Widefiled multiphoton excited florescence microcopy for animal study in vivo

L. C. Cheng*, C. Y. Chang, C. H. Lin, Y. D. Su, T. Y. Huang, Shean-Jen Chen

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingConference contribution

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 × 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.

Original languageEnglish
Title of host publicationNanobiosystems
Subtitle of host publicationProcessing, Characterization, and Applications III
DOIs
StatePublished - 26 Oct 2010
EventNanobiosystems: Processing, Characterization, and Applications III - San Diego, CA, United States
Duration: 4 Aug 20105 Aug 2010

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume7765
ISSN (Print)0277-786X

Conference

ConferenceNanobiosystems: Processing, Characterization, and Applications III
CountryUnited States
CitySan Diego, CA
Period4/08/105/08/10

Keywords

  • Multiphoton excited fluorescence microscopy
  • Neural cell
  • Temporal focusing
  • Widefield

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