Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures

Techniques based on two-photon excitation (TPE) allow three-dimensional (3D ) imaging in highly localized volumes, of the order of magnitude of a fract ion of a femtolitre up to single-molecule detection. In TPE microscopy a fu ndamental advantage over conventional widefield or con focal 3D fluorescenc e microscopy is given by the use of infrared (IR) instead of ultraviolet (U V) radiation to excite those fluorophores requiring UV excitation, hence ca using little damage to the specimen or to fluorescent molecules outside the volume of the TPE event and allowing a deeper penetration within the sampl e compared with conventional one-photon excitation of fluorescence. In our laboratory, within the framework of a national INFM project, we have realiz ed a TPE fluorescence microscope, part of a multipurpose architecture also including lifetime imaging and fluorescence correlation spectroscopy module s. The core of the architecture is a mode-locked Ti:sapphire infrared pulse d laser pumped by a high-power (5 W, 532 nm) solid-state laser and coupled to an ultracompact scanning head. For the source we have measured a pulse w idth from 65 to 95 fs as a function of wavelength (690-830 nm). The scannin g head allows conventional and two-photon confocal imaging. Point spread fu nction measurements are reported with examples of applications to the study of biological systems. (C) 2000 Elsevier Science S.A. All rights reserved.