Spectrally Resolved Super Resolution Raman Imaging of pH Probes
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Date
2025-05
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The Ohio State University
Abstract
Spectrally resolved super resolution wide field Raman spectroscopy is an emerging tool for studying chemical changes with nanometer spatial resolution. This thesis applies super resolution algorithms to the spectral regions of wide field spectroscopic images to improve frequency assignments and provide a powerful new tool within the field of Raman microscopy. The ability to simultaneously acquire precise spatial and spectral information is demonstrated by monitoring the pH-sensitive frequency shift in 4-mercaptobenzoic acid (4-MBA) surface enhanced Raman spectra. Spectral stochastic optical reconstruction microscopy (STORM) was achieved through gaussian fitting of intensity values in the first order diffraction signal, or spectrum, recorded from a qCMOS detector, calibration of the horizontal-axis pixel position to Raman shift, quantification of spectral fluctuations throughout the spectrum using histograms, and consequent spectral reconstruction. The precision of this spectral STORM analysis was compared to traditional second derivative and curve-fitting methods of improving frequency assignments and determined to yield the most precise pH determination when analyzing the frequency difference between two vibrational modes in 4-MBA. The difference between the two prominent modes in the 4-MBA SERS spectra was correlated to the pH of different buffer solutions to create Raman titration curves using both spectrally resolved wide field Raman spectroscopy and confocal Raman spectroscopy. Similar frequency differences were obtained with both methods, but more precise characterization of the nanoparticles’ pH was obtained with spectral STORM wide field Raman spectroscopy. This approach was used to investigate the 4-MBA SERS spectra within in-vitro cell samples, where spectral STORM analysis successfully determined the frequency differences in 4-MBA. Future experiments are necessary to better understand aggregation effects of gold nanospheres within cell samples and their effects on the SERS spectra.
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Keywords
Raman, Surface Enhanced Raman Spectroscopy, Cellular Imaging, Microscopy, 4-Mercaptobenzoic Acid, pH Sensing