Sectioning part: I Theory

It’s bound to happen sooner or later, particularly in a classroom setting where the microscope is not a chosen pursuit, one will run out of things to look at. There’s only so many things that naturally exist in a form that’s suitable for observation with the compound light microscope. Newsprint, onion skin, insect wings, pollen, pond water, and blood, are enough to occupy the interested for a lifetime while others are sure to tire much sooner. Lucky for the instructor, most pupils can be trusted with a knife.

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Principles of Sectioning

Transmitted light microscopy is all about the specimens capacity to permit the passage of light from the illumination source through itself and onwards into the objective, ocular, and finally the eye. Some materials are able to permit the transmission of light, more or less, in their natural state, onion skin and Elodea leaves are great examples. Onion skin is comparatively easy to separate from the whole in a sheet one layer of cells thick and many types of Elodea form leaves that are only a single layer of cells. Other materials must be mechanically manipulated to form a suitable specimen. Insect exoskeletons may be macerated, pressed, and cleared. Blood may be smeared. Minerals may be ground. Much else in the natural world, most organic materials, may be thinly sliced.

The thinly slicing of materials as a practice is called sectioning and those materials after sectioning are called sections. An ideal section is thin and uniform with a thickness that is matched to the depth of field of the objective which is used to observe it. In practice a section is apt to be rather thicker than the depth of field but this is generally an acceptable defect provided that the section is uniform and as thin as possible. One may set themselves up for success in regards to uniformity by beginning in sectioning with small objects—a pine needle rather than a branch, the thin tip of a carrot rather than the thick root.

The thinner the section the better the resolution of the resulting visual image. When one focuses on the section the microscope may only focus on the materials that exist within the depth of field of the particular objective, all else exists outside the narrow range of the objective and obscures the image—one will not see the fuzzy and out of focus layers, they will merely lower the sharpness of the image.

Practice of Sectioning

In a professional setting specialized, and nowadays fully automated, devices take care of sectioning; a sample goes in one end as a complete object and comes out the other as a finished slide. Manual apparatus for the preparation of sections are called microtomes (micro for small and tome from the Greek through Latin and French for section) which hold the material and assist the operator in obtaining the thinest and most uniform sections possible.

Microtomes are more often than not used with specimens which have previously undergone a series of preparatory treatments. The object to be sectioned is first dissected from the whole into a manageable portion. It’s then dehydrated and fixed in alcohol to completely remove all moisture and stop all biological processes of the cells. From here the alcohol is displaced by a solvent of paraffin wax which is in turn displaced by the paraffin itself. The paraffin (or other medium) acts to enclose and infiltrate the specimen supporting both the internal and external structures of the object. Properly infiltrated specimens are preserved perfectly and may be stored indefinitely—tissue samples treated by this means have been used successfully in genetic paternity testing decades after preparation and centuries later in the case of infectious disease research!

With their cytoplasm replaced by a supportive and preservative media infiltrated specimens are placed into a microtome and sectioned with exceptionally sharp blades called microtome knives. Some microtomes make use of what’s called a chisel microtome knife which is in essence a large, wedge shaped, razor blade. In some types of microtome the blade is held in ones hand, or by the microtome and moved against the specimen. In other types of microtome the blade is stationary and the specimen is moved against the knife. The microtome knife may also take the form of a cut-throat razor (although the blades have a specific cross section that differs from a shaving razor) or even a disposable razor blade.

The individual wishing to section specimens at home need not obtain a complex and expensive microtome or exotic knife, a simple disposable razor blade and a steady hand is all that’s required. The specimen to be sectioned is held securely in place in one hand over a glass slip. A few drops of water are introduced to the slip and one then slices the specimen with a smooth and speedy motion of the razor. The water serves to maintain the integrity of the sectioned portions which would otherwise dry out in an instant and the quick motion of the blade contributes to minimal distortion.

The razors that work best for sectioning are the thin and precise type sold for use with a safety razor—look for them in the shaving aisle of the grocery. Because razors of that type have a blade on both long surfaces one is forced to hold them delicately and is not able to apply excess force with sectioning. Far from making the activity more dangerous this contributes to safety by ensuring that one is not sectioning tough and obstinate materials that will lead one to slip and slice a finger. After a few sections are made, a likely one is transferred in a drop of water to a second slip and a coverslip is introduced.

 

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