Surface-enhanced hyper Raman hyperspectral imaging and probing in animal cells

Abstract

scattering, that is, spontaneous, two-photon excited Raman scattering, of organic molecules becomes strong when it occurs as surface-enhanced hyper Raman scattering (SEHRS), in the proximity of plasmonic nanostructures. Its advantages over one-photon excited surface-enhanced Raman scattering (SERS) include complementary vibrational information resulting from different selection rules, probing of very small focal volumes, and beneficial excitation with long wavelengths. Here, imaging of macrophage cells by SEHRS is demonstrated, using SEHRS labels consisting of silver nanoparticles and two different molecules, 2-naphthalenethiol and para-mercaptobenzoic acid, that are excited offresonance. The vibrational signatures of the molecules are discriminated using hyperspectral analysis and provide information about the subcellular localization of the SEHRS probes. The SEHRS based hyperspectral imaging approach presented here uses principal component analysis (PCA) to localize the reporter molecules inside the cells and is augmented by hierarchical cluster analysis (HCA). The high sensitivity of SEHRS spectra with respect to small environmental changes can be utilized for mapping of physiological parameters in the endosomal system of the cells. This is illustrated by discussing the spatial distribution of endosomes of varying pH inside the cytosol.

A Review on Surface-Enhanced Raman Scattering

“…General overviews on SERS can be found in books [6,8,80,81,82,83,84], Faraday discussions [85,86,87,88,89], special issues [90,91,92,93], and reviews [7,9,36,76,94,95,96,97,98]. Finally, it is worth mentioning that the plasmonic amplification of the optical response has been exploited to enhance also coherent anti-Stokes Raman scattering (CARS) [99,100], stimulated Raman scattering (SRS) [101,102], hyper Raman scattering (HRS) [75,103,104,105], fluorescence [106,107,108,109], and infrared absorption [8,110].…”

Theoretical investigation of a plasmonic substrate with multi-resonance for surface enhanced hyper-Raman scattering

“… 6 – 10 . With the development of experimental technology and theoretical research, SEHRS spectrum has attracted great attention and been used in many fields 11 – 14 , such as single molecule detection 11 , cell PH sensors 15 , spectral imaging like other nonlinear optical imaging 16 , 17 .…”

Bio‐probing with nonresonant surface‐enhanced hyper‐Raman scattering excited at 1,550 nm

“…Interestingly, compared with the spectra of 2‐NAT obtained with the same Ag and Au50 nanoparticles outside the cells (Figure 1c,d), the signals obtained inside cells are stronger. Results from previous work on SEHRS in cells [ 21 ] indicate that the larger signal can be the result of two effects. (i) The SEHRS nanosensors could be present at high concentration, as a result of the cellular processing by endocytosis, which can yield both endosomes with high numbers of particles in individual endosomes or high numbers of particle‐containing vesicles in the focal volume.…”

“…[ 19 ] The high cross sections suggest the applicability [ 19 ] of SEHRS vibrational labels using SEHRS for microscopic probing and imaging of biological samples. [ 21,23 ] This is extremely attractive, because of the different selection rules of hyper‐Raman scattering and SEHRS in particular, [ 24–26 ] additional, complementary information from vibrational probes can be gained for fingerprinting and imaging. Moreover, because of the special sensitivity of SEHRS to small changes in surface potential and adsorbate orientation, [ 18,27,28 ] small changes to reporter molecules may be detected, [ 29,30 ] thereby improving chemical probing.…”