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Multiphoton Fluorescence Microscopy

Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with highly specific fluorophores.

Introduction to Multiphoton Microscopy - Multiphoton microscopy features attractive advantages over confocal microscopy for imaging living cells and tissues with three-dimensionally resolved fluorescence imaging. Two-photon excitation, which occurs only at the focal point of the microscope, minimizes the photobleaching and photodamage that are the ultimate limiting factors in imaging live cells. This advantage allows investigations on thick living tissue specimens that would not otherwise be possible with conventional imaging techniques.

Fundamentals and Applications in Multiphoton Microscopy - Two-photon excitation microscopy offers great utility for dynamic imaging of living cells in thick specimens, such as intact tissue. The technique makes possible many experiments in which conventional imaging cannot be performed, or would not provide the information desired, as discussed in this Nikon MicroscopyU review article. Relying on mode-locked (pulsed) laser illumination to produce sufficient photon density at the focal point, two-photon excitation occurs only in the focal plane. The benefit of localized excitation is that emission is restricted to the narrow focal region, providing sectioning ability without the use of a pinhole. Furthermore, the limited excitation region reduces phototoxicity because photodamage is largely confined to the focal volume.

Laser Fundamentals - Non-linear optical excitation, an essential element of multiphoton microscopy, requires specialized laser systems capable of pulsed operation. An understanding of the general pinciples behind continuous and pulsed laser action is necessary in order to fully appreciate many aspects of multiphoton illumination. This section discusses how basic laser systems operate through stimulated emission, and how they are designed to amplify this form of light to create intense and focused beams. Interactive tutorials explore many of the fundamental concepts in laser technology and demonstrate the properties of selected individual systems.

Laser Systems for Optical Microscopy - The lasers commonly employed in optical microscopy are high-intensity monochromatic light sources, which are useful as tools for a variety of techniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. In addition, lasers are also the most common light source for scanning confocal fluorescence microscopy, and have been utilized, although less frequently, in conventional widefield fluorescence investigations.

Interactive Java Tutorials

Multiphoton Jablonski Diagram - Two-photon and three-photon excitation occurs as the result of simultaneous fluorophore absorption by either two or three photons in a single quantitized event. At high photon densities, the photons can be simultaneously absorbed (mediated by a virtual state) by combining their energies to provoke the electronic transition of a fluorophore to the excited state. This tutorial explores how fluorescence excitation events occur in multiphoton microscopy utilizing the classical Jablonski diagram.

Excitation Region Events - In multiphoton fluorescence microscopy, excitation of fluorophores is localized to a narrow region (approximately a micron thick at high numerical aperture) encompassing the microscope focal point, thus eliminating background fluorescence and out-of-focus flare that typically limits the effective sensitivity in confocal microscopy. This interactive tutorial examines events occurring in the microscope focal region during specimen excitation using long wavelength visible and near infrared laser illumination.

Excitation Photobleaching Patterns - Multiphoton fluorescence microscopy utilizes diffraction-limited focusing by a high numerical aperture objective to localize the spatial concentration of excitation light to narrow region near the focal point. This tutorial compares excitation-induced photobleaching patterns that occur near the focal region in both multiphoton and confocal microscopy systems.

Ti:Sapphire Mode-Locked Lasers - The self mode-locked Ti:sapphire pulsed laser is currently one of the preferred laser excitation sources in a majority of multiphoton fluorescence microscopy investigations. This tutorial explores the operation of Ti:sapphire lasers over a broad range of near-infrared wavelengths with variable pulse widths and an adjustable applet speed control.

Nd:YLF Mode-Locked Pulsed Lasers - An increasing number of applications, including new illumination techniques in multiphoton fluorescence optical microscopy, require a reliable high average-power laser source that enables efficient frequency conversion to ultraviolet and visible wavelengths. Several variants of the diode-pumped solid state laser have been developed, and of these, the Nd:YLF (neodymium: yttrium lithium fluoride) laser produces the highest pulse energy and average power in the repetition rate ranging from a single pulse up to approximately 6 kHz. This tutorial explores the operation of a Nd:YLF multi-pass slab laser side-pumped by two collimated diode-laser bars.

Literature References and Web Resources

Selected Literature References - A number of high-quality review articles on multiphoton fluorescence microscopy have been published by leading researchers in the field. This section contains periodical location information about these articles, as well as providing a listing of selected original research reports from this cutting-edge field of research.

ZEISS Campus Multiphoton Microscopy Reference Library - The application of nonlinear excitation techniques to the imaging of synthetic fluorophores and fluorescent proteins in biology and medicine has witnessed increasing attention over the past several years, primarily due to the introduction of turnkey pulsed laser systems coupled to advanced instrumentation. The references described in this section contain review articles and original research reports on multiphoton microscopy with emphasis on the theoretical background, microscope configuration, specimen preparation, deep tissue imaging, and numerous applications.

Multiphoton Resources on the Web - Optical microscopy is virtually the only means by which living cells and tissues can be studied with high spatial resolution. This has recently led to a return of light microscopy to the frontlines of biological research, with confocal and multiphoton fluorescence applications leading the way. Listed in this section are links to resources on the web for multiphoton fluorescence microscopy including microscope and laser manufacturers, university laboratories, industrial imaging laboratories, technical white papers and tutorials.

Contributing Authors

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37212.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

John C. Long, Brian O. Flynn, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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