Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 μJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 μm were observed in real-time with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.