Multiphoton microscopy: a personal historical review, with some future predictions.

The historical development of multiphoton microscopy is described, starting with a review of two-photon absorption, and including two- and three-photon fluorescence microscopies, and second- and third-harmonic generation microscopies. The effects of pulse length on signal strength and breakdown are considered. Different contrast mechanisms, including use of nanoparticles, are discussed. Two new promising techniques that can be applied to multiphoton microscopy are described.

Maria Goeppert-Mayer (1906­1972): two-photon effect on dermatology.

Maria Goeppert-Mayer (1906­1972) created the theoretic basis for investigations using the double-photon effect. She was also involved in work on the Manhattan Project for the development of the atomic bomb. In 1963, she received the Nobel Prize in physics for her discoveries concerning nuclear shell structure. Her theoretic results on double-photon absorption are directly used today in dermatology in nonlinear microscopy, multiphoton tomography, and photodynamic therapy.

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.

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.