Spectral precision improvement with demagnifying spectral images in spectroscopic nanoscopy.

Spectroscopic nanoscopy (SN) has been recognized as a key functional imaging tool in cell biology and chemistry because it offers the unique capability to simultaneously obtain the spatial and spectral information for single molecules. However, it has an intrinsic issue in using the limited photon budget from single emitters divided into two imaging channels to concurrently acquire spatial and spectral images. Accordingly, this issue lowers the spatial localization and spectral precision. Although several techniques have been introduced to improve the spatial precision in SN, improving the spectral precision has been overlooked so far. Here we propose a method to improve the spectral precision by optically manipulating the width of the spectroscopic signatures using a demagnifier. We evaluate its performance using numerical simulations with systematic investigations of several underlying optimal parameters such as the demagnification factor and the integration width in the proposed configuration. We also present achievable spectral precision values with different signal and background levels. Compared to the existing SN system, the 3× demagnifier-based configuration shows an approximate 35% improvement, from 2.9 nm to 1.9 nm, in the spectral precision at the 1000 photons signal level.

Theoretical and experimental study on the detection limit of the micro-ring resonator based ultrasound point detectors.

Combining the diffusive laser excitation and the photoacoustic signals detection, photoacoustic computed tomography (PACT) is uniquely suited for deep tissue imaging. A diffraction-limited ultrasound point detector is highly desirable for maximizing the spatial resolution and the field-of-view of the reconstructed volumetric images. Among all the available ultrasound detectors, micro-ring resonator (MRR) based ultrasound detectors offer the lowest area-normalized limit of detection (nLOD) in a miniature form-factor, making it an ideal candidate as an ultrasound point detector. However, despite their wide adoption for photoacoustic imaging, the underlying signal transduction process has not been systematically studied yet. Here we report a comprehensive theoretical model capturing the transduction of incident acoustic signals into digital data, and the associated noise propagation process, using experimentally calibrated key process parameters. The theoretical model quantifies the signal-to-noise ratio (SNR) and the nLOD under the influence of the key process variables, including the quality factor (Q-factor) of the MRR and the driving wavelength. While asserting the need for higher Q-factors, the theoretical model further quantifies the optimal driving wavelength for optimizing the nLOD. Given the MRR with a Q-factor of 1 × 105, the theoretical model predicts an optimal SNR of 30.1 dB and a corresponding nLOD of 3.75 × 10-2 mPa mm2/Hz1/2, which are in good agreement with the experimental measurements of 31.0 dB and 3.39 × 10-2 mPa mm2/Hz1/2, respectively. The reported theoretical model can be used in guiding the optimization of MRR-based ultrasonic detectors and PA experimental conditions, in attaining higher imaging resolution and contrast. The optimized operating condition has been further validated by performing PACT imaging of a human hair phantom.

Switchable and Functional Fluorophores for Multidimensional Single-Molecule Localization Microscopy.

Multidimensional single-molecule localization microscopy (mSMLM) represents a paradigm shift in the realm of super-resolution microscopy techniques. It affords the simultaneous detection of single-molecule spatial locations at the nanoscale and functional information by interrogating the emission properties of switchable fluorophores. The latter is finely tuned to report its local environment through carefully manipulated laser illumination and single-molecule detection strategies. This Perspective highlights recent strides in mSMLM with a focus on fluorophore designs and their integration into mSMLM imaging systems. Particular interests are the accomplishments in simultaneous multiplexed super-resolution imaging, nanoscale polarity and hydrophobicity mapping, and single-molecule orientational imaging. Challenges and prospects in mSMLM are also discussed, which include the development of more vibrant and functional fluorescent probes, the optimization of optical implementation to judiciously utilize the photon budget, and the advancement of imaging analysis and machine learning techniques.

Design strategy for a dual-wedge prism imaging spectrometer in spectroscopic nanoscopy.

Spectroscopic single-molecule localization microscopy (sSMLM, or spectroscopic nanoscopy) has been established as a key tool in functional super-resolution imaging by providing spatial and spectral information of single molecules at nanoscale resolution. A recently developed dual-wedge prism (DWP) imaging spectrometer, a monolithic optical component, has broadened the accessibility of sSMLM with an improved imaging resolution of more than 40%. It also improved the system reliability by reducing the number of discrete optical components. However, achieving its optimal performance requires the comprehensive understanding of the underlying constraints of the key system parameters, such as the refractive index of the DWP, spectral dispersion (SD), axial separation for three-dimensional (3D) biplane reconstruction, and the overall dimensional constraints. In this work, we present a generalized design principle for the DWP imaging spectrometer. Specifically, we develop the theoretical framework capturing the influence of the primary design parameters, including the achievable SD and localization performance, for different design cases. It further establishes the workflow to design and optimize the DWP imaging spectrometer for better multi-color functional imaging. This will give practical guidance for users to easily design the DWP imaging spectrometer, allowing for straightforward 3D sSMLM implementation.

Monolithic dual-wedge prism-based spectroscopic single-molecule localization microscopy.

By manipulating the spectral dispersion of detected photons, spectroscopic single-molecule localization microscopy (sSMLM) permits concurrent high-throughput single-molecular spectroscopic analysis and imaging. Despite its promising potential, using discrete optical components and managing the delicate balance between spectral dispersion and spatial localization compromise its performance, including non-uniform spectral dispersion, high transmission loss of grating, high optical alignment demands, and reduced precision. We designed a dual-wedge prism (DWP)-based monolithic imaging spectrometer to overcome these challenges. We optimized the DWP for spectrally dispersing focused beam without deviation and with minimal wavefront error. We integrated all components into a compact assembly, minimizing total transmission loss and significantly reducing optical alignment requirements. We show the feasibility of DWP using ray-tracing and numerical simulations. We validated our numerical simulations by experimentally imaging individual nanospheres and confirmed that DWP-sSMLM achieved much improved spatial and spectral precisions of grating-based sSMLM. We also demonstrated DWP-sSMLM in 3D multi-color imaging of cells.

Improving spatial precision and field-of-view in wavelength-tagged single-particle tracking using spectroscopic single-molecule localization microscopy.

Spectroscopic single-molecule localization microscopy (sSMLM) generates super-resolution images of single molecules while simultaneously capturing the spectra of their fluorescence emissions. However, sSMLM splits photons from single-molecule emissions into a spatial channel and a spectral channel, reducing both channels' precisions. It is also challenging in transmission grating-based sSMLM to achieve a large field-of-view (FOV) and avoid overlap between the spatial and spectral channels. The challenge in FOV has further significance in single-molecule tracking applications. In this work, we analyzed the correlation between the spatial and spectral channels in sSMLM to improve its spatial precision, and we developed a split-mirror assembly to enlarge its FOV. We demonstrate the benefits of these improvements by tracking quantum dots. We also show that we can reduce particle-identification ambiguity by tagging each particle with its unique spectral characteristics.

Investigating Single-Molecule Fluorescence Spectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule Spectroscopy.

We experimentally investigated several intramolecular coordinate and environmental changes as potential causes of single-molecule fluorescence spectral heterogeneities (smFSH). We developed a high-throughput single-molecule spectroscopy method to analyze more than 5000 single-molecule emission spectra from each of 9 commonly used fluorophores with different structural rigidities and deposited on substrates with different polarities. We observed an unexpectedly high smFSH from structurally rigid Rhodamine B compared with a structurally flexible Cyanine dye-Alexa Fluor 647. Based on experimentally measured smFSH, we ruled out the system's noise uncertainty, single-molecule spectral diffusion, and environmental polarity as the primary causes of the high smFSH. We found that the rotational flexibility of N,N-dialkylated groups contributed to the smFSH. With the high smFSH observed in structurally more rigid model fluorophores, we speculated that other intramolecular coordinate and environmental changes might also contribute to the high smFSH in Rhodamines.

Super-resolution imaging of flat-mounted whole mouse cornea.

Super-resolution microscopy revolutionized biomedical research with significantly improved imaging resolution down to the molecular scale. To date, only limited studies reported multi-color super-resolution imaging of thin tissue slices mainly because of unavailable staining protocols and incompatible imaging techniques. Here, we show the first super-resolution imaging of flat-mounted whole mouse cornea using single-molecule localization microscopy (SMLM). We optimized immunofluorescence staining protocols for ß-Tubulin, Vimentin, Peroxisome marker (PMP70), and Histone-H4 in whole mouse corneas. Using the optimized staining protocols, we imaged these four intracellular protein structures in the epithelium and endothelium layers of flat-mounted mouse corneas. We also achieved simultaneous two-color spectroscopic SMLM (sSMLM) imaging of ß-Tubulin and Histone-H4 in corneal endothelial cells. The spatial localization precision of sSMLM in these studies was around 20-nm. This work sets the stage for investigating multiple intracellular alterations in corneal diseases at a nanoscopic resolution using whole corneal flat-mount beyond cell cultures.

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.

Symmetrically dispersed spectroscopic single-molecule localization microscopy.

Spectroscopic single-molecule localization microscopy (sSMLM) was used to achieve simultaneous imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from a reduced photon budget because the photons from individual stochastic emissions are divided into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically dispersed sSMLM, or SDsSMLM, to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Compared with the existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multicolour imaging of fixed cells and three-dimensional single-particle tracking using SDsSMLM. SDsSMLM enables more precise spectroscopic single-molecule analysis in broader cell biology and material science applications.

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.

Three-dimensional biplane spectroscopic single-molecule localization microscopy.

Spectroscopic single-molecule localization microscopy (sSMLM) captures the full emission spectra of individual molecules while simultaneously localizing their spatial locations at a precision greatly exceeding the optical diffraction limit. To achieve this, sSMLM uses a dispersive optical component to separate the emitted photons into dedicated spatial and spectral imaging channels for simultaneous acquisition. While adding a cylindrical lens in the spatial imaging channel enabled three-dimensional (3D) imaging in sSMLM, the inherent astigmatism leads to technical hurdles in spectral calibration and nonuniform lateral resolution at different depths. We found that implementing the biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows for more efficient use of the limited photon budget and provides homogeneous lateral resolution compared with the astigmatism-based method using a cylindrical lens. Here we report 3D biplane sSMLM and demonstrate its multi-color 3D imaging capability by imaging microtubules and mitochondria in fixed COS-7 cells immunostained with Alexa Fluor 647 and CF 660C dyes, respectively. We showed a lateral localization precision of 20 nm at an average photon count of 550, a spectral precision of 4 nm at an average photon count of 1250, and an axial localization resolution of 50 nm.

Far-Red Photoactivatable BODIPYs for the Super-Resolution Imaging of Live Cells.

The photoinduced disconnection of an oxazine heterocycle from a borondipyrromethene (BODIPY) chromophore activates bright far-red fluorescence. The high brightness of the product and the lack of autofluorescence in this spectral region allow its detection at the single-molecule level within the organelles of live cells. Indeed, these photoactivatable fluorophores localize in lysosomal compartments and remain covalently immobilized within these organelles. The suppression of diffusion allows the reiterative reconstruction of subdiffraction images and the visualization of the labeled organelles with excellent localization precision. Thus, the combination of photochemical, photophysical and structural properties designed into our fluorophores enable the visualization of live cells with a spatial resolution that is inaccessible to conventional fluorescence imaging.

Theoretical analysis of spectral precision in spectroscopic single-molecule localization microscopy.

Spectroscopic single-molecule localization microscopy (sSMLM) is a novel super-resolution imaging technology, which simultaneously records the nanoscopic location and the corresponding full emission spectrum of every stochastic single-molecule emission event. This spectroscopic imaging capability of sSMLM necessitates the establishment of a theoretical foundation of the newly introduced spectral precision and to guide the system design and optimization. Based on numerical simulation and analytical solution, we introduced such a theoretical model to analyze spectral precision by considering the main system parameters, including signal and background shot noises, readout noise, and the spectral calibration procedure. Using this model, we demonstrated the delicate balance among these parameters in achieving the optimal spectral precision and discovered that the best spectral precision can only be achieved at a particular system spectral dispersion. For example, with a given signal of 3000 photons and a readout noise of 2 e-, a system spectral dispersion of 1.6 nm/pixel is required for sSMLM to achieve the highest spectral precision of 1.31 nm.

Formation and decay of charge carriers in aggregate nanofibers consisting of poly(3-hexylthiophene)-coated gold nanoparticles.

Thin nanofibers (NFs) of J-dominant aggregates with a thickness of 15 nm and thick NFs of H-dominant aggregates with a thickness of 25 nm were fabricated by the self-assembly of poly(3-hexylthiophene)-coated gold nanoparticles. The formation and decay dynamics of the charge carriers, which are dependent on the aggregate types of NFs, was investigated by time-resolved emission and transient-absorption spectroscopy. With increasing excitation energy, the fraction of the fast emission decay component decreased, suggesting that the fast formation of polaron pairs (PP), localized (LP), and delocalized polarons (DP) results from higher singlet exciton states produced by the singlet fusion. The faster decay dynamics of DP and LP in the thick NFs than in thin NFs is due to the increased delocalization of DP and LP. As the interchain aggregation is weaker than intrachain aggregation, PP decays faster in thin NFs than in thick NFs. In both thin and thick NFs, although triplet (T1) excitons were barely observed with excitation at 532 nm on a nanosecond time scale, they were observed with excitation at 355 nm, showing that T1 excitons within NFs are generated mainly through the singlet fission from a higher singlet exciton state rather than through intersystem crossing.

Quantum Beats and Phase Shifts in Two-Dimensional Electronic Spectra of Zinc Naphthalocyanine Monomer and Aggregate.

The origin of quantum coherence in two-dimensional (2D) electronic spectra of molecular aggregates and light-harvesting complexes still remains an open question. In particular, it could be challenging to distinguish between electronic and vibrational coherences for a coupled system, where both degrees of freedom can be simultaneously excited. In this Letter, we examine quantum beats in the 2D spectra of zinc naphthalocyanine (ZnNc) aggregate and monomer, and compare their characteristic features in terms of the frequency and relative phase of diagonal and off-diagonal amplitude oscillations. The long-lasting oscillating components (>1 ps) at 600-700 cm(-1) observed in both the aggregate and monomer are found to be attributed to the vibrational coherence. The wide phase variations of the 2D spectral amplitude oscillations are observed not just in the aggregate but also in the monomer state. This suggests that the unusual 90° phase shift may be attributed to neither quantum population-to-coherence transfer nor vibronic exciton coupling.