretion, where exchange of materials across a barrier has to be quick and occasionally massive. The simple cuboidal epithelium usually lines kidneys, salivary glands, or the pancreas. The simple columnar epithelium lines the surface of the gastrointestinal tract, and it is designed to aid the process of digestion. Finally, the pseudo-stratified ciliated columnar epithelial cells owes its name to the random arrangement of the nuclei of the simple columnar cells.
9. Categories of Epithelial Tissue (II)
The epithelial tissues illustrated here are characteristic of areas where there is lots of "wear and tear", so the thickness of the epithelial wall protects its function as a barrier. Such cells can be found on the skin, the trachea, the mouth, etc. The above two images simply illustrate the different classifications of endothelial cells. Please remember that endothelial tissue lines all organs, hollow cavities, and skin.
10. Epithelial Tissue (E.M.)
This is an image of pseudostratified epithelium of the trachea. Note the cilia (hairlike extensions on the top surface of the tissue).
11. Mitochondrion (E.M.)
We will soon see this image in our lecture (compare this image with fig. 2-10, page 35 in your textbook). Please try and identify the inner and outer mitochondrial membrane, the matrix, and the cristae. What are these "tentacles" on the bottom part of the picture? What characteristic can help you identify them?
This is a cross-section of bone, one type of connective tissue. You should consult the textbooks in the desk near you to identify the key characteristics of bone.
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