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Proceedings Paper

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning of thick tissues
Author(s): Li-Chung Cheng; Chia-Yuan Chang; Wei-Chung Yen; Shean-Jen Chen
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Paper 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.

Paper Details

Date Published: 15 October 2012
PDF: 8 pages
Proc. SPIE 8520, Unconventional Imaging and Wavefront Sensing 2012, 85200N (15 October 2012); doi: 10.1117/12.930324
Show Author Affiliations
Li-Chung Cheng, National Cheng Kung Univ. (Taiwan)
Chia-Yuan Chang, National Cheng Kung Univ. (Taiwan)
Wei-Chung Yen, Chung-Shan Institute of Science and Technology (Taiwan)
Shean-Jen Chen, National Cheng Kung Univ. (Taiwan)

Published in SPIE Proceedings Vol. 8520:
Unconventional Imaging and Wavefront Sensing 2012
Jean J. Dolne; Thomas J. Karr; Victor L. Gamiz, Editor(s)

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