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.

A directly diode-laser-pumped Ti:Al(2)O(3) laser is demonstrated. Using a 1 W, 452 nm GaN diode laser, 19 mW of cw output power is achieved in a potentially portable format. Pumping at this short wavelength induces a loss at the laser wavelength that is not seen for the more typical green pump wavelengths. This effect is characterized and discussed.

We report on the introduction of active optical elements into confocal and multiphoton microscopes in order to reduce the sample-induced aberration. Using a flexible membrane mirror as the active element, the beam entering the rear of the microscope objective is altered to produce the smallest point spread function once it is brought to a focus inside the sample. The conventional approach to adaptive optics, commonly used in astronomy, is to utilise a wavefront sensor to determine the required mirror shape. We have developed a technique that uses optimisation algorithms to improve the returned signal without the use of a wavefront sensor. We have investigated a number of possible optimisation methods, covering hill climbing, genetic algorithms, and more random search methods. The system has demonstrated a significant enhancement in the axial resolution of a confocal microscope when imaging at depth within a sample. We discuss the trade-offs of the various approaches adopted, comparing speed with resolution enhancement.

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