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Two-photon fluorescence microscope with a hollow-core photonic crystal fiber

Shih-Peng Tai, Ming-Che Chan, Tsung-Han Tsai, Shi-Hao Guol, Li-Jin Chen, and Chi-Kuang Sun

Open Access Open Access

Abstract

Self-phase-modulation and group velocity dispersion of near IR femtosecond pulses in fibers restrict their use in two-photon fluorescence microscopy (TPFM). Here we demonstrate a hollow-core photonic crystal fiber based two-photon fluorescence microscope with low nonlinearity and dispersion effects. We use this fiber-based TPFM system to take two-photon fluorescence (chlorophyll) images of mesophyll tissue in the leaf of Rhaphidophora aurea. With less than 2mW average power exposure on the leaf at 755nm, the near zero-dispersion wavelength, chloroplasts distribution inside the mesophyll cells can be identified with a sub-micron spatial resolution. The acquired image quality is comparable to that acquired by the conventional fiber-free TPFM system, due to the negligible temporal pulse broadening effect.

©2004 Optical Society of America

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Figures (6)
Fig. 1.
Fig. 1. Experiment setup of the TPFM with a hollow-core photonic crystal fiber.

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Fig. 2.
Fig. 2. Power dependent output spectra of the hollow-core PCF at central wavelengths of (a) 750nm, (b) 755nm, (c) 780nm, and (d) 795nm. Input spectra are also provided for comparison.

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Fig. 3.
Fig. 3. (a)–(d) Intensity autocorrelation trace with 80mW average power transmitted through the hollow core PCF at different central wavelengths. (e) The relation between temporal pulse width and center wavelength at 80mW and 120mW average power transmitted through the hollow core PCF.

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Fig. 4.
Fig. 4. 670nm fluorescence power (rectangle) vs. 755nm excitation power for the mesophyll tissue in the leaf of Rhaphidophora aurea. The well-matched solid line is the slope=2 fitting, confirming its two-photon nature

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Fig. 5.
Fig. 5. (a)–(e) Two-photon fluorescence images of mesophyll tissues in the leaf of Rhaphidophora aurea, taken with the conventional fiber-free system. Image size: 160µm×160µm. (f)–(j) Two-photon fluorescence images of mesophyll tissues in the leaf of Rhaphidophora aurea, taken with the system based on a hollow-core PCF. Image size: 160µm×160µm.

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Fig. 6.
Fig. 6. Wavelength dependent temporal pulse broadening ratio (squares) and two-photon fluorescence image degradation ratio (triangles).

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