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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, and Shean-Jen Chen

Open Access Open Access

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 μJ/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 μm2 at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 μm were observed in real-time with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

©2012 Optical Society of America

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Supplementary Material (2)

Media 1: MOV (706 KB)     
Media 2: MOV (622 KB)     

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Figures (6)
Fig. 1
Fig. 1 Optical setup of the widefield multiphoton microscope based on spatiotemporal focusing.

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Fig. 2
Fig. 2 Spatial resolution of the widefield multiphoton excitation microscope: (a) axial resolutions under different laser powers at 10, 20, 30, and 40 mW are 2.5, 2.8, 3.1, and 3.4 μm, respectively; and, (b) the point spread function showing 0.5 μm FWHM.

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Fig. 3
Fig. 3 TPEF intensities of the PMMA thin film doped with R6G dye as a function of time under different excitation powers. Circles: ultrafast oscillator; squares: ultrafast amplifier.

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Fig. 4
Fig. 4 (a) Brownian motion of 1.0 μm fluorescent beads at 100 Hz frame rate (Media 1). The excitation laser power is 40 mW with a 9 ms exposure time. (b) Displacements of the bead at the top of Fig. 4(a) along the x and y axes as a function of time recorded at 10 ms intervals from Media 1.

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Fig. 5
Fig. 5 (a) Brownian motions of 0.5 μm fluorescent beads at 100 Hz frame rate with an exposure time of 9 ms (Media 2). (b) Sequentially images captured from Fig. 5(a).

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Fig. 6
Fig. 6 SHG images of chicken tendon: (a) 10 Hz frame rate at the full pixel number of 1000 × 1000 pixels and exposure time of 100 ms, (b) 100 Hz frame rate at 4x4 binning with pixel number of 250 × 250 pixels and exposure time of 9 ms, (c) image of Fig. 6(a) red square region at 100 Hz frame rate, 256 × 256 pixels, and 9 ms exposure time, and (d) image of Fig. 6(a) red square region at 10 Hz frame rate by accumulating 10 shots in one frame.

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Equations (2)

Equations on this page are rendered with MathJax. Learn more.

F W H M z ∝ k 1 + τ k 2 ⋅ τ l + k 3 ⋅ M ⋅ N A 2 ,
Δ d r m s = 6 D × Δ t ,
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