Techniques Used to Study Neurons at the Cellular Level
Techniques Used to Study Neurons at the Cellular Level
Objective 2: Describe some of the microscopic techniques used to examine nerve cells.
Light cannot penetrate more than a few thousands of a millimeter into most tissues. For this reason, light microscopy depends on the thin sectioning (slicing) of tissues. However, tissue is also too soft to slice very thin without some sort of modification. Two methods of thin slicing became popular among scientists studying tissues (histologists).
The first of these was fixation in formaldehyde followed by removal of water and its replacement with organic solvents and then paraffin (the wax used in candles). Fixation is a necessary step for preserving tissues. It kills bacteria and fungi, and chemically modifies the proteins in a tissue so that they are not dissolved by the organic solvents. Paraffin wax is not soluble in water, so the water must be removed by a series of increasing concentrations of alcohol, then replacement of the alcohol with xylene or another organic solvent that can be mixed with paraffin so that the paraffin molecules can penetrate into the tissue. Once this embedding step is completed, the tissue can be sliced very thin (typically, about 1/250 of a millimeter, or 3-5 μm) using an instrument called a microtome (Latin, “small cutter“).
However, it quickly became apparent that by solving one technical problem, histologists created another. Now light could penetrate the tissue, but in general, the constituents of cells were clear and so there was nothing to see. One solution is to alter the properties of light to visualize tissues, but that had yet to be developed. The other is to stain the tissue with dyes which revealed specific cell components.
In the 19th century, chemists began to study the properties of coal tar, a toxic sludge which was being produced in large quantities as a waste product of burning coal. We now know it’s a complex mixture of carbon ring structures. When chemists reacted coal tar with nitrogen, colorful dyes (aniline dyes) were produced which were immediately popular as fabric dyes in the fashion industry. These dyes were also popular with German histologists of the late 19th century. These dyes are still in use today to stain tissues that the pathologist will examine under the light microscope. The limit of resolution (smallest thing one can see) of the light microscope is an unchangeable property of the wavelength of light. The limit of resolution of light microscopy is about 1 μm, but as most cell bodies are about 10 times larger than that, histologists could at least see the nuclei and some of the larger parts of cells.
Neurons were still a problem, because the dendrites and especially the axon were often too small to see — for example, the axons carrying pain and temperature information are about 0.1 μm in diameter.
The Nissl Stain
Franz Nissl used one of these aniline dyes, cresyl violet, to stain neurons. The chemical constituents of cells were not known