Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning

Li Chung Cheng*, Chia Yuan Chang, Chun Yu Lin, Keng Chi Cho, Wei Chung Yen, Nan Shan Chang, Chris Xu, Chen Yuan Dong, Shean Jen Chen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

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 μ J/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 μ m2 at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 μ m were observed in real-time with a lateral spatial resolution of less than 0.5 μ m and an axial resolution of approximately 3.5 μ m. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

Original languageEnglish
Pages (from-to)8939-8948
Number of pages10
JournalOptics Express
Volume20
Issue number8
DOIs
StatePublished - 9 Apr 2012

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