Development of Selective Plane Illumination Surface Enhanced Raman Scattering Spectral Microscopy for Improved...

The combination of optical microscopy and spectroscopy is a powerful tool for accessing both spatial and chemical information from a diverse range of samples. Applications of spectroscopic imaging techniques in biological samples are often limited by high backgrounds and autofluorescence. Surface enhanced Raman scattering (SERS) provides highly specific signals that can be used as labels or to monitor chemical interactions in complex environments. Previous work establishing a spectrally resolved SERS imaging approach demonstrated the presence of high backgrounds when imaging biological samples, causing interference with clear detection and identification of SERS signals. To minimize this out of focus scattering, this work demonstrates the incorporation of selective plane illumination microscopy (SPIM) with wide-field SERS spectral imaging, an approach we term selective plane illumination surface enhanced Raman scattering (SPI-SERS) spectral imaging. The generated light sheet avoids exposure outside of the focal plane altogether, which simultaneously improves image contrast and signal-to-noise ratios (SNRs) in the observed spectra. These improvements enable molecular identification in both 3-dimensional (3D) matrices and biological cellular samples, advancements which hold promise for the ability of this methodology to achieve high-contrast SERS imaging in complex biological samples.