Three-Dimensional Imaging of Sulfides in Silicate Rocks at Submicron Resolution with Multiphoton Microscopy Antoine Bénard, 1, * Sabine Palle, 2 Luc Serge Doucet, 1 and Dmitri A. Ionov 1 1 Université de Lyon & Université Jean Monnet, 23, rue Dr. Paul Michelon, 42023 Saint-Étienne & UMR 6524, CNRS, France 2 Centre de Microscopie Confocale Multiphotonique, 18, Rue Pr Benoît Lauras, 42000 Saint-Étienne, France Abstract: We report the first application of multiphoton microscopy ~MPM! to generate three-dimensional ~3D! images of natural minerals ~micron-sized sulfides! in thick ~;120 mm! rock sections. First, reflection mode ~RM! using confocal laser scanning microscopy ~CLSM!, combined with differential interference contrast ~DIC!, was tested on polished sections. Second, two-photon fluorescence ~TPF! and second harmonic signal ~SHG! images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences. Key words: 3D imaging, rock, multiphoton microscopy ~MPM!, microstructure, morphology, sulfide I NTRODUCTION Three-dimensional ~3D! studies of rock structures ~spatial distribution and shapes of fractures and minerals! and mineral morphology ~surface microtexture! remain a tech- nological challenge. Optical microscopy can be applied to geomaterial analysis to provide information on morpholog- ical properties, but it is usually limited to two-dimensional ~2D! observations. Scanning electron microscopy ~SEM! combined with focused ion beam can provide 3D structural and textural information on a micron scale but only on the surface of geomaterials. Transmission electron microscopy ~TEM! can be used for 3D imaging ~3D-TEM! only on a nanometer scale ~e.g., Ersen et al., 2007!, such that this technique is not suitable to obtain 3D morphological infor- mation on minerals. Furthermore, TEM requires electron- transparent specimens produced using destructive and time-consuming procedures to penetrate the sample ~e.g., Wirth, 2009!. New technologies, such as X-ray or neutron computed tomography and magnetic resonance imaging, generate 3D imagery and permit rapid visualization of optically opaque objects without destruction of the sample ~Lindquist & Venkatarangan, 1999; Gualda & Rivers, 2006; Barnes et al., 2008!. However, typical resolution ~minimum resolved dis- tance r between two points! of these techniques ~10 mm to 1 mm; Carlson, 2006! is not sufficient for detailed character- ization of very fine-grained geological materials, such as microinclusions in minerals. By contrast, confocal imaging techniques, like confocal laser scanning microscopy ~CLSM! and multiphoton microscopy ~MPM!, offer the highest res- olution of any 3D imaging methods currently available ~0.1–0.4 mm!. The CLSM technique emerged recently in geosciences and has proven to be a powerful tool for 3D imaging thanks to its ability to obtain optical sections of a sample at various depths, at high resolution and without sample destruction. Few applications of the technique have been reported in geoscience research; however, these are mainly fluorescence imaging of microfractures in granite rocks ~Menendez et al., 1999; Onishi & Shimizu, 2005! or in sandstones ~Fredrich et al., 1995; Fredrich, 1999!. This lack of interest is due to the fact that CLSM is generally used in the fluorescent mode, while rock components usually have very low natural fluorescence. Consequently, a rock must be first impreg- nated with a low-viscosity resin doped with a fluorescent dye to be studied. This process applies well to porous media, but is usually limited to studies of fracturing in compact materials and, importantly, gives no 3D information on minerals that remain nonfluorescent. Furthermore, the z-axis extent of the image volume ~typically ;15 mm in 3D volume rendering of igneous rock with CSLM, e.g., Fre- drich, 1999! is limited due to absorption and scattering of the laser beam by the material overlying the optical plane, even in transparent minerals. MPM has been mainly applied to biology where it has provided important advantages over CSLM, such as inher- ent 3D resolution, negligible out-of-focus photobleaching and reduced light scattering or photodamage ~Denk et al., 1990!. A review of recent literature reveals that MPM has Received December 1, 2010; accepted May 24, 2011 *Corresponding author. E-mail: antoine.benard@univ-st-etienne.fr Microsc. Microanal. 17, 937–943, 2011 doi:10.1017/S1431927611011883 Microscopy AND Microanalysis © MICROSCOPY SOCIETY OF AMERICA 2011