Temporal focusing (TF) microscopy is a wide-field two-photon excitation fluorescence (2PEF) microscopy technique, the optical sectioning capability of which is lower than that of point-scanning 2PEF microscopy. Here we demonstrate TF microscopy using three-photon excitation fluorescence (3PEF), which enhances the optical sectioning capability. As an excitation light source for the 3PEF, we developed an Yb-fiber chirped pulse amplifier, which produces 92-fs 9.0-μJ 1060-nm pulses at a repetition rate of 200 kHz. The optical sectioning capability was improved by a factor of 1.3 compared with that of 2PEF-TF microscopy. We also demonstrate dual-color imaging with both 2PEF and 3PEF.
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