2019 Michigan Microscopy and Microanalysis Society Meeting

2019 Michigan Microscopy and Microanalysis Society Meeting

Rankin attended the 2019 Michigan Microscopy and Microanalysis Society (MMMS) Meeting held at Kellogg Hotel and Conference Center in East Lansing, MI on October 31, 2019.

Rankin’s tie-in to the MMMS meeting was by invitation from Victoria Kimler, PhD, CMA, the lab manager of the Ocular Structure and Imaging Lab at Oakland University’s Eye Research Institute and President of the Michigan Microscopy and Microanalysis Society. We got to know Dr. Kimler a few years ago, when she brought one of Oakland University’s ultramicrotomes in for repair.

At the tradeshow, and when not at my exhibit booth, I was able to attend a few of the presentations. I enjoyed the presentation of keynote speaker Sara E. Miller, PhD from the Department of Pathology at Duke University School of Medicine. Her topic was It’s a Small, Small World in which she spoke about tools, methods, and techniques for handling and processing miniscule specimens for EM examination.

To view pictures of the 2019 MMMS meeting go to http://michmicroscopy.org/2019_pictures.php

Dalen Agnew, DVM, from the College of Veterinary Medicine at Michigan State University also presented at the meeting. His topic was Hyrax Under Glass: Microscopy Tools to Solve Some Exotic Cases.

After the meeting, Dr. Agnew lead a tour of the MSU Veterinary Diagnostic Laboratory (VDP), a premiere, full-service, fully accredited lab for all species. The histology department, managed by Tom Wood, was impressively equipped with high-end laboratory equipment, with as much diagnostic capability as most human hospital labs. This tour was the highlight of my day.

 

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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

Microscopes

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.