Microscopes

Question: As a histologist, where would you be without your microscope?

Answer: Probably without a job.

The great grandfather of the modern light microscope is considered to be Anthony van Leeuwenhoek in 1674, a little more than 500 years ago. Even though Robert Hooke hand shaved thin slices of cork to view under a magnifying glass in 1665, and coined the word “cell”, Leeuwenhoek perfected the art of grinding and matching lenses to be able to visualize individual cells and bacteria in water droplets, which he called “animalcules”. Leeuwenhoek’s optical principles have stood the test of time, and have provided the basis for the light microscopes that we use today.

In fact, many of the stains we use today in special stains, including the H&E (hematoxylin and eosin) were developed in the “golden years” of histology stain development from 1858 to the early 1900’s. Single stain solutions were used in the late 1800’s. However, in the early 1900’s, histologists developed multi component stains (i.e. Masson (1929) and silver stains (i.e. Warthin-Starry (1920). To this day, purified hematoxylin for histology use is still obtained from the logwood tree Haematoxylum campechianum, located in the Yucatan peninsula of Central America. Many of you will remember the hematoxylin shortage of 2008-2009, when supplies from this area were disrupted. Efforts to develop a “synthetic” hematoxylin have been somewhat successful; but its use is currently limited.

Electron microscopy (EM) was developed by Ernst Ruska in 1933. While the procedure was (and still is) labor intensive, the resolution was such that individual nonliving objects could be investigated down to the atomic level. EM was used in pathology to help diagnose a number of diseases. However, while there are still a few diagnoses that require EM confirmation, the use of immunohistochemistry has replaced the electron microscope, for the most part.

During the 1970’s and 1980’s, immunohistochemical staining using fluorescent and chromogenic labels was developed. The standard light microscope could be used to visualize immunoperoxidase staining with DAB and other colored chromogens. Dark field microscopy using ultraviolet light was needed to see immunofluorescent staining of tissues.

Up to this point, microscopic images were obtained using light that reflects off the object. In 2000, researchers began the use of fluorescent proteins to tag cell parts which, when illuminated by a laser, radiate their own light. In that way, people could now view living cells under study. However, the resolution remained the same, equal to approximately 200 nanometers (nm). Resolution is the shortest distance that can be identified between two points.

In 2005 and 2006, “Super Resolution Optical Microscopy” (STORM) was developed by Xiaowei Zhuang. This technology allowed resolution down to 10-20 nm. Similar technology developed by Eric Betzig and Harald Hess, referred to as PALM (“Photoactivated Localization Microscopy”), allowed the composition of images below the diffraction limit.

This diffraction limit had previously prevented the viewing of cell membranes, mitochondria and bacteria. STORM and PALM employ the use of fluorescent molecules which emit colored light when exposed to certain wavelengths of excitation light. This allows the localization of individually identifiable proteins within the cell itself. Studies performed by Lippincott-Schwartz focused on the mechanism of how the cell membrane protrudes out from a cell, then pulls the cell behind it. This is how cancer cells metastasize into healthy tissue.

Ethan Gardner of Harvard University is using PALM and STORM to investigate the cell wall of the bacterium Bacillus. Some antibiotics work by preventing the growth of the cell wall. Bacteria that become antibiotic resistant are able to recover their ability to build the cell wall. The hope is that by studying the molecules involved in cell wall synthesis and repair, this may lead to the development of new and better antibiotics.

Clearly, the light microscope has evolved tremendously since 1665, when Robert Hooke observed thin slices of cork and coined the term “cell”. Biologists are now able to look into living cells to determine the mechanisms of life itself, with positive effects on human health care.

References

Massachusetts General Hospital, Protomag.com, Fall 2013, pp. 26-31.

Histologic. Recent Hematoxylin Shortage and Evaluation of Commercially

Available Substitutes. Ashley Groover, BS; Carlette Geddis, BS, HTL(ASCP);

Amanda Finney, BS. Vol. XLII No. 1, June 2009.

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