Optical fiber based ultrashort pulse multispectral nonlinear optical microscopy

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC?s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.