Multimodal nonlinear microscope by a compact fiber-laser system

Summary form only given. Nonlinear optical (NLO) microscopy [1-3] allows for intrinsic confocal (three-dimensional) high-speed imaging with deep penetration and high molecular contrast. Two-photon excitation fluorescence (TPEF) has been widely employed in biological imaging, taking advantage of either the intrinsic fluorescence or the extrinsic labeling of cells and tissues. Coherent Raman scattering (CRS), comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has been established as the most powerful chemically selective label-free high-speed imaging tool. Multimodal approaches, combining TPEF and CRS into a NLO microscope, can bring a very rich biological information on the sample under study. Up to now, NLO microscopes were based either on two synchronized Ti:sapphire oscillators or a bulk solid-state oscillator driving an optical parametric oscillator [2]. The complexity and cost of the overall apparatus, from the laser source to the detection systems, has so far hindered its mainstream use in biology. In this paper, we introduce a home-built NLO microscope, combining TPEF, SRS and CARS, based on an Erbium-fiber laser source, thus greatly simplifying the excitation scheme and reducing the costs and maintenance of the system. It provides two synchronized picosecond pulse trains at 40-MHz repetition rate: the pump pulses (at 778 nm, with 120-mW power, modulated at 1 MHz) and the Stokes pulses (tunable from 840 to 1100nm, with power up to 10 mW). A dichroic mirror combines the two pulses and a pair of galvanometric mirrors scans the beam over the sample area, at the focus of the microscope objective, thanks to a relay imaging system made of a scan lens and a tube lens in a standard 4-f configuration. After the sample, a dichroic beam splitter transmits the Stokes beam and reflects the CARS or TPEF (at shorter wavelengths). A photomultiplier tube reads CARS or TPEF signals, while a photodiode and a high-speed lock-in amplifier detect the SRS signal. Another set of lenses in 4-f configuration are placed after the sample to conjugate the back aperture of the microscope with the detectors, thus guaranteeing a proper light collection. Our NLO microscope can also be readily configured in epi (back scattering) detection in case of need. SRS spectra can be rapidly recorded in the 2800-3100 cm
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Raman region, thus providing a detailed chemical information on the sample.We tested our system by simultaneously acquiring SRS and TPEF images of an unprocessed fresh leaf of Elodea aquatic plant, with 1-ms pixel dwell time. Fig. 1(b) shows a linear transmission image of the leaf at the Stokes wavelength (1004 nm) collected by recording the DC voltage of the SRS photodiode. Fig. 1(c) shows a composite image of TPEF and SRS. TPEF (in green, in the 600-750nm wavelength region) is due to the excitation of chlorophylls by two-photon absorption of the pump pulse and thus mainly targets chloroplast, scattered within the intracellular spaces. SRS at 2890 cm
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Raman frequency (in red) mainly targets cellulose molecules, abundant in the cellular walls. Other imaging modalities can be easily added, such as second- or third-harmonic generation, making this a compact nonlinear microscopy platform.