We developed a transmission diffraction grating-based spectroscopic single-molecule localization microscopy (sSMLM) to collect the spatial and spectral information of single-molecule blinking events concurrently. We characterized the spectral heterogeneities of multiple far-red emitting dyes in a high-throughput manner using sSMLM. We also investigated the influence of spectral dispersion on the single-molecule identification performance of fluorophores with large spectral overlapping. The carefully tuning of spectral dispersion in grating-based sSMLM permitted simultaneous three-color super-resolution imaging in fixed cells with a single objective lens at relatively low photon budget. Our sSMLM has a compact optical design and can be integrated with conventional localization microscopy to provide add-on spectroscopic analysis capability.

Laser speckle contrast imaging has become a widely used tool for dynamic imaging of blood flow, both in animal models and in the clinic. Typically, laser speckle contrast imaging is performed using scientific-grade instrumentation. However, due to recent advances in camera technology, these expensive components may not be necessary to produce accurate images. In this paper, we demonstrate that a consumer-grade webcam can be used to visualize changes in flow, both in a microfluidic flow phantom and in vivo in a mouse model. A two-camera setup was used to simultaneously image with a high performance monochrome CCD camera and the webcam for direct comparison. The webcam was also tested with inexpensive aspheric lenses and a laser pointer for a complete low-cost, compact setup ($90, 5.6 cm length, 25 g). The CCD and webcam showed excellent agreement with the two-camera setup, and the inexpensive setup was used to image dynamic blood flow changes before and after a targeted cerebral occlusion.

A borondipyrromethene (BODIPY) chromophore is connected to a benzoxazole, benzothiazole, or nitrobenzothiazole heterocycle through an olefinic bridge with trans configuration. Rotation about the two [C−C] bonds flanking the olefinic bridge occurs with fast kinetics in solution, leading to the equilibration of four conformational isomers for each compound. Ensemble spectroscopic measurements in solutions fail to distinguish the coexisting isomers. They reveal instead averaged absorption and emission bands with dependence of the latter on the excitation wavelength. Using high-throughput single-molecule spectroscopy, two main populations of single molecules with distinct spectral centroids are observed for each compound on glass substrates. Computational analyses suggest the two populations of molecules to be conformational isomers with antiperiplanar and periplanar arrangements of the BODIPY chromophores about its [C−C] bond to the olefinic bridge. Thus, statistical analysis of multiple single-molecule emission spectra can discriminate stereoisomers that would otherwise be impossible to distinguish by ensemble measurements alone.

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Self-assembled nanocarriers have inspired a range of applications for bioimaging, diagnostics, and drug delivery. The noninvasive visualization and characterization of these nanocarriers are important to understand their structure to function relationship. However, the quantitative visualization of nanocarriers in the sample's native environment remains challenging with the use of existing technologies. Single-molecule localization microscopy (SMLM) has the potential to provide both high-resolution visualization and quantitative analysis of nanocarriers in their native environment. However, nonspecific binding of fluorescent probes used in SMLM can introduce artifacts, which imposes challenges in the quantitative analysis of SMLM images. We showed the feasibility of using spectroscopic point accumulation for imaging in nanoscale topography (sPAINT) to visualize self-assembled polymersomes (PS) with molecular specificity. Furthermore, we analyzed the unique spectral signatures of Nile Red (NR) molecules bound to the PS to reject artifacts from nonspecific NR bindings. We further developed quantitative spectroscopic analysis for cluster extraction (qSPACE) to increase the localization density by 4-fold compared to sPAINT; thus, reducing variations in PS size measurements to less than 5%. Finally, using qSPACE, we quantitatively imaged PS at various concentrations in aqueous solutions with ∼20 nm localization precision and 97% reduction in sample misidentification relative to conventional SMLM.

. The existence of fluorescent impurities has been a long-standing obstacle in single-molecule imaging, which results in sample misidentification and higher localization uncertainty. Spectroscopic single-molecule localization microscopy can record the full fluorescent spectrum of every stochastic single-molecule emission event. This capability allows us to quantify the spatial and spectral characteristics of fluorescent impurities introduced by sample preparation steps, based on which we developed a method to effectively separate fluorescent impurities from target molecules.

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.

Summary Spectroscopic single-molecule localization microscopy (sSMLM) simultaneously captures the spatial locations and full spectra of stochastically emitting fluorescent single-molecules. It provides an optical platform to develop new multi-molecular and functional imaging capabilities. While several open-source software suites provide sub-diffraction localization of fluorescent molecules, software suites for spectroscopic analysis of sSMLM data remain unavailable. RainbowSTORM is an open-source ImageJ/FIJI plug-in for end-to-end spectroscopic analysis and visualization for sSMLM images. RainbowSTORM allows users to calibrate, preview, and quantitatively analyze emission spectra acquired using different reported sSMLM system designs and fluorescent labels. Availability RainbowSTORM is a java plug-in for ImageJ (https://imagej.net)/FIJI (http://fiji.sc) freely available through: https://github.com/FOIL-NU/RainbowSTORM. RainbowSTORM has been tested with Windows and Mac operating systems and ImageJ/FIJI version 1.52. Supplementary information Supplementary data are available at Bioinformatics online.