Pulse Front Adaptive Optics in Multiphoton Microscopy

The accurate focusing of ultrashort laser pulses is important in multiphoton microscopy, in order to achieve high resolution imaging in biological specimens. Recent advances have shown the improvements to imaging through the inclusion of an adaptive optic element in the microscope. Using a device such as a liquid crystal spatial light modulator or a deformable mirror to modify the wavefront of the incident ultrafast laser beam, it is possible to nullify aberrations induced by the specimen, leading to improved resolution and efficiency [1]. In an alternative configuration, the adaptive optical element may be used to shape the incident beam in the spectral domain to compensate for any dispersion in the optical system and minimise the pulse duration at the focus, leading to an improved fluorescence yield. Such systems however only provide the same correction of group velocity dispersion across the whole beam. Another phenomenon that can detrimentally affect focussing of ultrashort pulses is pulse front distortion, which arises when the group delay varies across the beam. The effect of this is that pulse components in different parts of the beam arrive at the focus at different times, leading to a temporal spreading of the pulse. In order to overcome this problem we demonstrate the new concept of pulse front adaptive optics as a further mechanism for control in a multiphoton microscope [2,3]. A deformable mirror and a spatial light modulator are operated in concert to give control over the incident pulse fronts (contours of constant intensity in space and time within an ultrashort pulse). By this approach, pulse fronts can be generated which are spatially variant across the beam. Rotationally symmetric time delays in the pulse front are of particular interest in multiphoton microscopy since they are equivalent to the deformation of ultrashort pulses caused by many lenses. By varying the amount of pulse distortion in the incident beam using adaptive optics, we demonstrate an enhancement in the measured fluorescence from a two photon microscope with non-uniform pulse fronts. This method will be beneficial to maintain short pulses at the focus of a microscope when focussing through dispersive specimens or using ultrashort (10s of femtoseconds) pulses.