Non-Linear Microscopy

Non-linear interactions between light and matter have been extensively used for spectroscopic analysis of biological, natural and synthetic samples [1–5]. In the 1990s, the development in laser technology allowed to apply these principles to the light microscopy field [6–8]. In this context multi-photon excitation (MPE) fluorescence microscopy and second harmonic generation (SHG) imaging are representative of the continuing growth of interest in optical microscopy. Although other modern imaging techniques like scanning near-field microscopy [9], scanning probe microscopy [10] or electron microscopy [11] provide higher spatial resolution, light microscopy techniques have unique characteristics for the three-dimensional (3D) investigation of biological structures in hydrated states, including the direct observation of living samples [12–14]. Multi-photon microscopy relies on the property of fluorescent molecules to simultaneously absorb two or more photons [15]. In this context, the advances in fluorescence labelling and the development of new fluorescent/luminescent probes like the so-called quantum dots [16] and the visible fluorescent proteins (VFPs), that can be expressed permanently bound to proteins of interest by genetically modified cells, allow the study of the complex and delicate relationships existing between structure and function in the four-dimensions (x-y-z-t) biological systems domain [17,18]. MPE shares with the confocal microscopy the intrinsical 3D exploration capability and provides some additional interesting features. First, MPE greatly reduces photo-interactions and permits to image living samples for long time periods. Second, it allows high sensitivity measurements due to the low background signal. Third, since most of the fluorescent molecules show a wide two-photon absorption spectrum, MPE allows simultaneous excitation of multiple fluorescent molecules with only one excitation wavelength, reducing the effects of chromatic abberations of the optical path. Fourth, two-photon microscopy can penetrate into thick and turbid media up to a depth of some 100 μm. Fifth, MPE can induce chemical rearrangement and photochemical reactions within a sub-femtoliter volume in solutions, culture cells and living tissues.