Multiphoton Fluorescence Light Microscopy

Abstract Multiphoton fluorescence microscopy is a powerful imaging technique that depends on complex quantum mechanical interactions between photons and matter for fluorophore excitation. In conventional fluorescence microscopy, a fluorescent molecule is pumped to an excited state by absorbing a single photon. The molecule subsequently falls back to its ground state by emitting a less energetic photon. This is a linear process of absorbing and emitting energy in the form of single photons. By contrast, multiphoton microscopy is based on nonlinear interactions between light and matter, whereby multiple photons are absorbed to bring single fluorophore molecules to an excited state. Two‐photon fluorescence microscopy is the most commonly used multiphoton imaging technique. In two‐photon microscopy, the fluorescent molecule absorbs two photons simultaneously in a single event, and their combined energies provoke the electronic transition of the molecule to the excited state. Advantages of two‐photon fluorescence, compared to typical single‐photon epifluorescence microscopy, include reduced autofluorescence, deeper tissue penetration, inherent confocality and three‐dimensional (3D) imaging as well as minimised photobleaching and photodamage. Thus, two‐photon microscopy facilitates optical sectioning of thick biological specimens in vivo , which would not be possible with conventional imaging techniques. Recent advances in fluorescence microscopy have expanded the application spectrum and usability of multiphoton imaging, which has become an important and versatile tool in modern biomedical research. Key Concepts Multiphoton microscopy is based on nonlinear interactions between light and matter. Two‐photon excitation transpires only at the objective lens focal volume, where photon density is high enough to generate sufficient absorption events. Multiphoton microscopy requires the use of ultrafast femtosecond, pulsed laser light sources to achieve appropriate fluorophore excitation conditions at the focal point. Because of sharply focused excitation in two‐photon microscopy, photodamage is reduced and viability of the biological specimen is increased. Multiphoton microscopy uses photons with near infrared wavelengths that are poorly absorbed and less scattered by biological material, allowing deeper light penetration into specimens. As a result of using photons of longer wavelength for excitation, two‐photon fluorescence microscopy is limited to slightly lower resolution, compared with single‐photon confocal microscopy. In two‐photon excitation, photodamage and thermal damage are considerably reduced and become significant only at the focal point. Photoconvertible fluorophores have been developed for multiphoton microscopy applications that allow localised photochemical reactions in a biological sample. Conventional fluorophores exhibit distinct two‐photon absorption spectra, which are not directly related to their single‐photon excitation properties. Two‐photon microscopy is suitable for in vivo imaging and monitoring of neuronal function in freely moving and behaving animals.