Simultaneous atomic-force and two-photon fluorescence imaging of biological specimens in vivo.

We describe in this paper a home-built scanning-probe setup that combines the high spatial resolution of a commercial atomic-force microscope (AFM) with the high sensitivity and the discriminative power of a confocal two-photon fluorescence microscope. This scheme offers the ability of acquiring simultaneous, directly correlated topography and optical images with high sensitivity and resolution, and was successfully tested using model systems, such as dye-loaded latex beads. As a first biological application, we studied the (un)stacking of grana membranes in the envelope-free plant chloroplasts. The topographs showed two grana layers attached together in a "native unit" 15-16 nm thick and 4 nm protrusions on their surface, which we assign to Photosystem II reaction center. The optical imaging did not resolve single photosynthetic proteins, but helped in identifying the grana and indicated that the protein conformation and the chromophore binding are intact. Furthermore, our instrument allowed a direct comparison between the cell morphology and the distribution of the signaling protein H-Ras in living cells, i.e. mouse fibroblasts. With our approach the nanometer-scale resolving power of AFM is improved with the chemical identification capabilities of optical techniques, thus opening up interesting possibilities in various areas of research, including material and life sciences.