Abstract
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.
Highlights
Temporal focusing (TF) microscopy is a wide-field two-photon excitation fluorescence (2PEF) microscopy technique that is capable of optical sectioning [1,2]
TF microscopy can be combined with structured illumination microscopy to reject the out-of-focus background fluorescence, which is eliminated by post-processing after the data acquisition [5,6]
We report on the development of an Yb-fiber chirped pulse amplifier (CPA) system for 3PEF-TF microscopy that produces 92-fs pulses with an energy of 9.0 μJ at a repetition rate of 200 kHz and a center wavelength of 1060 nm
Summary
Temporal focusing (TF) microscopy is a wide-field two-photon excitation fluorescence (2PEF) microscopy technique that is capable of optical sectioning [1,2]. The observation of a large field of view with TF microscopy requires high excitation power, and excitation with a high average power may induce sample heating This problem can be solved by utilizing an amplifier that produces pulses with high peak power and ultrashort pulse duration at a low repetition rate (< 1 MHz) as the excitation light source. We report on the development of an Yb-fiber CPA system for 3PEF-TF microscopy that produces 92-fs pulses with an energy of 9.0 μJ at a repetition rate of 200 kHz and a center wavelength of 1060 nm. By compensating for gain narrowing using a long-pass filter and for higher-order dispersion using a 4-f pulse shaper [24], an FTL pulse duration of 92 fs is obtained at a gain of 48 dB Using this Yb-fiber laser system, we demonstrate 3PEF-TF microscopy as well as dual-color imaging of a blue fluorophore and a red fluorophore.
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