Biomedical Imaging Using Ultrashort Laser Pulses

The field of optical microscopy experienced significant gains in resolution and speed following the introduction of lasers. Unfortunately, these gains came at the expense of sample degradation caused by the continuous flux of intense light. Taking advantage of the two-photon absorption process, Webb and Denk implemented a microscope based on the use of near-IR light pulses capable of causing simultaneous multiple fluorophore excitation. Two-photon microscopy is now widely applied in the biomedical imaging field due to the absence of out-of-focus photobleaching and reduced photodamage and fluorescence scattering. These advantages are brought about collectively by the inherent instantaneous peak intensity and narrow focal plane of excitation. Given that peak intensity increases with decreasing laser pulse duration, one would expect extensive use of available ultrashort (sub-10 fs) pulse laser systems in the field of biomedical imaging. However, most two-photon microscopes still use the same pulse duration that Webb and Denk used in 1990 (≈150 fs).