RainbowSTORM: an open-source ImageJ plug-in for spectroscopic single-molecule localization microscopy (sSMLM) data analysis and image reconstruction.

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 multimolecular and functional imaging capabilities. While several open-source software suites provide subdiffraction 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 AND IMPLEMENTATION: 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.

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

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.

Sub-10-nm distance measurements between fluorophores using photon-accumulation enhanced reconstruction (PACER).

Single-molecule localization microscopy (SMLM) precisely localizes individual fluorescent molecules within the wide field of view. However, the localization precision is fundamentally limited to around 20 nm due to the physical photon limit of individual stochastic single-molecule emissions. Using spectroscopic SMLM (sSMLM) to resolve their distinct fluorescence emission spectra, we can specifically distinguish and identify individual fluorophore, even the ones of the same type. Consequently, the reported photon-accumulation enhanced reconstruction (PACER) method accumulates photons over repeated stochastic emissions from the same fluorophore to significantly improve the localization precision. This work showed the feasibility of PACER by resolving quantum dots that were 6.1 nm apart with 1.7-nm localization precision. Next, a Monte Carlo simulation is used to investigate the success probability of PACER's classification process for distance measurements under different conditions. Finally, PACER is used to resolve and measure the lengths of DNA origami nanorulers with inter-molecular spacing as small as 6 nm. Notably, the demonstrated sub-2-nm localization precision bridges the detection range between Förster Resonance Energy Transfer (FRET) and conventional SMLM. Fully exploiting the underlying imaging capability can potentially enable high-throughput inter-molecular distance measurements over a large field of view.

High-Throughput Single-Molecule Spectroscopy Resolves the Conformational Isomers of BODIPY Chromophores.

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.

Multicolor super-resolution imaging using spectroscopic single-molecule localization microscopy with optimal spectral dispersion.

We developed 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 careful tuning of spectral dispersion in grating-based sSMLM permitted simultaneous three-color super-resolution imaging in fixed cells with a single objective lens at a 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.

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes.

Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. DM is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of DM in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2, which is characterized by nucleotide repeat expansions often greater than 5,000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, in the presence of muscleblind-like 1 (MBNL1) foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls and DM1 and DM2 subjects, and we differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. iPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High-resolution imaging revealed tight association between MBNL clusters and RNA foci in DM1. Ca2+ transients differed between DM1- and DM2 iPSC-derived cardiomyocytes, and each differed from healthy control cells. RNA-sequencing from DM1- and DM2 iPSC-derived cardiomyocytes revealed distinct misregulation of gene expression, as well as differential aberrant splicing patterns. Together, these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.

Method to identify and minimize artifacts induced by fluorescent impurities in single-molecule localization microscopy.

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.

Spectroscopic analysis beyond the diffraction limit.

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.

Low-cost laser speckle contrast imaging of blood flow using a webcam.

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.