Optimizing a three-photon microscope by direct monitoring of group delay dispersion at the sample plane.

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Poster Session 1
Sanjeev Kaushalya (1), Hans Fried (1)
1. CRFS, Light Microscope Facility, Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE) - Bonn

Three-photon microscopy, autocorrelator, deep-tissue intravital microscopy, multi-photon microscopy

Abstract text

In multiphoton microscopy, laser pulse width measurements at the setup plays an important role in optimizing the fluorescence output with least laser power applications. For example, in deep two- and three-photon intravital microscopy it is very crucial to have shortest possible laser pulses for efficient excitation with lowest possible laser power at the sample. Laser pulses used in multi-photon microscopy have a large spectral bandwidth and therefore dispersion broadens the pulse width significantly. A broadened pulse at the sample results in much lower fluorescence and usage of higher laser power to increase the fluorescence signal can quickly result in photo damage. Here, we used a self-built and portable optical collinear autocorrelator, to probe the beam anywhere in the beam path and after the objective to measure the pulse width, and to optimize with a pre-chirp (dispersion compensation) unit. We demonstrate that fluorescence excitation efficiency could be increased by many fold in three-photon microscopy.