Frequency-modulation stimulated Raman scattering microscopy with an acousto-optic tunable filter.

Stimulated Raman scattering (SRS) microscopy is increasingly employed for highly specific, label-free, and high-speed bioimaging. Despite its benefits, SRS is susceptible to spurious background signals caused by competing effects, which lower the possible imaging contrast and sensitivity. An efficient approach to suppress these undesired background signals is frequency-modulation (FM) SRS, which exploits the competing effects' weak spectral dependence compared to the SRS signal's high spectral specificity. We propose an FM-SRS scheme realized with an acousto-optic tunable filter, which presents a few advantages compared to other solutions presented in the literature. In particular, it can perform automated measurements from the fingerprint to the CH-stretching region of the vibrational spectrum without any manual adjustment of the optical setup. Moreover, it allows simple all-electronic control of the spectral separation and relative intensities of the pair of probed wavenumbers.

Hadamard-transform spectral acquisition with an acousto-optic tunable filter in a broadband stimulated Raman scattering microscope.

We present a novel configuration for high spectral resolution multiplexing acquisition based on the Hadamard transform in stimulated Raman scattering (SRS) microscopy. The broadband tunable output of a dual-beam femtosecond laser is filtered by a fast, narrowband, and multi-channel acousto-optic tunable filter (AOTF). By turning on and off different subsets of its 8 independent channels, the AOTF generates the spectral masks given by the Hadamard matrix. We demonstrate a seamless and automated operation in the Raman fingerprint and CH-stretch regions. In the presence of additive noise, the 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.

When microplastics are not plastic: Chemical characterization of environmental microfibers using stimulated Raman microspectroscopy.

The abundance of anthropogenic debris dispersed in the environment is exponentially growing, raising concerns about marine life and human exposure to microplastics. Microfibers are the most abundant microplastic type in the environment. However, recent research suggests that most microfibers dispersed in the environment are not made of synthetic polymers. In this work, we systematically tested this assumption by determining the man-made or natural origin of microfibers found in different environments, including surface waters, sediments at depths >5000 m and highly sensitive habitats like mangroves and seagrass, and treated water using stimulated Raman scattering (SRS) microscopy. Our findings show that ¾th of analyzed microfibers are of natural origin. One plastic fiber is estimated per every 50 L in surface seawater, every 5 L in desalinated drinking water, every 3 g in deep sea sediments and every 27 g in coastal sediments. Synthetic fibers were significantly larger in surface seawaters compared to organic fibers due to higher resistance to solar radiation. These results emphasize the necessity of using spectroscopical methods to assess the origin of environmental microfibers to accurately estimate the abundance of synthetic materials in the environment.

Stimulated Raman microspectroscopy as a new method to classify microfibers from environmental samples.

Microfibers are reported as the most abundant microparticle type in the environment. Their small size and light weight allow easy and fast distribution, but also make it challenging to determine their chemical composition. Vibrational microspectroscopy methods as infrared and spontaneous Raman microscopy have been widely used for the identification of environmental microparticles. However, only few studies report on the identification of microfibers, mainly due to difficulties caused by their small diameter. Here we present the use of Stimulated Raman Scattering (SRS) microscopy for fast and reliable classification of microfibers from environmental samples. SRS microscopy features high sensitivity and has the potential to be faster than other vibrational microspectroscopy methods. As a proof of principle, we analyzed fibers extracted from the fish gastrointestinal (GIT) tract, deep-sea and coastal sediments, surface seawater and drinking water. Challenges were faced while measuring fibers from the fish GIT, due to the acidic degradation they undergo. However, the main vibrational peaks were still recognizable and sufficient to determine the natural or synthetic origin of the fibers. Notably, our results are in accordance to other recent studies showing that the majority of the analyzed environmental fibers has a natural origin. Our findings suggest that advanced spectroscopic methods must be used for estimation of the plastic fibers concentration in the environment.

Fingerprint-to-CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope.

Stimulated Raman scattering (SRS) microscopy is a label-free method generating images based on chemical contrast within samples, and has already shown its great potential for high-sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio-samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub-cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto-optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon-Hydrogen (CH)-stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm-1 , is demonstrated.