Abstract
Hyperspectral stimulated Raman scattering (hsSRS) microscopy has recently emerged as a powerful non-destructive technique for the label-free chemical imaging of biological samples. In most hsSRS imaging experiments, the SRS spectral range is limited by the total bandwidth of the excitation laser to ~300 cm-1 and a spectral resolution of ~25 cm-1. Here we present a novel approach for broadband hsSRS microscopy based on parabolic fiber amplification to provide linearly chirped broadened Stokes pulses. This novel hsSRS instrument provides >600 cm-1 spectral coverage and ~10 cm-1 spectral resolution. We further demonstrated broadband hsSRS imaging of the entire Raman fingerprint region for resolving the distribution of major biomolecules in fixed cells. Moreover, we applied broadband hsSRS in imaging amyloid plaques in human brain tissue with Alzheimer's disease.
Highlights
Optical microscopy has become a fundamental and indispensable tool for biomedical research
We showed that with spectral broadening, Hyperspectral stimulated Raman scattering (hsSRS) imaging could cover up to ~680 cm−1 without the need to change wavelengths
In addition to increased spectral coverage, we demonstrate an improvement in spectral resolution compared to traditional spectral-focusing hsSRS setups
Summary
Optical microscopy has become a fundamental and indispensable tool for biomedical research. In parallel development, stimulated Raman scattering (SRS) microscopy has emerged as an alternative to CARS as a powerful non-destructive and label-free chemical imaging technique [13,14,15,16,17,18,19,20,21] It has shown tremendous potential in detecting tumor margins [22,23,24], unraveling dysregulated lipid metabolism [25,26], and tracing small molecule metabolites and drugs [27,28,29,30,31]. To differentiate and quantify different molecules based on their unique vibrational signatures, multiple Raman transitions must be probed with SRS, necessitating the use of multiplex SRS or hsSRS imaging [32,33,34,35,36]
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