We combine an all-fiber dual wavelength, self-synchronized laser and a dedicated multi-channel detection unit to perform state-of-the-art multiplex Stimulated Raman Scattering (SRS) microscopy. The system covers the full CH spectrum in 1 μs reaching shot-noise limited performances with 25 μW per detection channel. This all-inone solution is based on a passively synchronized dual-wavelength laser source with shot-noise limited relative intensity noise from 600 kHz and a modular multi-channel lock-in detection unit. The synergistic design between laser source and detection system simplifies multiplex SRS implementation for real-time full-chemical imaging.
We present a novel approach to balanced-detection stimulated Raman scattering (SRS) spectroscopy and microscopy, called In-line Balanced Detection (IBD). IBD-SRS not only completely removes high-frequency laser fluctuations, a crucial ingredient to improve signal-to-noise ratio in modulation-transfer techniques, but also passively and automatically balances the low-frequency signal variation due to spatially varying sample transmission. It takes advantage of polarization multiplexing and an inherently stable common-path geometry. A birefringent crystal before the sample creates two orthogonally polarized and collinear replicas of the Stokes pulse, with ~10-ps relative delay. The first serves as a reference pulse; the second, temporally overlapped with the pump pulse, probes the Raman response of the sample. As reference and probe pulses cross the sample at the same position, they maintain their balance during image acquisition. IBD can be implemented in any conventional SRS setup, by simply adding a few passive optical elements in the beam. We have tested its performances on a home-built multimodal laser-scanning microscope, coupled with a compact fiber-format laser source. We obtain common-mode noise rejection up to 30 dB with respect to the unbalanced case, thus reaching shot-noise-limited detection, without the need of any electronic (active) auto-balancing. We have employed IBD-SRS to distinguish different polymer beads, to locate lignin and cellulose in the walls of plant cells and to visualize the three-dimensional distribution of lipids in HuH7 and HepaRG hepatic cells. To demonstrate the suitability of IBD-SRS in scattering environments, we show a significant image-quality improvement also when measuring lipids in thick bovine liver tissues.
Stimulated Raman scattering spectroscopy is a powerful nonlinear optical technique for label-free identification of molecules, based on their characteristic vibrational fingerprint. Current implementations of SRS, while achieving extremely high acquisition speeds up to the video rate, mostly work at a single frequency, thus providing limited chemical information. Broadband implementation of SRS is technically challenging, as for imaging dilute species in biological microscopy applications one must detect very small (approx. 10^-5) signals sitting on a large background via modulation transfer technique. We introduce and experimentally demonstrate a novel approach to broadband SRS spectroscopy based on photonic time stretch (PTS). The broadband femtosecond Stokes pulse, after interacting with the sample, is stretched by a telecom fiber to 15ns duration, mapping its spectrum in time. The signal is sampled through a fast analog-to-digital converter, providing single-shot spectra at 80-kHz rate. We demonstrate 10^-5 sensitivity, over 500 cm-1 bandwidth in the C-H region with high resolution. These performances are already suitable for a number of applications, such as monitoring microfluidic flows, the onset of chemical reactions or solid-state samples such as pharmaceutical products. As the acquisition speed of PTS does not depend upon the covered spectral region, we are planning to extend the spectral coverage of SRS to the fingerprint region. Furthermore, using commercially available lasers with higher rep-rates, we could shorten the acquisition time considerably. This will pave the way to high-speed broadband vibrational imaging for materials science and biophotonics.
We introduce a home-built laser-scanning nonlinear optical microscope, combining two-photon excitation fluorescence (TPEF), stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS). Narrowband pump and tunable Stokes pulses at 40-MHz are delivered by an Erbium-fiber laser source, thus greatly simplifying the excitation scheme and reducing the costs and maintenance with respect to standard bulk free-space lasers. After the sample, a dichroic beam splitter transmits the Stokes beam for SRS imaging and reflects the CARS or TPEF (at shorter wavelengths). Signal-to-noise ratio in SRS imaging is greatly enhanced (by up to 30 dB, reaching shot-noise-limited detection without the need of any electronic auto-balancing) thanks to the use of an innovative scheme that we call In-line Balanced Detection (IBD). IBD-SRS not only completely removes high-frequency laser fluctuations but also passively and automatically balances the low-frequency signal variation due to spatially varying sample transmission. We record SRS/CARS spectra in the 2800-3100 cm-1 Raman vibrational spectrum, thus providing a detailed chemical information on the sample in the C-H stretching region. We report various bioimaging applications of our instrument: the study of breast tumour cells using CARS, three-dimensional visualization of lipid distribution in HuH7 and in HepaRG hepatic cells using SRS and a combined TPEF/SRS study of plant cells. Microscopy in scattering media such as a bovine liver tissue is as well demonstrated.
Stimulated Raman scattering spectroscopy is a powerful technique for label-free molecular identification, but its broadband implementation is technically challenging. We introduce and experimentally demonstrate a novel approach based on photonic time stretch. The broadband femtosecond Stokes pulse, after interacting with the sample, is stretched by a telecom fiber to 15ns, mapping its spectrum in time. The signal is sampled through a fast analog-to-digital converter, providing single-shot spectra at 80-kHz rate. We demonstrate 10^-5 sensitivity over 500 cm-1 in the C-H region. Our results pave the way to high-speed broadband vibrational imaging for materials science and biophotonics.
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