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1. Super-resolution SERS spectral bioimaging

   https://doi.org/10.1117/12.2632824  

   Schultz, Zachary D.; Shoup, Deben N.; Smith, Abigail E. (October 2022, SPIE Nanoscience + Engineering)

   Verma, Prabhat; Suh, Yung Doug (Ed.)

   Advances in nanotechnology enable the detection of trace molecules from the enhanced Raman signal generated at the surface of plasmonic nanoparticles. We have developed technology to enable super-resolution imaging of plasmonic nanoparticles, where the fluctuations in the surface enhanced Raman scattering (SERS) signal can be analyzed with localization microscopy techniques to provide nanometer spatial resolution of the emitting molecule’s location. Additional work now enables the super-resolved SERS image and the corresponding spectrum to be acquired simultaneously. Here we will discuss how this approach can be applied to provide new insights into biological cells. 

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2. Reduction of Spectral Overlap in Spectral Surface-Enhanced Raman Spectroscopy Imaging Using a Dove Prism

   https://doi.org/10.1177/00037028251322540  

   Shoup, Deben N; Smith, Abigail E; Schultz, Zachary D (September 2025, Applied Spectroscopy)

   The ability to combine microscopy and spectroscopy is beneficial for directly monitoring physical and biological processes. Spectral imaging approaches, where a transmission diffraction grating is placed near an imaging sensor to collect both the spatial image and spectrum for each object in the field of view, provide a relatively simple method to simultaneously collect images and spectroscopic responses on the same sensor. Initially demonstrated with fluorescence spectroscopy, the use of spectral imaging in Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) can provide a vibrational spectrum containing molecularly specific information that can inform on chemical changes. However, a major complication to this approach is the spectral overlap that occurs when objects are spaced closely together horizontally. In this work, we add a dove prism to a spectral imaging instrument developed for SERS imaging, enabling rotation of the collected SERS image and dispersed spectrum onto the imaging complementary metal-oxide semiconductor (CMOS) sensor. We demonstrate that this effectively reduces spectral overlap for emitters with clear separation between them and emitters with slightly overlapping point spread functions thereby facilitating collection of unambiguous spectra from each emitter. 

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3. Comparison of Gap-Enhanced Raman Tags and Nanoparticle Aggregates with Polarization Dependent Super-Resolution Spectral SERS Imaging

   https://doi.org/10.1021/acs.analchem.4c01564  

   Shoup, Deben N; Fan, Sanjun; Zapata-Herrera, Mario; Schorr, Hannah C; Aizpurua, Javier; Schultz, Zachary D (July 2024, Analytical Chemistry)

   Strongly confined electric fields resulting from nanogaps within nanoparticle aggregates give rise to significant enhancement in surface-enhanced Raman scattering (SERS). Nanometer differences in gap sizes lead to drastically different confined field strengths, so much attention has been focused on the development and understanding of nanostructures with controlled gap sizes. In this work, we report a novel petal gap-enhanced Raman tag (GERT) consisting of bipyramid core and a nitrothiophenol (NTP) spacer to support the growth of hundreds of small petals and compare its SERS emission and localization to a traditional bipyramid aggregate. To do this, we used super resolution spectral SERS imaging that simultaneously captures the SERS images and spectra while varying the incident laser polarization. Intensity fluctuations inherent of SERS enabled super resolution algorithms to be applied which revealed sub-diffraction limited differences in the localization with respect to polarization direction for both particles. Interestingly, however, only the traditional bipyramid aggregates experienced a strong polarization dependence in their SERS intensity and in the plasmon-induced conversion of NTP to dimercaptoazobenzene (DMAB), which was localized with nanometer precision to regions of intense electromagnetic fields. The lack of polarization dependence (validated through electromagnetic simulations) and surface reactions from the bipyramid-GERTs suggest that the emissions arising from the bipyramid-GERTs are less influenced by confined fields. 

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4. Hyperspectral Raman Imaging Using a Spatial Heterodyne Raman Spectrometer with a Microlens Array

   https://doi.org/10.1177/0003702820906222  

   Allen, Ashley; Waldron, Abigail; Ottaway, Joshua M.; Chance Carter, J.; Michael Angel, S. (August 2020, Applied Spectroscopy)

   null (Ed.)

   A new hyperspectral Raman imaging technique is described using a spatial heterodyne Raman spectrometer (SHRS) and a microlens array (MLA). The new technique enables the simultaneous acquisition of Raman spectra over a wide spectral range at spatially isolated locations within two spatial dimensions ( x, y) using a single exposure on a charge-coupled device (CCD) or other detector types such as a complementary metal-oxide semiconductor (CMOS) detector. In the SHRS system described here, a 4 × 4 mm MLA with 1600, 100 µm diameter lenslets is used to image the sample, with each lenslet illuminating a different region of the SHRS diffraction gratings and forming independent fringe images on the CCD. The fringe images from each lenslet contain the fully encoded Raman spectrum of the region of the sample “seen” by the lenslet. Since the SHRS requires no moving parts, all fringe images can be measured simultaneously with a single detector exposure, and in principle using a single laser shot, in the case of a pulsed laser. In this proof of concept paper, hyperspectral Raman spectra of a wide variety of heterogeneous samples are used to characterize the technique in terms of spatial and spectral resolution tradeoffs. It is shown that the spatial resolution is a function of the diameter of the MLA lenslets, while the number of spatial elements that can be resolved is equal to the number of MLA lenslets that can be imaged onto the SHRS detector. The spectral resolution depends on the spatial resolution desired, and the number of grooves illuminated on both diffraction gratings by each lenslet, or combination of lenslets in cases where they are grouped. 

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5. Monocular depth estimation based on deep learning for intraoperative guidance using surface-enhanced Raman scattering imaging

   https://doi.org/10.1364/PRJ.536871  

   Juhong, Aniwat; Li, Bo; Liu, Yifan; Yao, Cheng-You; Yang, Chia-Wei; Atique_Ullah, A_K M; Liu, Kunli; Lewandowski, Ryan P; Harkema, Jack R; Agnew, Dalen W; et al (January 2025, Photonics Research)

   Imaging of surface-enhanced Raman scattering (SERS) nanoparticles (NPs) has been intensively studied for cancer detection due to its high sensitivity, unconstrained low signal-to-noise ratios, and multiplexing detection capability. Furthermore, conjugating SERS NPs with various biomarkers is straightforward, resulting in numerous successful studies on cancer detection and diagnosis. However, Raman spectroscopy only provides spectral data from an imaging area without co-registered anatomic context. This is not practical and suitable for clinical applications. Here, we propose a custom-made Raman spectrometer with computer-vision-based positional tracking and monocular depth estimation using deep learning (DL) for the visualization of 2D and 3D SERS NPs imaging, respectively. In addition, the SERS NPs used in this study (hyaluronic acid-conjugated SERS NPs) showed clear tumor targeting capabilities (target CD44 typically overexpressed in tumors) by anex vivoexperiment and immunohistochemistry. The combination of Raman spectroscopy, image processing, and SERS molecular imaging, therefore, offers a robust and feasible potential for clinical applications. 

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