Improving the penetration depth in multiphoton excitation laser scanning microscopy

Abstract

More than a threefold increase in multiphoton laser scanning microscopy depth penetration using a passive predispersion compensation system is reported. Using dispersion-controlled pulses to counteract the effects of positive group delay dispersion in the imaging platform, optical sectioning of fluorescent samples to depths in excess of 800 microm was observed, compared with only 240 microm using a noncompensated setup. Experimental results obtained from both the predispersion compensated and noncompensated systems are compared with theoretical values of pulse broadening in a laser scanning microscope. The observed improvement in depth profiling potentially widens the applications and user base of nonlinear microscopy techniques.