Review
New developments in multiphoton microscopy

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Abstract

Multiphoton laser-scanning microscopy is still developing rapidly, both technologically and by broadening its range of application. Technical progress has been made in the optimization of fluorophores, in increasing the imaging depth of multiphoton microscopy, and in microscope miniaturization. These advances further facilitate the study of neuronal structure and signaling in living and even in behaving animals, in particular in combination with the expression of fluorescent proteins. In addition, nonlinear optical contrast mechanisms other than multiphoton excitation of fluorescence are being explored.

Introduction

Nonlinear optical microscopy (NLOM) techniques rely on nonlinear interactions of (pulsed) laser light with particular molecules in the biological sample, causing for example the emission of fluorescent light. Such molecules are either naturally present or have been introduced by genetic or other means. The most commonly used nonlinear process is two-photon excitation (2PE) of fluorescence, which involves the near-simultaneous absorption of two (usually near-infrared) photons, but other processes, such as three-photon excitation and second harmonic generation (SHG), have recently gained interest (reviewed in [1]).

Since its inception over a decade ago [2], two-photon-excited fluorescence microscopy (2PM) has developed into a powerful tool in biological research. The key advantage of 2PM over single-photon microscopy techniques is that it provides high-resolution images from deep within living tissue [3], which is particularly important for neuroscience. Two general features of nonlinear processes are crucial for the performance benefits of NLOM. First, because multiple low-energy photons act together, excitation is achieved using relatively long illumination wavelengths. Compared to microscopy techniques that employ one-photon excitation illumination, light therefore scatters less and can penetrate deeper into biological tissue. Second, because of the nonlinear dependence on illumination intensity, signal is generated only in the vicinity of the focal point. This results in intrinsic optical sectioning, reduces or eliminates out-of-focus effects, and permits efficient signal detection [4].

In combination with fluorescent indicators, 2PM has proven especially useful for the study of dendritic processing and synaptic physiology in various cell types in brain slices (reviewed in 5., 6.). The range of biological structures and processes that are investigated using 2PM is continually expanding. Because of the large depth penetration of 2PM, imaging of individual neuronal processes and their activity is possible in the intact brain [7]. This enables high-resolution optical experiments in living animals while preserving native physiological conditions as much as possible and leaving network structures intact. Particularly promising is the combination of 2PM with genetically encoded fluorescent proteins (FPs). Expression of FPs or FP-tagged proteins solves the problem of labeling cells deep in tissue, provides excellent specificity when labeling neuronal subpopulations, marks key signaling proteins (reviewed in [8]), and provides functional indicators of neural activity (reviewed in 9., 10.). Multiphoton microscopy is thus one key technology that enables studies of cellular and subcellular activity in the context of the intact brain [11]. In this review, we first give an overview of technological developments in multiphoton microscopy, followed by a summary of recent applications to brain slices and intact animals.

Section snippets

Technological developments

Although two-photon microscopy is a relatively mature technique by now and has been established in many laboratories, a continuous stream of technological developments is refining its technology. Recent work pushes further the limits of speed, resolution and, in particular, imaging depth of the technique. Fiber optics has been used to miniaturize two-photon microscopy, thus increasing its flexibility and mobility. Together with progress in dye development, these advances have extended the range

Applications of multiphoton microscopy

Besides being routinely used in brain slice preparations, 2PM is well suited for deep measurements in various other intact tissues and in particular for imaging in living animals.

Conclusions

NLOM (mostly in the form of two-photon microscopy) has found widespread applications in biological research, providing high spatial resolution deep within tissue and covering a wide range of time scales reaching from milliseconds to months. These features make NLOM an excellent tool for studying dynamic processes in the brain, including biochemical signaling, development, and structural reorganization. Ongoing technological developments not only continue to improve existing microscopy tools but

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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