Living Cells and Dynamic Molecules Observed with the Polarized Light Microscope: the Legacy of Shinya Inoué.

In 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research at the Marine Biological Laboratory (MBL), which quickly became Inoué's scientific home. Primed by his Japanese mentor, Katsuma Dan, Inoué followed Dan's mantra to work with healthy, living cells, on a fundamental problem (mitosis), with a unique tool set that he refined for precise and quantitative observations (polarized light microscopy), and a fresh and brilliant mind that was unafraid of challenging current dogma. Building on this potent combination, Inoué contributed landmark observations and concepts in cell biology, including the notion that there are dynamic, fine structures inside living cells, in which molecular assemblies such as mitotic spindle fibers exist in delicate equilibrium with their molecular building blocks suspended in the cytoplasm. In the late 1970s and 1980s, Inoué and others at the MBL were instrumental in conceiving video microscopy, a groundbreaking technique which married light microscopy and electronic imaging, ushering in a revolution in how we know and what we know about living cells and the molecular mechanisms of life. Here, we recount some of Inoué's accomplishments and describe how his legacy has shaped current activities in polarized light imaging at the MBL.

Forgotten research from 19th century: science should not follow fashion.

The fine structure of cross-striated muscle and its changes during contraction were known already in considerable detail in the 19th century. This knowledge was the result of studying birefringence properties of muscle fibres under the polarization microscope, a method mainly established by Brücke (Denk Kais Akad Wiss Math Naturwiss Cl 15:69-84, 1858) in Vienna, Austria. The knowledge was seemingly forgotten in the first half of the 20th century before it was rediscovered in 1954. This rediscovery was essential for the formulation of the sliding filament theory which represents the commonly accepted concept of muscle contraction (A.F. Huxley and Niedergerke, Nature 173:971-973, 1954; H.E. Huxley and Hanson, Nature 173:973-976, 1954). The loss of knowledge was the result of prevailing views within the scientific community which could be attributed to "fashion": it was thought that the changes of cross-striations, which were observed under the microscope, were inconsequential for contraction since other types of movements like cell crawling and smooth muscle contraction were not associated with similar changes of the fine structure. The basis for this assumption was the view that all types of movements associated with life must be caused by the same mechanisms. Furthermore, it was assumed that the light microscopy was of little use, because the individual molecules that carry out life functions cannot be seen under the light microscope. This unfortunate episode of science history teaches us that the progress of science can severely be retarded by fashion.

Gamete competence assessment by polarizing optics in assisted reproduction.

BACKGROUND: The purpose of this study was first to give an overview of the historical development of polarization microscopy, second to describe the various applications of this technique in assisted reproduction techniques (ART) and third to discuss the potential benefit of polarization microscopy as a predictor for IVF success. METHODS: The history of polarization microscopy was undertaken by performing a backward search in the scientific literature using Google and internet sites of several Societies for Microscopy and Cell Biology. Studies of polarization microscopy in ART were identified by using a systematic literature search in PubMed and Scopus. RESULTS: A total of 62 articles were identified by the direct search and further relevant articles were found by screening the cited literature in these articles. The topics relevant for assisted reproduction were spindle and zona imaging in combination with IVF success, meiotic cell cycle progression, pharmaceutical studies and cryopreservation. A separate topic was the use of sperm birefringence in ART. CONCLUSIONS: The majority of studies are observational studies and were not performed in a randomized manner and there is no direct comparison of techniques using other gamete selection markers. Despite this, most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis. Whether certain applications such as spindle or zona imaging may lead to an increase in IVF success is unclear at present. Publications on the use of polarization microscopy on sperm are still very limited.

George Romhányi (1905-1991): pioneer in amyloid research and polarization microscopy, on the 100th anniversary of his birth.

George Romhányi was an outstanding pathologist, university teacher and a renowned scientist of the 20th century. After studying medicine and pathology, his scientific interest focused on haemochromogenic reactions and submicroscopic structure research. He was among the first to describe the fibrillar-micellar structure of amyloid, as well as the helical-fibrillar structure of elastic fibres. Between 1951 and 1976 Romhányi held the chair of pathology at the University of Pécs. During this time he published numerous topo-optical reactions leading to a renaissance of polarization microscopy. Romhányi's topo-optical reactions possess a high molecular specificity and enabled submicroscopic structural analysis. He and his disciples analysed the biomembrane, collagen fibres, the extracellular matrix of connective tissues, cell walls of bacteria and fungi, the ergastoplasmatic membrane, RNA and DNA. Romhányi's investigation of a range of amyloid depositions was a milestone and led to intensive immuno-biologic research in this area. He firmly held the opinion that polarization microscopy is, along with the electron and fluorescent microscopy, indispensable for solving fundamental questions of molecular biology.

A tribute to Shinya Inoue and innovation in light microscopy.

The 2003 International Prize for Biology was awarded to Shinya Inoue for his pioneering work in visualizing dynamic processes within living cells using the light microscope. He and his scientific descendants are now pushing light microscopy even further by developing new techniques such as imaging single molecules, visualizing processes in living animals, and correlating results from light and electron microscopy.

Dr. McCrone's teaching methods in forensic microscopy, their nature, history, and durability.

The microscopy teaching activities of Walter C. McCrone started long before the McCrone Research Institute (McRI) was incorporated as a not-for-profit research institute in Chicago. McCrone obtained his first microscopy training at Cornell University, with Emile Monnin Chamot, and was shortly thereafter appointed a full instructor in chemical microscopy before obtaining his Ph.D. (in 1941). After leaving Cornell, he had classes at the Armour Research Foundation (now Illinois Institute of Technology Research Institute--IITRI) from 1942-1956 and founded McRI in 1960. The course and student totals from McCrone's educational activities are impressive. As of January, 1, 2002, the cumulative for McRI (1942-2002) is 2,130 courses for 22,557 students. There has been an average of 600 students in an average of 60 classes for the last several years. Nearly all of the courses contain one week of intensive hands-on microscopy training with usually only one instructor for the entire duration of the class, making it a unique teaching experience for both student and Instructor. Thousands of students have successfully completed at least one of McCrone's specialized forensic microscopy (trace evidence) courses and the number will steadily increase as a result of McRI's continued efforts to interest forensic investigators in microscopy.

Dr. Walter C. McCrone's contribution to the characterization and identification of explosives.

Dr. McCrone was an amazing individual, possessing many talents and having many interests. He especially loved applying polarized light microscopy (PLM) to answering the question-at-hand and solving problems. He applied PLM to many different fields including the identification of air pollution particles, asbestos identification, art conservation, pharmaceuticals, industry problems and forensic sciences. A field that I believe he enjoyed the most was the characterization and identification of explosives. Throughout his life he worked on, gave presentations and published articles on the characterization and identification of explosives. Also, he encouraged other scientists to give presentations and publish on the subject by providing "behind the scene" advice and/or be a co-author on a paper. He unselfishly taught others how to apply PLM and incorporate this invaluable tool into their analytical scheme.

Dr. Walter C. McCrone--his contributions to environmental microscopy.

This paper briefly highlights Dr. McCrone's contributions to the recently emerging field of forensic environmental microscopy. Few, if any, criminalists are not familiar with Dr. Walter C. McCrone's voluminous contributions to the field of forensic microscopy and the analyses of micro and ultra micro transfer (trace) evidence. Dr. McCrone was renowned for his life long efforts in promoting the application of the Polarized Light Microscope (PLM) to problem solving. It is therefore not surprising that Dr. McCrone would also apply his analytical and deductive skills employing the PLM to problems in environmental analysis. He is well known for his many publications dealing with the analysis of asbestos and asbestos like materials by PLM. His philosophy of presenting intense professional training courses stressing the practical applications of the PLM carried over to a series of courses offered to students requiring education in other areas of microscopical analysis. Through McCrone Research Institute, Dr. McCrone can be said to have been responsible for the training of a large majority of microscopists who literally analyzed tens of millions of samples. These analyses were performed utilizing methodologies developed predominately by him and adopted by regulatory agencies in the United States and abroad. The methods he fostered are a major part of the arsenal of microscopical techniques employed by forensic environmental microscopists in their efforts to identify a manufacturer of an insulation product for the purpose of litigation.