Chemically imaging bacteria with super-resolution SERS on ultra-thin silver substrates

Abstract

Plasmonic hotspots generate a blinking Surface Enhanced Raman Spectroscopy (SERS) effect that can be processed using Stochastic Optical Reconstruction Microscopy (STORM) algorithms for super-resolved imaging. Furthermore, by imaging through a diffraction grating, STORM algorithms can be modified to extract a full SERS spectrum, thereby capturing spectral as well as spatial content simultaneously. Here we demonstrate SERS and STORM combined in this way for super-resolved chemical imaging using an ultra-thin silver substrate. Images of gram-positive and gram-negative bacteria taken with this technique show excellent agreement with scanning electron microscope images, high spatial resolution at <50 nm, and spectral SERS content that can be correlated to different regions. This may be used to identify unique chemical signatures of various cells. Finally, because we image through as-deposited, ultra-thin silver films, this technique requires no nanofabrication beyond a single deposition and looks at the cell samples from below. This allows direct imaging of the cell/substrate interface of thick specimens or imaging samples in turbid or opaque liquids since the optical path doesn’t pass through the sample. These results show promise that super-resolution chemical imaging may be used to differentiate chemical signatures from cells and could be applied to other biological structures of interest.

Advances in Imaging Specific Mediators of Inflammatory Bowel Disease

“…A framework satisfying these criteria was recently developed by Earle et al [ 58 ], where high-resolution quantification of the spatial organization of the gut microbiome revealed that changes in the proximity of microbes to the epithelium are sufficient to induce increased expression of key inflammatory markers, despite negligible shifts in microbiota composition. Most recently, chemical imaging techniques exploiting a blinking surface enhanced Raman spectroscopy (SERS) have been used to visualize bacteria in vitro with exquisite resolution, and have demonstrated the ability to differentiate between the chemical signatures of different bacteria [ 59 ]. MRI, CT, fluorescence/bioluminescence imaging, and PET have also been applied to investigate the activity of bacteria in vivo; however, these typically only provide indirect information about bacterial activity by inference from immune function [ 60 , 61 ].…”

Recent progress of surface-enhanced Raman spectroscopy for subcellular compartment analysis

“…Lindquist's group 149 combined SERS direct detection with STORM by a 700 nm hexagonal periodic metal nanopore SERS hotspot array with a unit diameter of 150 nm to match a 660 nm laser, which causes the hotspot scintillation locating at 10 nm regions of the substrate to achieve the SERS-STORM imaging. They further employed an ultrathin silver island film to identify the molecular structures of the cell walls of two types of bacteria (Gram-negative bacteria and Gram-positive bacteria) 150 . So far, there are no reports on SERS supra-resolution imaging to visualize the distributions of intracellular compartments with the help of SERS tags.…”

Surface‐Enhanced Raman Scattering for Rapid Detection and Characterization of Antibiotic‐Resistant Bacteria

“…Recently, a technique employing stochastic optical reconstruction microscopy (STORM) algorithms was used to process SERS spectra, enabling chemical‐specific, super‐resolution images . The spatial location of SERS “hot‐spots” were dynamically shifted over nanohole arrays or roughened Ag films with subdiffraction‐limited resolution using a spatial light modulator . The dynamically shifted SERS “hot‐spots” generated “blinking” events where SERS spectral information was collected.…”

“…Over the timescale of minutes, about 4000 frames of SERS “blinking” events were collected, and processed with STORM to generate an image. SERS‐STORM images were collected for B. subtilis , E. coli , and M. luteus , and compared against conventional diffraction‐limited SERS and SEM imaging . Excellent agreement between the SEM and SERS‐STORM images was observed.…”