Modern Laser Scanning Confocal Microscopy.

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.

A historical overview of the study and representation of uterine microvascular structures.

The concept of anatomical modelling of the internal vascular structures of organs dates back to the Middle Ages by way of corrosion casting. The first to apply this classic injection technique in the reproductive arena was John Hunter (1754), who undertook to establish the independence of the maternal and fetal circulations in the placenta. The first detailed microscopic study of the endometrial vessels was undertaken a century later. Endometrial inoculation studies in the 1930s with coloured fluids such as India ink have provided the basis of our current understanding of the complex sequence of morphological vascular changes which occur in the endometrial tissue leading up to and during the process of menstruation. Classic injection techniques were limited in that they were often associated with artefacts due to injection-induced vessel breakages and variability in size of the suspended particles in the injection material. Following this, the smallest blood vessels were better demonstrated using Gomori's alkaline phosphate method. An adaptation of this method in the early 1960s demonstrated the uterine vasculature in a more detailed way than ever before. In the early 1970s, novel microradiography studies involved the injection of warmed radio-opaque medium into both arterial and venous microvasculature of the human uterus. Early 1980s investigators also utilized corrosion casting of uterine microvessels combined with scanning electron microscopy. The last 20 years have seen the dawn of the computer age, immunohistochemistry, advanced microscopy (laser scanning confocal and multiphoton emission), and stereological methods to obtain quantitative measurements of 3-dimensional endometrial vascular structures. This review article contains a historical overview of uterine microanatomical vascular visualisation from the early beginnings to the latest computerised techniques.

David Maurice's contributions to optical ophthalmic instrumentation: roots of the scanning slit clinical confocal microscope.

This paper explores the seminal contributions of David Maurice to the field of ophthalmic instrumentation. His development of the specular microscope, the scanning slit optical confocal microscope, and the corneal microfluorometer resulted in advances in our understanding of corneal morphology, physiology, and pathology. The development of the scanning slit, clinical confocal microscope is not a new paradigm or a paradigm shift, but a continuous series of interlinked technical advances from the early work of Vogt to Thaer's development of a clinical confocal microscope. For each instrument both the connection to the prior work of others and the unique advances are discussed and contrasted. This paper develops the connections and parallel developments in the instrument developments of Goldmann, Maurice, Svishchev, Baer, Koester, Masters, and Thaer. The evidence in support of the thesis consists of published papers, patents, personal communication, and study of Goldmann's book collection in Bern. A second theme is that knowledge of physics is a prerequisite for optical instrument development in ophthalmology. David Maurice had a university degree in physics and Hans Goldmann learned physics from his books. The contributions of David Maurice to optical instrumentation follow the major contributions of Goldmann and facilitated and stimulated other scientists who acknowledged their important intellectual debt to David Maurice.

Antecedents of two-photon excitation laser scanning microscopy.

In 1931, Maria Göppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria Göppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.

Notes on theory and experimental conditions behind two-photon excitation microscopy.

This report deals with the fundamental quantum physics behind two-photon excitation also providing a link to the experimental consequences exploited in microscopy. The optical sectioning effect is demonstrated as well as the distribution of excitation and of fluorescence emission.

How the confocal laser scanning microscope entered biological research.

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.