Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning of thick tissues

Li-Chung Cheng; Chia-Yuan Chang; Wei-Chung Yen; Shean-Jen Chen

doi:10.1117/12.930324

15 October 2012 Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning of thick tissues

Li-Chung Cheng, Chia-Yuan Chang, Wei-Chung Yen, Shean-Jen Chen

Author Affiliations +

Proceedings Volume 8520, Unconventional Imaging and Wavefront Sensing 2012; 85200N (2012) https://doi.org/10.1117/12.930324
Event: SPIE Optical Engineering + Applications, 2012, San Diego, California, United States

Abstract

Conventional multiphoton microscopy employs beam scanning; however, in this study a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. The microscope integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) with a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled device camera. This configuration can produce multiphoton excited images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm have been instantaneously observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Moreover, we combine the widefield multiphoton microscopy with structure illuminated technique named HiLo to reject the background scattering noise to get better quality for bioimaging.

Citation Download Citation

Li-Chung Cheng, Chia-Yuan Chang, Wei-Chung Yen, and Shean-Jen Chen "Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning of thick tissues", Proc. SPIE 8520, Unconventional Imaging and Wavefront Sensing 2012, 85200N (15 October 2012); https://doi.org/10.1117/12.930324

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