Fourier Transform Coherent Anti-Stokes Raman Scattering Spectroscopy: A Comprehensive Review.

Fourier transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy is a powerful spectroscopic method that combines the principles of Fourier transform spectroscopy with coherent anti-Stokes Raman scattering (CARS). This method stands out in spectroscopy for its ability to rapidly acquire coherent Raman spectra, achieving an impressive rate of over 10 000 spectra per second. The method involves scanning the optical delay between two femtosecond pulses; the initial pulse induces a vibrational coherence in the sample, while the subsequent pulse probes this coherence over increasing delays. The anti-Stokes scattering intensity generated is modulated by the vibrational dynamics of the sample, enabling the retrieval of Raman spectra through Fourier transformation. Over the past two decades, FT-CARS spectroscopy has undergone substantial evolution, paving the way for its application in a wide array of fields, including material analysis and flow cytometry. In this comprehensive Review, we explore the fundamental principles and diverse applications of FT-CARS spectroscopy and delve into the potential future advances and challenges associated with this emerging method.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay.

Amyloid-beta (Aß42) aggregates are characteristic Alzheimer's disease signatures, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aß42 fibrils. We correlate fibril architectures measured by SMOLM with their growth and decay over the course of 5 to 20 min visualized by single-molecule localization microscopy (SMLM). We discover that stable Aß42 fibrils tend to be well-ordered and signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order and disorder are signatures of growing and decaying fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve ß-sheet organization. SMOLM reveals that increased fibril architectural heterogeneity is correlated with dynamic remodeling and that large-scale fibril remodeling tends to originate from strongly heterogeneous local regions.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay.

Amyloid-beta (Aß42) aggregates are characteristic signatures of Alzheimer's disease, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aß42 fibrils. We quantify correlations between fibril architectures, measured by SMOLM, and their growth and decay visualized by single-molecule localization microscopy (SMLM). We discover that stable Aß42 fibrils tend to be well-ordered, signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order and disorder are signatures of growing and decaying Aß42 fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve ß-sheet organization. SMOLM reveals that increased heterogeneity in fibril architectures is correlated with more dynamic remodeling and that large-scale fibril remodeling tends to originate from local regions that exhibit strong heterogeneity.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets.

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Resolving the Nanoscale Structure of ß-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-ß fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8L and KFE8D and the pathological amyloid-beta peptide Aß42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aß42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-ß-rich fibrils.

Resolving the nanoscale structure of ß-sheet assemblies using single-molecule orientation-localization microscopy.

Synthetic peptides that self-assemble into cross-ß fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8L and KFE8D, and the pathological amyloid-beta peptide Aß42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aß42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross ß-rich fibrils.

Is AI essential? Examining the need for deep learning in image-activated sorting of Saccharomyces cerevisiae.

Artificial intelligence (AI) has become a focal point across a multitude of societal sectors, with science not being an exception. Particularly in the life sciences, imaging flow cytometry has increasingly integrated AI for automated management and categorization of extensive cell image data. However, the necessity of AI over traditional classification methods when extending imaging flow cytometry to include cell sorting remains uncertain, primarily due to the time constraints between image acquisition and sorting actuation. AI-enabled image-activated cell sorting (IACS) methods remain substantially limited, even as recent advancements in IACS have found success while largely relying on traditional feature gating strategies. Here we assess the necessity of AI for image classification in IACS by contrasting the performance of feature gating, classical machine learning (ML), and deep learning (DL) with convolutional neural networks (CNNs) in the differentiation of Saccharomyces cerevisiae mutant images. We show that classical ML could only yield a 2.8-fold enhancement in target enrichment capability, albeit at the cost of a 13.7-fold increase in processing time. Conversely, a CNN could offer an 11.0-fold improvement in enrichment capability at an 11.5-fold increase in processing time. We further executed IACS on mixed mutant populations and quantified target strain enrichment via downstream DNA sequencing to substantiate the applicability of DL for the proposed study. Our findings validate the feasibility and value of employing DL in IACS for morphology-based genetic screening of S. cerevisiae, encouraging its incorporation in future advancements of similar technologies.

In Situ Imaging of Catalytic Reactions on Tungsten Oxide Nanowires Connects Surface-Ligand Redox Chemistry with Photocatalytic Activity.

Semiconductor nanocrystals are promising candidates for generating chemical feedstocks through photocatalysis. Understanding the role of ligands used to prepare colloidal nanocrystals in catalysis is challenging due to the complexity and heterogeneity of nanocrystal surfaces. We use in situ single-molecule fluorescence imaging to map the spatial distribution of active regions along individual tungsten oxide nanowires before and after functionalizing them with ascorbic acid. Rather than blocking active sites, we observed a significant enhancement in activity for photocatalytic water oxidation after treatment with ascorbic acid. While the initial nanowires contain inactive regions dispersed along their length, the functionalized nanowires show high uniformity in their photocatalytic activity. Spatial colocalization of the active regions with their surface chemical properties shows that oxidation of ascorbic acid during photocatalysis generates new oxygen vacancies along the nanowire surface. We demonstrate that controlling surface-ligand redox chemistry during photocatalysis can enhance the active site concentration on nanocrystal catalysts.

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble Using a Polarized Vortex Point Spread Function.

Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule's wobble, simultaneously from a single image. Even when imaging dim emitters (∼500 photons detected), the polarized vortex PSF can obtain 12 nm localization precision, 4°-8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive by using molecular positions alone.

Single-Molecule Colocalization of Redox Reactions on Semiconductor Photocatalysts Connects Surface Heterogeneity and Charge-Carrier Separation in Bismuth Oxybromide.

The surface structure of semiconductor photocatalysts controls the efficiency of charge-carrier extraction during photocatalytic reactions. However, understanding the connection between surface heterogeneity and the locations where photogenerated charge carriers are preferentially extracted is challenging. Herein we use single-molecule fluorescence imaging to map the spatial distribution of active regions and quantify the activity for both photocatalytic oxidation and reduction reactions on individual bismuth oxybromide (BiOBr) nanoplates. Through a coordinate-based colocalization analysis, we quantify the spatial correlation between the locations where fluorogenic probe molecules are oxidized and reduced on the surface of individual nanoplates. Surprisingly, we observed two distinct photochemical behaviors for BiOBr particles prepared within the same batch, which exhibit either predominantly uncorrelated activity where electrons and holes are extracted from different sites or colocalized activity in which oxidation and reduction take place within the same nanoscale regions. By analyzing the emissive properties of the fluorogenic probes, we propose that electrons and holes colocalize at defect-deficient regions, while defects promote the selective extraction of one carrier type by trapping either electrons or holes. Although previous work has used defect engineering to enhance the activity of bismuth oxyhalides and other semiconductor photocatalysts for useful reductive half-reactions (e.g., CO2 or N2 reduction), our results show that defect-free regions are needed to promote both oxidation and reduction in fuel-generating photocatalysts that do not rely on sacrificial reagents.

Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy.

The resolution and accuracy of single-molecule localization microscopes (SMLMs) are routinely benchmarked using simulated data, calibration rulers, or comparisons to secondary imaging modalities. However, these methods cannot quantify the nanoscale accuracy of an arbitrary SMLM dataset. Here, we show that by computing localization stability under a well-chosen perturbation with accurate knowledge of the imaging system, we can robustly measure the confidence of individual localizations without ground-truth knowledge of the sample. We demonstrate that our method, termed Wasserstein-induced flux (WIF), measures the accuracy of various reconstruction algorithms directly on experimental 2D and 3D data of microtubules and amyloid fibrils. We further show that WIF confidences can be used to evaluate the mismatch between computational models and imaging data, enhance the accuracy and resolution of reconstructed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.

Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils.

Simultaneous measurements of single-molecule positions and orientations provide critical insight into a variety of biological and chemical processes. Various engineered point spread functions (PSFs) have been introduced for measuring the orientation and rotational diffusion of dipole-like emitters, but the widely used Cramér-Rao bound (CRB) only evaluates performance for one specific orientation at a time. Here, we report a performance metric, termed variance upper bound (VUB), that yields a global maximum CRB for all possible molecular orientations, thereby enabling the measurement performance of any PSF to be computed efficiently (~1000× faster than calculating average CRB). Our VUB reveals that the simple polarized standard PSF provides robust and precise orientation measurements if emitters are near a refractive index interface. Using this PSF, we measure the orientations and positions of Nile red (NR) molecules transiently bound to amyloid aggregates. Our super-resolved images reveal the main binding mode of NR on amyloid fiber surfaces, as well as structural heterogeneities along amyloid fibrillar networks, that cannot be resolved by single-molecule localization alone.

Single-Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes.

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules µm-2 ) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single-molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus "wobble") of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme-induced compositional heterogeneity within membranes, where NR within liquid-ordered vs. liquid-disordered domains shows a ≈4° difference in polar angle and a ≈0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid-lipid, lipid-protein, and lipid-dye interactions with single-molecule, nanoscale resolution.

Competing Activation and Deactivation Mechanisms in Photodoped Bismuth Oxybromide Nanoplates Probed by Single-Molecule Fluorescence Imaging.

Oxygen vacancies in semiconductor photocatalysts play several competing roles, serving to both enhance light absorption and charge separation of photoexcited carriers as well as act as recombination centers for their deactivation. In this Letter, we show that single-molecule fluorescence imaging of a chemically activated fluorogenic probe can be used to monitor changes in the photocatalytic activity of bismuth oxybromide (BiOBr) nanoplates in situ during the light-induced formation of oxygen vacancies. We observe that the specific activities of individual nanoplates for the photocatalytic reduction of resazurin first increase and then progressively decrease under continuous laser irradiation. Ensemble structural characterization, supported by electronic-structure calculations, shows that irradiation increases the concentration of surface oxygen vacancies in the nanoplates, reduces Bi ions, and creates donor defect levels within the band gap of the semiconductor particles. These combined changes first enhance photocatalytic activity by increasing light absorption at visible wavelengths. However, high concentrations of oxygen vacancies lower the photocatalytic activity both by introducing new relaxation pathways that promote charge recombination before photoexcited electrons can be extracted and by weakening binding of resazurin to the surface of the nanoplates.

Imaging the three-dimensional orientation and rotational mobility of fluorescent emitters using the Tri-spot point spread function.

Fluorescence photons emitted by single molecules contain rich information regarding their rotational motions, but adapting single-molecule localization microscopy (SMLM) to measure their orientations and rotational mobilities with high precision remains a challenge. Inspired by dipole radiation patterns, we design and implement a Tri-spot point spread function (PSF) that simultaneously measures the three-dimensional orientation and the rotational mobility of dipole-like emitters across a large field of view. We show that the orientation measurements done using the Tri-spot PSF are sufficiently accurate to correct the anisotropy-based localization bias, from 30 nm to 7 nm, in SMLM. We further characterize the emission anisotropy of fluorescent beads, revealing that both 20-nm and 100-nm diameter beads emit light significantly differently from isotropic point sources. Exciting 100-nm beads with linearly polarized light, we observe significant depolarization of the emitted fluorescence using the Tri-spot PSF that is difficult to detect using other methods. Finally, we demonstrate that the Tri-spot PSF detects rotational dynamics of single molecules within a polymer thin film that are not observable by conventional SMLM.

Super-resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin†T Molecules.

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super-resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid-ß aggregation, as well as the structural remodeling of amyloid-ß fibrils by the compound epi-gallocatechin gallate.