We present a new architecture for frequency-modulation stimulated Raman Scattering (FM-SRS) that allows broadband background-free acquisitions. The system is based on a femtosecond laser oscillator (Stokes beam), used to pump a narrowband picosecond optical parametric oscillator (pump beam). Using an electro-optic modulator and polarizing beam splitter, we separate the broadband Stokes beam into two intensity-modulated Stokes beams that are spectrally filtered using a high-speed, narrowband acousto-optic tunable filter (AOTF) and a narrowband etalon. The two Stokes sub-beams and the pump beam are then recombined. By detecting the signal as the difference between in-Raman-resonance and off-Raman-resonance wavenumbers, we achieve background-free SRS measurements. Our scheme significantly simplifies and gives flexibility in selecting the pair of wavenumbers for FM-SRS, and allows background-free acquisitions from the fingerprint to the CH-stretch region of the Raman spectrum.
We propose a new broadband stimulated Raman scattering microscopy scheme. A high-efficient spectral compression method is used to frequency double a solid-state femtosecond laser oscillator and pump a picosecond optical parametric oscillator. The broadband femtosecond beam, used as Stokes beam, is intensity modulated by an electro-optical modulator, and spectrally filtered by a multichannel acousto-optic tunable filter. We implement Hadamard transform acquisitions by applying spectral masks on the broadband Stokes beam to perform compressive sensing for sparse wavenumber analysis to classify environmental micro-fibers, for fast discrimination of natural and synthetic origin materials.
Stimulated Raman Scattering (SRS) microscopy is a Coherent Raman technique that emerged in recent years as a powerful tool for biomedical imaging due to its high specificity, high speed, and label-free capability. Despite its advantages, SRS microscopy can be affected by nonlinear competing phenomena, namely two-photon absorption (TPA), cross-phase modulation (XPM), and thermal lensing (TL), which generate a background signal that reduces the achievable specificity and sensitivity. These competing processes are quasi-instantaneous and spatially non-uniform in heterogeneous samples and require customized setups to be canceled in SRS acquisitions. A robust approach for background-free SRS measurements is the frequency-modulation (FM) SRS, which is based on the broad spectral dependence of the parasitic effects (typically tens of nanometers) compared to the narrower band of the SRS effect (~ 1 nm).
Performing a differential measurement at two different wavenumbers, respectively on- and off- Raman resonance, it is possible to selectively detect the SRS process.
Different solutions for background cancellation via FM have been reported in the literature, but they present various drawbacks, such as the limited applicability over certain ranges of the vibrational spectrum or the necessity to modify the optical setup when performing measurements at different Raman shifts.
We propose an FM-SRS configuration realized for the first time with an acousto-optic tunable filter, able to perform measurements from the fingerprint to the CH-stretch region of the spectrum without any modification of the optical setup. We determined its efficiency in canceling the background signal due to different types of competing effects on various samples: polymer beads, human hair, and human cells. These results underline the importance of an effective cancellation of background signals of diverse nature when collecting SRS images. Our FM-SRS setup demonstrated critical advantages compared to other FM configurations.
Stimulated Raman Scattering (SRS) is a label-free technique utilized to analyze the chemical nature of various substances with high-speed. However, SRS can be affected by parasitic effects that can distort the signal. In order to eliminate these spurious effects, we propose a frequency-modulation SRS scheme realized for the first time with an acousto-optic tunable filter, which can perform measurements from the fingerprint to the CH-stretch region of the spectrum without any modification of the optical setup. We demonstrate the efficient cancellation of the parasitic effects with our developed configuration on several samples: human hairs, polymer beads and human cells.
The assessment of man-made or natural origin of microscopic debris in the environment requires spectroscopical analysis. However, it is difficult to use commercial FT-IR and spontaneous Raman microspectrometers to analyze micro-fibers due to their small size and strong autofluorescence in some specimens. Using a custom stimulated Raman scattering (SRS) microscope, we assessed the origin of microfibers from different environments by comparing their SRS spectra to a home-built library. Our analysis shows that the majority of the analysed micro-fibers are of natural origin. We show that SRS microscopy overcomes the limitations of FT-IR and spontaneous Raman microscopy for classification of micro-fibers.
We present a high spectral resolution multiplexing acquisition mode for SRS microscopy based on a dual-beam femtosecond laser. A multi-channel Acousto Optical Tunable Filter (AOTF) generates spectral masks, given by the Hadamard matrix, by turning on and off different subsets of its 8 independent channels, corresponding to different wavelengths available within the broad bandwidth of the “pump” femtosecond laser. The SRS spectrum is retrieved by using the inverse Hadamard matrix. When additive noise is dominating, spectral measurements using the multiplexed method show the same signal to noise ratio of conventional single-wavenumber acquisitions performed with 4 times longer integration time.
We present a novel design for a Stimulated Raman Scattering (SRS) microscope based on a dual beam femtosecond laser in combination with spectral shaping through a fast and narrowband Acousto Optical Tunable Filter. This configuration allows the measurement of broad SRS spectra, all the way from fingerprint region to CH stretch region without any modification of the optical setup. High spectral resolution over a broad spectral region allows label-free quantitative imaging of biological samples. We will show the application of our SRS system to a quantitative study of lipid droplets in colon Cancer Stem Cells.
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