Introduction

For medical students, students of histotechnology, or other health care professionals, I want to stress the fact that this paper is written by an amateur microscopist for amateur microscopists. I welcome suggestions to improve this paper and I am thankful for all corrections the reader might have. But this is most certainly not an introduction into histology. With this paper, I want to wake up an interest in histology that does not require the lengthy study of this subject but will provide a reasonable starting block for future explorations in microscopic anatomy of the human tissue. What resources and tools can we use and where do we start? These are the main topics of this paper.

What Resources and Tools?

If we want to make our own human histology slides, we will be most likely limited to blood smears. In the USA, due to FDA regulations regarding the handling of biohazardous materials, it is not easily possible for private individuals to buy fresh organ sections (see http://www.fda.gov/). Furthermore, most of the stains used for histology slide preparation are not easily obtainable. Except for human blood smears, the amateur microscopist's study of human histology is therefore "limited" to already prepared, permanent slides. However, even having access to tissues and all equipment necessary, the preparation of good histology slides remains an art that is difficult to master for most of us. No wonder, it requires over two years to gather the expertise of a certified histotechnician. I have used the following two suppliers of histology slides:

1. Carolina Biological Supply Company, 2700 York Road, Burlington, NC 27215 (http://www.carolina.com)
2. Ward's Natural Science Establishment, POB 92912, Rochester, N.Y. 14642

Other resources about histology are books and information obtained from the World Wide Web. I frequently use the following books:

1. B. Young, J. W. Heath, Wheater's Functional Histology, 4th Edition, Churchill Livingstone, London (2001); and B. Young, Picture Tests in Histology, Churchill Livingstone, London (2001).
2. R. C. Curran and J. Crocker, Curran's Atlas of Histopathology, 4th Edition, Harvey Miller Publishers, London & Oxford University Press, Oxford (2000).
3. Erik Undritz, Hämatologische Tafeln, Sandoz AG Basel, 2nd Edition, Frobenius AG Basel, Switzerland (1972).
4. Maxwell M. Wintrobe et al., Clinical Hematology, 7th Edition, Lea & Febiger, Philadelphia (1975).

Where to Start?

The building blocks of life are cells. Cells form tissues, tissues form organs, organs form organ-systems, and organ-systems form an entire living and breathing organism (see the following table).

Atoms form simple molecules	Such as short peptides.
Simple molecules form macromolecules	Such as proteins (long, amino-acid polymers).
Macromolecules form membranes	Such as lipid bilayers.
Membranes form organelles	Parts of a eukaryotic cell, such as mitochondria.
Organelles form cells	These cells are eukaryotic cells. That's where we definitely know that life starts. We can study these cells inside a living organism (in vivo) or outside (in vitro), for instance in a cell culture.
Cells form tissues	Four basic tissue types exist: epithelial tissue, connective tissue (for example blood and bones), muscle tissue, and nervous tissue.
Tissues form organs	Organs are made out of different tissue types. Examples are liver, kidney, skin, heart, lung, brain.
Organs form organ-systems	Examples of systems are digestive system, cardio-vascular system, respiratory system.
Organ-systems form a complex living body	Such as humans.

Cells are structural units that make up plants, animals and single cell organisms. The cells of single cell organisms are called prokaryotic cells (prokaryotes). A prokaryotic cell does not have a membrane around its nuclear region (for example a bacterium). It has a cell wall, plasma membrane, nucleoid (region of DNA), and cytoplasm with ribosomes. Cells that make up plants and animals (including us!) are called eukaryotic cells (eukaryotes). This type of cell contains cell organelles. The parts that make up a eukaryotic cell are Golgi bodies (secretory systems), endoplasmic reticulum (transport system within the cell), nucleus, nucleolus, microtubule organizing centers (MTOC), mitochondria (the cell's powerhouses), ribosomes (small organelles that synthesize proteins), and a cell membrane. The term "eukaryote" comes from Greek and means "true nut".

Tissues are groups of cells that lie together to accomplish a common function. They are the basic building blocks of organs. Tissues are divided into four groups (epithelial tissue, connective tissue, muscle tissue, and nervous tissue). These groups are further subdivided into many subgroups. As an example, the epithelial tissue is subdivided into covering and lining epithelia (outer layer of skin, inner surface of heart and blood vessels, inner surface of respiratory cavities, etc.) and glandular epithelia (most of the glands in the body). One of the major tasks in histology is to clearly identify the various tissues when looking at a thin section. For instance, looking at a histology slide of healthy nonpigmented, human skin (slide number 31-4522 from histopathology of disease set 31-6986 [3]), we find at moderate magnification (objective 10x) many cells close to each other with little extracellular material between them. See Fig. 1. These epithelial tissue cells are arranged in a shape known as "stratified squamous". This kind of cell arrangement offers the most protection to the underlying tissue. These cells form the epithelium of our skin. As seen in Fig 1, the epidermal ridge, which is given by corrugation of the epithelium (indicated by a thick, blue line), is rather flat or less prominent, suggesting that this portion of skin is less subjected to high shearing forces. Since skin is an organ that consists of various tissues, it is no surprise that we find other tissues in Fig. 1, such as connective tissue. There is a "free surface", which is exposed. It is called the apical surface. The lower surface of the epithelium (called basal surface) rests on layers of non-living, adhesive material that has been secreted by the epithelium and the underlying connective tissue (called dermis). These layers form the basement membrane. This membrane can be thought of as a sticky layer to keep the epithelial cells attached to the tissue that underlies them. When going to a higher magnification (see Fig. 2), we clearly see the cell-nucleus of cells forming the epithelium. We can also see clearly the cornified layer that forms the top of the thin epidermis. But we cannot find any blood vessels within the epithelium layer. - A good histology slide contains a lot of information. With a good histology atlas, such as [5], we are able to gradually recognize more and more components. Suddenly, a "nice looking" histology slide is telling us an entire story - a story about ourselves.

To round up this small "crash-course", we should know how such wonderful slides are made and what contrast methods are used to help us to identify various tissues as well as to understand more about their condition. (I will neither talk about phase-contrast methods nor other purely optical contrast methods, but limit myself to staining methods to enhance contrast.)

I tried to "simplify" this rather lengthy process of making histology slides by drawing a flowchart. In Fig. 3, I illustrate the workflow for making histology slides with H&E staining and with a thickness of a few microns (3 to 10 microns usually). - Of course, there are many other special staining procedures available, which might require different dehydration and fixation steps. Two good web sites about staining techniques are 'StainsFile' (located at http://stainsfile.info/) and 'The Histology Page' (located at http://home.primus.com. au/royellis/histo.html). An example of a different famous staining technique is the Masson's trichrome technique. It is used to emphasize supporting tissue elements, principally collagen. Several modifications of Masson's trichrome are in use, such as the one used by a company called MICR-O-SLIDE. This company produces various slide sets for hobby microscopists. I got set number 59-5550 "Healthy Human Tissue II". Although the slides are informative, the sections are rather thick and the stains used are uncommon, which makes the interpretation of these slides quite difficult. However, there is a small booklet included in each of these sets. To illustrate this, I present 59-5550/10 from this collection called "Human kidney, t.s. of cortical zone". (The cortical zone is the outer zone of the kidney and "t.s." stands for tangential section.) See Fig. 4. Fig. 4a and 4b show an image of a section through a renal corpuscle. While in Fig. 4a I emphasize important parts that make up a renal corpuscle, in Fig. 4b I am investigating the kind of epithelial tissue that can be found inside a renal corpuscle.

Our Lung - Take a Deep Breath

When looking at a histology slide, we must keep in mind that we are most likely looking at various tissue types that form part of the organ under investigation, in this case the lung. Recognizing the tissues will be our first task. We should not attempt to understand a "fancy" feature that might just be an artifact of the process used to make this slide or a cut through a well-known portion at a somewhat unconventional angle. As I have already mentioned, I start by identifying components I am more familiar with, such as epithelial tissue. (BTW, I found the tutorial at http://www.pathguy.com/histo/000.htm very helpful.)

Let us look at a section of the healthy human lung. The slide is from [3] (slide number 31-5670). At lower magnification (objective 10x), we see a section of the respiratory alveoli (see Fig. 5a and 5b). Fig. 5a shows a very nice network of simple cubiodal epithelial tissue that covers the airspaces in the lung. Fig. 5b shows a beautiful pulmonary artery, which has a thin wall compared to its diameter. At a higher magnification (objective 40x), we can identify various different cell types. We can find two cell types that make up the epithelium, which provides a continuous lining to each alveolus. These are the large, elongated, squamous cells (alveolar lining cells or type I pneumocytes) and cells that are round in shape (type II pneumocytes) (see Fig. 6a). The diffusion of oxygen happens through the larger type I pneumocytes, while the type II cells secrete a surface-active material called surfactant, which reduces surface tension and avoids that the alveoli collapse during expiration. - We can also find alveolar macrophages. In Fig. 6b, we find a clearly visible blood vessel. This is a capillary, 7 to 10 micrometer in diameter, which contains deformed erythrocytes. The erythrocytes must deform to be able to squeeze through such tiny capillaries. I hope the reader can see the thin membrane that separates the blood cells from the actual airspace. Such a thin separation is very important to allow oxygen diffusion to take place easily.

Summary and Acknowledgements

I hope the reader enjoyed reading this simple introduction into a rather complex field. Hopefully this paper is not oversimplifying the challenges ahead but proves to be useful for the reader starting to learn about microscopic anatomy of the human tissue. I want to thank Drs. Fei Liu and Jerry Dowell for stimulating discussions.

Comments to the author, Gregor Overney, are welcomed.

References

[2] A. Potter and M. Smith, A Summary Study of Blood, Micscape Magazine, England, November 1995.

[3] Carolina Biological Supply Company, 2700 York Road, Burlington, NC 27215 (http://www.carolina.com).

[4] Deborah W. Vaughan, A Learning System in Histology, Oxford University Press, Oxford (2002).

[5] B. Young, J. W. Heath, Wheater's Functional Histology, 4th Edition, Churchill Livingstone, London (2001).

[6] Herbert Spencer, Pathology of the Lung (2 Volumes), 3rd Edition, Pergamon Press, Oxford (1977).

Figure Captions and Figures