Widefield multiphoton excited fluorescence microscopy for animal study in vivo

L.-C. Cheng; C.-Y. Chang; C.-H. Lin; Y.-D. Su; T.-Y. Huang; S.-J. Chen

doi:10.1117/12.864107

24 August 2010 Widefield multiphoton excited fluorescence microscopy for animal study in vivo

L.-C. Cheng, C.-Y. Chang, C.-H. Lin, Y.-D. Su, T.-Y. Huang, S.-J. Chen

Proceedings Volume 7765, Nanobiosystems: Processing, Characterization, and Applications III; 77650Y (2010) https://doi.org/10.1117/12.864107
Event: SPIE NanoScience + Engineering, 2010, San Diego, California, United States

Abstract

Unlike conventional multiphoton excited microscopy according to pixel-by-pixel point scanning, a widefield multiphoton excited microscopy based on spatiotemporal focusing has been developed to construct three-dimensional (3D) multiphoton fluorescence images only with the need of an axial scanning. By implementing a 4.0 W 10 kHz femtosecond laser amplifier with an instant strong peak power and a fast TE-cooled EMCCD camera with an ultra-sensitive fluorescence detection, the multiphoton excited fluorescence images with the excitation area over 100 μm x 100 μm can be achieved at a frame rate up to 80 Hz. A mechanical shutter is utilized to control the exposure time of 1 ms, i.e. average ten laser pulses reach the fluorescent specimen, and hence an uniform enough multiphoton excited fluorescence image can be attained with less photobleaching. The Brownian motion of microbeads and 3D neuron cells of a rat cerebellum have been observed with a lateral spatial resolution of 0.24 μm and an axial resolution of 2.5 μm. Therefore, the developed widefield multiphoton microscopy can provide fast and high-resolution multiphoton excited fluorescence images for animal study in vivo.

Citation Download Citation

L.-C. Cheng, C.-Y. Chang, C.-H. Lin, Y.-D. Su, T.-Y. Huang, and S.-J. Chen "Widefield multiphoton excited fluorescence microscopy for animal study in vivo", Proc. SPIE 7765, Nanobiosystems: Processing, Characterization, and Applications III, 77650Y (24 August 2010); https://doi.org/10.1117/12.864107

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