A dedicated two-photon microscope incorporating adaptive-optic correction of specimen-induced aberrations is presented. Wavefront alteration of the scanning laser beam was achieved by use of a micromachined deformable mirror. Post scan head implementation produces a compact module compatible with the Bio-Rad MRC-600 scan head. Automatic aberration correction using feedback from the multiphoton fluorescence intensity allowed the adaptive optic to extend the imaging depth attainable in both artificial and biological refractive-index mismatched samples. With a 1.3-NA, x40, Nikon oil immersion objective, the imaging depth in water was extended from approximately 3.4 to 46.2 microm with a resolution defined by a FWHM axial point-spread function of 1.25 microm.

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We report on the integration of active optical elements in a multiphoton microscope to improve theresolution and overall image quality when imaging deeply into biological samples. Optical models were generated of sample systems and these have been compared with the performance of thecomplete imaging system. The active elements used were commercially available flexible membranemirrors controlled by custom, home written, software. Significant improvements in image qualityhave been demonstrated using a range of optimisation routines based on the analysis of the imagesproduced by the system, rather than with a wavefront sensor. A three-fold increase in the resolution100µm into the sample was achieved