Spectroscopic analysis beyond the diffraction limit

Abstract

The recent surge in spectroscopic Single-Molecule Localization Microscopy (sSMLM) offers exciting new capabilities for combining single molecule imaging and spectroscopic analysis. Through the synergistic integration of super-resolution optical microscopy and single-molecule spectroscopy, sSMLM offers combined strengths from both fields. By capturing the full spectra of single molecule fluorescent emissions, sSMLM can distinguish minute spectroscopic variations from individual fluorescent molecules while preserving nanoscopic spatial localization precision. It can significantly extend the coding space for multi-molecule super-resolution imaging. Furthermore, it has the potential to detect spectroscopic variations in fluorescence emission associated with molecular interactions, which further enables probing local chemical and biochemical inhomogeneities of the nano-environments. In this review, we seek to explain the working principle of sSMLM technologies and the status of sSMLM techniques towards new super-resolution imaging applications.

Multimodal single-molecule microscopy with continuously controlled spectral resolution

“…As a result, the FOV sizes of single-molecule spectral imaging schemes are small, ranging between 100 and 515 μ m 2 ( 7 , 9 , 10 , 17 , 18 , 24 ), 30–144-fold smaller compared with sequential color channel imaging. The limit on marker density can be removed by applying super-resolution techniques in which only a sparse subset of markers is emitting in each frame of a multiframe acquisition ( 18 , 19 , 25 , 26 , 27 ). This, however, is achieved at the cost of significantly increased acquisition time per FOV because of the numerous frames required for reproducing the final image.…”

“…Moreover, the spectral range of detected molecules still needs to be contained to small pixel areas to avoid overlaps in the sparse image and to maintain workable single-molecule sensitivity. Therefore, previous spectral super-resolution works were restricted to the spectral resolution of dyes within the same spectral window (usually far-red) excited with a single excitation laser ( 7 , 8 , 18 , 19 , 22 , 26 ), not exploiting the range of available dyes and limited to working with high spectral resolutions and lower throughputs.…”

Accelerating multicolor spectroscopic single-molecule localization microscopy using deep learning

“…Typically, conventional SMLM requires excellent emission spectral separation (∼100 nm) between dyes to obtain sequential multicolor imaging with minimal cross-talk [5,6]. Recently developed spectroscopic SMLM (sSMLM) simultaneously extracts the spatial locations as well as corresponding spectral information of single-molecule blinking events, offering simultaneous multicolor imaging of multi-stained samples [5,[7][8][9][10][11][12][13]. In sSMLM, a dispersive optical component, such as a grating or prism, is used to obtain the single-molecule emission spectrum while corresponding spatial information is collected in a separate optical path [5,8].…”

“…Here, we present a novel approach to achieve fast multicolor sSMLM imaging using deep learning. The experimental setup, data acquisition, and spectral classification methods remain the same as previously reported [8,12,13], except that fewer frames (spatial and spectral images) are acquired from multi-dye stained samples, which in turn accelerates the imaging speed. The lower number of frames provides less information on fluorophore localizations and corresponding spectra, not enough for the existing method to extract the fine structures in the sample properly.…”

Super-Resolution Imaging of Self-Assembled Nanocarriers Using Quantitative Spectroscopic Analysis for Cluster Extraction

“…Recently developed spectroscopic SMLM or sSMLM − techniques distinguish fluorescent signals from individual molecules according to their emission spectra. They provide new strategies for multicolor ,,− and functional super-resolution imaging. ,,− Furthermore, spectral signatures have been used to identify and remove the influence from fluorescent impurities .…”