As histologists, you should be familiar with the light microscope. It is generally accepted that in 1674, Anthony van Leeuwenhoek invented the modern light microscope. Though Robert Hooke hand-shaved thin slices of cork to view under a magnifying glass in 1665 (hence the coining of the word “cell”), van Leeuwenhoek perfected the art of grinding and matching lenses. His lenses allowed the visualization of individual cells and bacteria in water droplets, which he called “animalcules”. Van Leeuwenhoek’s optical principles have stood the test of time, and have provided the basis for the light microscopes that we use today. Then, as now, microscopic images are obtained using light that reflects off the object.
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 has been used in pathology to help diagnose many diseases. While there are still diagnoses that require EM confirmation, the use of immunohistochemistry has replaced the electron microscope in histology, for the most part.
It was during the 1970’s and 1980’s that immunohistochemical staining using fluorescent and chromogenic labels was developed to visualize stained tissue with ultraviolet light from a dark field microscope. It is noteworthy that immunoperoxidase staining with DAB and other colored chromogens can be visualized with a standard light microscope.
In 2000, researchers began the use of fluorescent proteins to tag cell parts which, when illuminated by a laser, radiate their own light. Through this methodology people could view living cells under study. 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 brought resolution down to 10-20 nm. “Photoactivated Localization Microscopy” PALM, a similar technology developed by Eric Betzig and Harald Hess, improved the composition of images below the diffraction limit.
In 2015, Chen et al. developed a technique called “expansion microscopy” ExM, a type of superresolution microscopy. The scientists synthesized a swellable polymer network within the specimen under study that physically expanded the specimen, resulting in physical magnification. Specific labels were covalently bonded to the specimen, separated and optically resolved, resulting in expansion microscopy. This process is used to perform scalable superresolution microscopy, resulting in 70 nm lateral resolution.
The procedure of expansion microscopy, explained by Beniot Kornmann, “is to ‘inflate’ the specimen before imaging such that it becomes big enough for standard microscopy, instead of trying to image small objects. The procedure starts like a standard immuno-fluorescence, but before imaging the sample, it is infused with a resin. During polymerization, the fluorophore that is on the secondary antibody becomes covalently linked to the polymer. All proteins are digested away and the polymer is dilated to make an isotropic enlargement of the imprint. The imprint can then be imaged at superresolution using a standard microscope.”
Clearly, the light microscope has evolved tremendously since its discovery, to where biologists can now look into living cells to determine the mechanisms of life itself, vastly enhancing human health care.
REFERENCES:
Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill Livingstone, NY. Fourth edition. 1996
Theory and Practice of Histotechnology. DC Sheehan, BB Hrapchak. CV Mosby Company, St. Louis. First edition. 1980.
Luna L. AFIP. Manual of Histologic Staining Methods. Third Edition. McGraw-Hill. p39. 1968. As modified by CM Chapman
Dermatopathology Laboratory Techniques. CM Chapman, I Dimenstein.
Author’s note: Information taken from original research reported in Optical imaging. Expansion microscopy. will be in quotation marks.
Chen F1, Tillberg PW2, Boyden ES3.
Science. 2015 Jan 30;347(6221):543-8. doi: 10.1126/science.1260088. Epub 2015 Jan 15.
http://www.ncbi.nlm.nih.gov/pubmed/25592419
