Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Abstract

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free-space delivery is difficult. The delivery of high-peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided.

Fig. 1

An experimental design for measuring ultrafast optical pulse characteristics through optical fibers and the resultant two-photon excitation fluorescence microscopy performance. The experimental apparatus consists of a pulse compressor, an optical fiber coupling system including a fiber focuser and a fiber collimator, a two-photon excitation laser scanning fluorescence microscope. The pulse compressor provides linear prechirping to compensate for chromatic dispersion in the system. The optical pulse profile can be measured with an optical autocorrelator and a spectrum analyzer. The delivered optical pulses enter into a two-photon excitation fluorescence microscope where the TPE and the optical resolution are measured.

Fig. 2

The maximum coupling efficiencies for different types of fibers are plotted. The x-axis represents the fiber output optical power on a logarithmic scale. Fiber coupling efficiency is observed to be power-independent. All the small-core fibers have 40-50%, whereas most of the large-core fibers have 80-90%. Note that DCPCF16um has similar coupling efficiency to small-core ones due to its small fiber core NA, even though it has relatively large core.

Fig. 3

(a) Temporal pulse width and (b) spectral bandwidth of the optical pulse after various fiber delivery methods are plotted. Free space delivery is included as a reference. The x-axis represents fiber output optical power on a logarithmic scale for both (a) and (b). HCPCF6um demonstrates intact optical pulse delivery from the experimental results.

Fig. 4

TPE efficiency in a two-photon excitation fluorescence microscope with light pulses delivered by different fibers. Quadratic dependence of sample fluorescence as a function of power indicates TPE process. Fiber delivery is much less efficient for TPE than for free space delivery. Among all the fibers tested, HCPCF6um is the most efficient fiber delivery medium.

Fig. 5

PSFs in a two-photon excitation fluorescence microscope using different fiber delivery methods. The top figures show both the lateral and the axial PSF measurements with light delivered through various fibers. Free space delivery is included for comparison. Bottom figures represent statistical analysis of the lateral and the axial PSF FWHMs for each delivery method. The lateral resolution is dependent on the number of modes that the fiber supports, whereas the axial resolution is almost independent of delivery method. The symbol, *, on top of bars, indicates that optical resolution is statistically different from free space delivery, based on a t-test with P<0.05.

Fig. 6

(a) Two-photon excitation fluorescence images for H&E-stained ex-vivo human skin with different fiber delivery methods. From left to right: AIR, HCPCF6um, DCPCF16um, SISMF5um, SIMMF10um, SIMMF50um and GIMMF50um (b) The power spectra of images obtained with different fiber delivery methods. Power spectral image analysis was performed on a series of human skin images obtained in (a). At low spatial frequency range, the images acquired using fibers with small number of modes have higher power spectral densities than those with large number of modes. At high spatial frequency region, power spectral density levels for all the fiber delivery methods, including free space delivery, are same since noise dominates at this regime. The maximum spatial frequency for given NA is 2.3 μm^-1

Fig. 7

The effects of linear prechirping on (a) temporal pulse width and (b) spectral bandwidth of the optical pulse with respect to grating pair separation are shown. At the optimal grating pair separation in the pulse compressor, power dependences are also observed in terms of (c) temporal pulse width and (d) spectral bandwidth.

Fig. 8

The normalized TPE coefficients affected by the linear prechirping were measured in the function of grating pair separation. TPE coefficient for each fiber delivery was measured and normalized by the one for chirp-free optical pulse delivery in free space.