Investigating the Performances of Wide-Field Raman Microscopy with Stochastic Optical Reconstruction Post-Processing

Abstract

Super-resolution fluorescence microscopy based on localization algorithms has tremendously impacted the field of imaging by improving the spatial resolution of optical measurements with specific blinking fluorophores and concomitant reduction of acquisition time. In vibrational spectroscopy and imaging, various methods have been developed to surpass the diffraction limit including near-field scattering methods, such as in tip-enhanced Raman and infrared spectroscopies. Although these scanning-probe techniques can provide exquisite spatial resolution, they often require long acquisition times and tedious fabrication of nano-scale scanning probes. Herein, stochastic optical reconstruction microscopy (STORM) protocol is applied on Raman measurements acquired using a wide-field home-built microscopy setup. We explore how the fluctuations of the Raman signal acquired over a series of time-lapse images at specific spectral ranges can be exploited with STORM processing, possibly revealing details with improved spatial resolution, under lower irradiance and with faster acquisition speed that cannot be achieved in point scanning mode over the same field of view. Samples studied here include patterned silicon, polystyrene microspheres on a silicon wafer, and graphene on a silicon/silicon dioxide substrate. The outcome presents an effective way to collect Raman images at selected spectral ranges with spatial resolutions of ∼200 nm over a large field of view under 532 nm excitation together with an acquisition speed improved by two orders of magnitude and under a significantly reduced irradiance compared to confocal laser scanning acquisition.

Real-Time Observation of Photo-oxidation of Single MoS2 Flakes Using Stochastic Optical Reconstruction Microscopy

“…The open-source software RapidSTORM was used for the data treatment and Gaussian fitting of the point spread function (PSF) to localize the area where intensity fluctuations are present. Previous works show that the STORM postprocessing improves the spatial resolution to ∼20 nm for PL data, to ∼160 nm for Raman data, , and to 10 nm by using a surface-enhanced Raman spectroscopy (SERS) substrate . The intensity fluctuations in the case of Raman are related to molecular adsorption desorption, surface diffusion, or molecular reorientation at the surface in ambient conditions .…”

A Wide-Field Imaging Approach for Simultaneous Super-Resolution Surface-Enhanced Raman Scattering Bioimaging and Spectroscopy

“…Figure B,C shows the n = 0 and n = 1 FOV for a single frame and the average of 1000 frames, respectively. SIFs are a common occurrence not only with single-molecule SERS but also with single particles functionalized with a monolayer. ,, SIFs and temporal fluctuations associated with Raman scattering enable super-resolution/localization algorithms such as STORM to be applied by fitting the point spread function (PSF) of the nanoparticle signal fluctuations to a 2D-Gaussian. ,, By plotting the center of the PSF for each emitter, the position of the emitting center of the nanoparticle is localized to a few pixels. For particles with near-uniform coverage of the molecule, the emission is the weighted average of all emitters on the particle .…”