Abstract
Early nucleation events during α-synuclein (αS) aggregation remain challenging to investigate because conventional fluorescence assays lose sensitivity in highly concentrated biomolecular environments. Here, we present Thioflavin T (ThT) lasing spectroscopy in a Fabry-Pérot microcavity as a sensitive methodological approach for probing αS aggregation within condensate-like microenvironments that more closely mimic intracellular molecular crowding. Beyond conventional lasing-threshold analysis, we introduce the first automated frame-by-frame analysis of ThT lasing spectra using machine-vision algorithms, enabling statistical evaluation of individual lasing events throughout the aggregation process. As a demonstration of this methodology, we investigate how physiologically abundant monovalent and divalent cations (Na+, K+, Mg2+, and Ca2+) modulate the nucleation phase of highly concentrated αS. Ion-specific differences were identified in both lasing-threshold kinetics and spectral evolution. Monovalent cations produced complex, non-monotonic threshold behavior accompanied by transient spectral shifts, consistent with incomplete electrostatic screening and dynamic accessibility of ThT to early oligomeric species. In contrast, divalent cations generated more stable lasing wavelengths and distinct threshold regimes, reflecting stronger electrostatic screening. These results demonstrate that automated analysis of ThT lasing provides complementary information beyond conventional fluorescence, enabling sensitive interrogation of nucleation processes in highly crowded protein systems. The presented methodology establishes lasing spectroscopy combined with machine-vision analysis as a powerful platform for investigating early protein aggregation under physiologically relevant condensed conditions.
Bisulfite (HSO3-) is widely used as a preservative and antioxidant in the food industry and chemical manufacturing, yet its excessive residues pose risks to food quality, environmental safety, and human health. To achieve rapid, selective, and quantitative detection of HSO3-, a ratiometric fluorescent probe based on a benzothiazole-carbazole unit and a benzoindole ion was developed. Upon reaction with HSO3-, the probe exhibits a distinct dual-emission response, fluorescence intensity decreases at 612 nm and increases at 467 nm, enabling robust ratiometric sensing (F467/F612). Owing to its pronounced fluorescence modulation (emission signal change > 145 nm) and high sensitivity (limit of detection = 0.62μM), the probe demonstrates reliable performance in aqueous samples and has been successfully integrated into portable test strips and swabs. Furthermore, when coupled with a smartphone-based colorimetric application software, the probe allows convenient and real-time quantification of both gaseous SO2and aqueous HSO3-across environmental and food-related matrices. This work thus delivers a practical, instrument-free platform for sulfite monitoring in complex real-world settings.
The study demonstrates significant splitting of the impurity luminescence peak of cadmium sulfide quantum dots (QDs) doped with copper and manganese ions in polymer composites on substrates with silver nanoparticles obtained by chemical deposition. The influence of polymer concentration and QD type on the splitting is studied. Our results demonstrate that the probability of splitting occurrence and the spectral separation between the peaks are governed not by the optical density of the film or the size of the silver islands, but by the inter-island distance, highlighting the critical role of spatial arrangement in the collective optical response of the system. It is hypothesized that the observed effect can be caused by coupling between the luminescent nanoparticles, which have long luminescence lifetimes, and the silver nanoislands, which act as nanoresonators.
Cesium lead halide perovskite nanocrystals (NCs) are emerging as a promising material for next-generation optoelectronic systems. This work investigates how variations in particle size and halide composition affect the structural and optical properties of CsPbX3NCs. CsPbBr3NCs of different sizes were prepared using a hot-injection procedure, with the ability to regulate the quenching time, and it was found that larger NCs have a smaller band gap. The crystal structure was found to be cubic. The influence of the halide composition was analyzed by synthesizing CsPbCl3, CsPbBr3, and CsPbI3under the same conditions. CsPbCl3showed the broadest bandgap and shortest lifetime, CsPbI3the closest bandgap and longest lifetime, and CsPbBr3showed a good compromise between bandgap, luminescence intensity, and structural stability. Optical tuning was also confirmed with respect to size and composition, with reference to UV-visible absorption and photoluminescence tests; blue, green, and red emissions were observed in the case of CsPbCl3, CsPbBr3, and CsPbI3, respectively, with UV illumination. These findings demonstrate the synergistic nature of particle size and halide composition in designing the structural and optical characteristics of CsPbX3NCs to offer principles of perovskite-based devices.
Single-molecule orientation localization microscopy (SMOLM) is an optical means to measure complex transport in charged and crowded conditions, such as inside cells or polymer materials. SMOLM extracts time- and space-dependent three-dimensional orientation information from dipole emitters. Achieving simultaneous position-orientation resolution with high photon efficiency remains a central challenge in SMOLM instrument design. We developed an optical fluorescence microscope that uses the double-helix point spread function (DHPSF) to localize dipole emitters in six dimensions (6D), delineated by spatial and dynamic orientational parameters. Furthermore, we developed a fused deep learning approach based on existing neural network architectures to localize dipole emitters in 6D. Our microscope enables simultaneous 6D localization of single fluorophores, achieving a median spatial precision of 10 nm and angular precision below 10° across most of orientation space, except for the azimuthal angle at high polar angles where the DHPSF exhibits known optical degeneracies. We demonstrate our approach by localizing single rhodamine B molecules in poly(methyl methacrylate) films. The recovered orientations shownear 90° and small wobble angles. We also demonstrate 6D SMOLM of a spherical supported lipid bilayer, where despite the low signal, out-of-training distribution of the experimental data, we observe clearly ordered orientation of Nile red molecules within the membrane.
Fluorescence recovery after photobleaching (FRAP) is widely used to characterize diffusion in cells, but quantitative interpretation of the data in small prokaryotes requires explicitly accounting for cell geometry. While this has been successfully achieved for spherical and rod-shaped bacteria, analytical approaches developed in these cases are not directly applicable to cells with more complex morphologies. Here, we explore the application of FRAP to helical bacteria using simulations. We show that half-compartment FRAP experiments, where one-half of the cell is photobleached, provide a robust means of characterizing fast protein diffusion. To help with the practical implementation of this technique, we established the relationship between the diffusion coefficient and characteristic fluorescence recovery time as a function of cell length and helical parameters, and for two different ways of estimating the recovery time. As a first application, we report measurements of the diffusion coefficient of the fluorescent protein, mNeonGreen, in the helical bacteriumParamagnetospirillum magneticumAMB-1. We find it to bein isosmotic conditions, not significantly different from the value measured inEscherichia coli. Although developed for helical bacteria, including spirilla, spirochetes, and vibrios, our framework can readily be extended to cells or compartments with other geometries.
Many ecosystems thrive in near-0 °C conditions, and the mechanisms supporting life in these conditions remain understudied due to the challenges in reproducing such environments in laboratory conditions. One such example is polar organisms, that have adapted their entire lifecycle to operate below freezing temperatures through largely unknown cellular adaptations. As rapid polar warming threatens these species, elucidating their survival strategies is increasingly urgent. Fluorescence-based optical microscopy has been central to the understanding of the dynamic processes sustaining life at the cellular level, yet most imaging approaches have been developed and validated for conditions near mammalian physiological temperatures. Imaging at low temperature introduces a distinct physical regime in which molecular motion, membrane organisation, protein conformational dynamics, and fluorophore photophysics are fundamentally altered. As a result, imaging tools, fluorescent probes, and super-resolution methods optimised at 37 °C often fail when applied near 0 °C, or they report biased information. Here, we examine the conceptual, technical, and practical challenges associated with live-cell fluorescence microscopy at cold temperatures. We discuss when and why common imaging modalities and labelling strategies break down, and how probe behaviour becomes tightly coupled to local changes in physicochemical environment. We offer a perspective on new biological questions that become accessible for study with a microscopy platform optimised for imaging in cold conditions. We highlight trade-offs in current temperature-control strategies and identify unmet needs in fluorophore design, instrument engineering, and quantitative standards. By framing cold microscopy as a distinct operational regime rather than an extension of conventional live-cell imaging, this perspective aims to guide the development of robust tools for studying biological systems near-0 °C conditions.
In the present work, we developed a data-driven algorithm for the automated segmentation of fluorescence lifetime imaging microscopy (FLIM) images, enhancing the analysis of multifunctionalized nanoparticles (NPs) within living cancer cells. FLIM, a powerful microscopy technique, generates images that capture the fluorescence lifetime across a sample at the pixel level, revealing critical details about the molecular environment. Traditionally, FLIM image analysis has relied on manual segmentation with the phasor plot approach, a graphical representation of FLIM data in aGandScoordinate system, which is susceptible to user bias and inconsistency. Here, we present an automated and free of user-biased thresholding and segmentation algorithm that streamlines with clustering techniques to automatically identify phasor-clusters in the phasor plot space, reducing user dependency and providing a reproducible strategy under tested conditions image segmentation. We demonstrate its application in the context of FLIM images displaying a map of intensity heterogeneity where functionalized NPs affect the cellular metabolism of HeLa cells, reported by NADH, a bright and a dim fluorescent source, respectively, both of biological relevance. This algorithm provides a transparent and reproducible approach for FLIM image analysis, showing good agreement with expert-defined segmentation under sufficient contrast conditions, while presenting limitations in low-contrast or noisy regimes.
Near-infrared dye Cy7 is widely used as a fluorescent imaging probe but suffers from poor aqueous solubility, low stability, and limited bioavailability, which restricts its clinical translation. Here, we report a mechanochemical resonance mixing approach to construct noncovalent solid dispersions of Cy7 with poly(amidoamine) (PAMAM) dendrimers. Systematic optimization of milling parameters and drug/excipient ratios yielded Cy7-PAMAM dispersions with a 1000-fold increase in solubility and >98% retention rate. Physicochemical characterizations (Fourier transform infrared, differential scanning calorimetry, x-ray diffraction, scanning electron microscopy, and transmission electron microscopy) confirmed the amorphization of Cy7 and the formation of hydrogen-bonding and ionic interactions with PAMAM, resulting in stable nanoscale dispersions. Compared to raw Cy7, the Cy7-PAMAM formulation exhibited enhanced fluorescence quantum yield, improved thermal and photostability, and reduced photobleaching. Dissolution and permeability assays revealed rapid release (>85% within 120 min) and ∼10-fold higher mucosal penetration. Pharmacokinetic studies in rats demonstrated a 10-fold increase in oral bioavailability, along with elevated plasma levels and accelerated metabolism. Importantly, when integrated into a kidney-targeted nanoparticle platform (ibuprofen-modified Polygonatum sibiricum polysaccharide, IBU-PSP), Cy7-PAMAM enabled precise renal imaging with 3.8-fold higher accumulation and prolonged retention, while minimizing off-target signals in liver and heart. Molecular docking analysis supported the mechanistic role of hydrogen bonding and steric stabilization in Cy7-PAMAM assembly. Collectively, this study establishes a robust and scalable strategy for noncovalent solubilization of hydrophobic fluorophores, offering a promising formulation strategy to advance cyanine-based bioimaging agents with improvedin vivoefficacy.
Meso-fluorinated porphycenes reveal photophysical properties that vary significantly with the number and position of the substituents. 9,10-difluoro-2,7,12,17-tetra-tert-butylporphycene emits weakly, whereas two derivatives bearing the fluorines on the oppositemesopositions: 9,19- and 9,20- exhibit much higher fluorescence quantum yields and longer decay times. For weakly emitting porphycenes, fluorescence can be increased by placing the chromophore in a viscous solvent. The same effect is observed for two novel porphycenes in which a methyl group is placed next to a fluorine: 9-fluoro-2,7-di-tert-butyl-10,19-dimethylporphycene and 9,20-difluoro-2,7-tert-butyl-10,19-dimethylporphycene. These results can be explained by calculations that reveal, in the lowest excited singlet state, a highly nonplanar structure, from which rapid depopulation toS0can occur. The energy required to attain such geometry is of the order of a few kcal/mol. The relative energies calculated for the three difluorosubstituted porphycenes correlate well with the experimentally obtained fluorescence quantum yields and lifetimes. Based on these results, we propose a model that postulates that the nonradiative deactivation channel in the lowest excited singlet state of porphycenes originates from geometry distortion due to the loss of aromaticity.
Linear and superimposed diffraction gratings have been designed to selectively enhance light emission at specific wavelengths from thin films of lanthanide complexes at multiple luminescent transitions. Optimal grating pitches were determined through numerical modeling and the gratings were fabricated on photoactive azobenzene films, then transferred through nano-imprint lithography, to a bilayer of a europium dye-doped polymethyl methacrylate thin film mounted on an epoxy layer. The optimized gratings were found to increase emission intensity by up to ninefold at the emission wavelengths through angle-dependent out-coupling of waveguiding modes. This strategy was extended to simultaneously increase the emission intensity of multiple bands of the europium dye at the emission wavelengths by using a triple parallel-superimposed grating. This work demonstrates how diffraction gratings on thin films can be readily designed and manufactured for tunable angular control and wavelength-specific enhancement of emission bands from thin films doped with dyes.
We devise and experimentally validate a theoretical model to account for lost photon counts during the exposure time of a time-correlated single photon counting (TCSPC)-based QuantICAM single photon avalanche diode array camera. The work is motivated by the quest for TCSPC-based wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM), implemented by the acquisition of images at orthogonal polarization. For accurate, quantitatively correct TR-FAIM, the two images must be acquired under equivalent conditions and any photons lost during the camera exposures must be precisely quantified. Our model is based on a binomial distribution with a single adjustable parameter. We plot the recorded versus the true photon counts for exposure times of 250 µs and 1000 µs, using photons with random arrival times and from fluorescence decays. Our model describes the experimental data well and the correct number of excitation cycles during the exposure time is extracted from least-squares fits of the binomial model to the experimental data. On the basis of this model, we account for lost photons in TCSPC-based TR-FAIM and show that a compensation for lost photons is essential to obtain quantitatively correct steady-state anisotropy andG-factor histograms in TR-FAIM. We also show that, under the conditions used, the rotational correlation time, initial anisotropyr0and hindered rotation parameterr∞histograms are only marginally affected by lost photons. Our work thus paves the way for robust and reliable TCSPC-based TR-FAIM.
The increasing demand for monoclonal antibodies (mAbs) as therapeutic agents highlights the ever-growing necessity of optimizing sample-selection procedures; the rate-limiting step of therapy development. One such mAb property test is viscosity, which affects syringeability, concentration dose, and patient experience. Current viscometry methods, such as cone and plate rheometry, although accurate, are limited by low throughput, high sample volume requirements, and lack of automation, driving up the cost and time of mAb development. This paper introduces an innovative imaging-based viscometry application that significantly mitigates these issues. By integrating a wide-field camera with a fluorescence microscope and Python-based single particle tracking (SPT) software, this approach allows for rapid, low-volume (down to 2μl) viscosity measurements of mAb solutions. Using 200 nm yellow-green fluorescent polystyrene beads as tracers, the system ensures accurate macroviscosity assessments of protein solutions and the capability for high-throughput analysis. The platform's precision and sensitivity were validated using bovine serum albumin and viscosity standard solutions, followed by a single-blinded study using Immunoglobulin G1 and G2 (IgG1/IgG2) solutions of varying concentrations (⩽150 mg ml-1) and viscosities (2-31 cP). When compared to the unblinded values, the SPT blinded sample analysis resulted in a linear fit ofR2= 0.97 and an average error of 2.1 cP. Our findings suggest that this novel platform can substantially streamline and enhance the mAb development process, offering a feasible solution to one of the industry's pressing challenges.
Ergosterol is the main sterol in yeast and an important lipid constituent of the yeast plasma membrane (PM). Methods for analysis of ergosterol trafficking between PM and subcellular compartments often rely on fluorescence microscopy, but existing sterol probes either mimic ergosterol poorly or have inconvenient fluorescence properties. Here, we present a novel intrinsically fluorescent probe that differs from ergosterol only by having a 3'-keto group and two additional conjugated double bonds in the ring system. We show that this analog, named Erg-Tetraene, can order fatty acyl chains of phospholipids and partitions partially into the liquid-ordered phase in model membranes containing cholesterol. The Erg-Tetraene has a red-shifted emission and a much stronger two-photon absorption than the widely used analog dehydroergosterol, allowing for its convenient imaging on commercial microscope systems. Using multi-color confocal and two-photon microscopy, we show that uptake of Erg-Tetraene into yeast depends on the sterol transporters Aus1/Pdr11 and is followed by rapid transport to the vacuole and to lipid droplets. Together, we present a novel analogue of ergosterol with improved fluorescence properties for sterol trafficking studies in yeast and other model organisms.
Lipid accumulation has been implicated in the progression of chronic kidney disease, yet its role in glomerulonephritis (GN) remains poorly characterized. Here we use Nile Red (NR), a solvatochromic fluorophore that stains for lipid droplets (LDs) to examine LD distribution and lipid chemistry in glomerular diseases. Seventy-two kidney biopsy samples, including histologically diagnosed GN subtypes and control nephrectomy tissues, were stained with NR. LDs were quantified using a semi-automated MATLAB-based image analysis pipeline and spectral emission profiles were generated to assess the emission ratio, reflecting differences in lipid composition. We found that GN subtypes exhibit distinct patterns of lipid accumulation and lipid polarity as evaluated by using a NR emission ratio. Our study demonstrates the utility of NR fluorescence spectroscopy in detecting disease-specific lipid alterations in GN. The findings suggest that distinct lipid signatures may serve as pathological indicators, offering potential new diagnostic and mechanistic insights into glomerular disease.
Modern smartphones equipped with integrated red-green-blue (RGB) cameras are widely used for biomedical imaging and diagnostic assays. However, their limited spectral resolution prevents accurate fluorescence imaging and quantitative sensing. Recent advancements in smartphone-based multispectral imaging (MSI), such as multi-channel CMOS sensors and Bragg mirror-based spectral filters, provide direct multi-wavelength detection with enhanced spectral accuracy. The shift from RGB imaging to MSI smartphones significantly improves fluorescence imaging and sensing by allowing precise differentiation of overlapping emission spectra, true-color fluorescence observation, and quantitative assessment of fluorescent biomarkers. These characteristics are important for fluorescence-based diagnostics, point-of-care testing, and novel applications such as contact-lens sensors and fluorescence-guided imaging. Smartphone-based MSI platforms provide compact, user-friendly, and high-resolution devices that enhance the depth and accuracy of fluorescence imaging and optical sensing in biomedical research and clinical applications.
Atmospheric aerosols affect the climate, ecosystems and human well-being. Accurate detection and classification of aerosols, particularly bioaerosols are essential for effective environmental and public health management. This review presents an overview of laser-induced fluorescence (LIF) Spectroscopy as a powerful approach for real-time, non-destructive aerosol analysis. This study outlines the technological progression from foundational systems to modern commercial instruments such as the ultraviolet aerodynamic particle sizer, wideband integrated bioaerosol sensor, BioScout and Rapid-E. These technologies have provided detailed insights into aerosol size, composition and biological content, yet there are challenges in standardization and signal interpretation. By summarizing key findings and innovations, this study highlights the significance of expanding LIF applications in under-represented regions and encourages the development of robust, field-ready systems to advance air quality management and health safeguards.
The human immunodeficiency virus 1 (HIV-1) group specific antigen (Gag) polyprotein, the main structural protein of HIV-1 is sufficient to mimic the late stages of the viral replication cycle. When expressed in cells, Gag binds to RNAs and assembles at the plasma membrane to form virus-like particles (VLPs) with a morphology similar to that of HIV-1 virions. The nucleocapsid (NC) domain of Gag plays a critical role in RNA binding and selective encapsidation of the viral genome. This work focuses on the impact of NC deletion on Gag assembly, VLP formation, and intracellular trafficking. By combining several fluorescence-based quantitative microscopy techniques, we show that in the absence of the NC domain, Gag cytoplasmic oligomerization and formation of initial ribonucleoprotein complexes are impacted, resulting in a significant delay in the kinetics of VLP formation. Interestingly, VLPs formed from the Gag-ΔNC mutant display greater diversity in size and shape. Single particle tracking experiments revealed that VLPs formed at the plasma membrane are immobile, while intracellular VLPs are mostly mobile. For the latter, the motions and diffusion coefficients of VLPs formed by the Gag-ΔNC mutant were highly similar to those of VLPs formed with the wild-type Gag, indicating that the NC domain is not implicated in the intracellular trafficking. These findings illustrate how fluorescence-based microscopy techniques can provide quantitative insights into the role of the NC domain in Gag assembly.
This study presents a fluorescence-colorimetric dual-mode method for the detection of organophosphorus pesticides (OPPs). The method exploits the fact that products of organophosphorus pesticides oxidized by potassium persulfate can both quench the fluorescence of carbon dots and induce color changes. Using Dimethoate (Dim) as the model analyte, the linear detection ranges for the fluorescence and colorimetric modes were 0.05-4.6μg ml-1and 0.05-3.0μg ml-1, respectively, with limits of detection of 0.027μg ml-1and 0.0109μg ml-1. The synergistic dual-mode response significantly enhances the reliability of the measurements. In spiked recovery tests using actual water samples, both detection modes demonstrated high recovery rates, confirming that the method maintains excellent accuracy and precision even in complex matrices. The dual-mode sensing probe developed in this study features a straightforward preparation process and achieves efficient, sensitive, and reliable detection of multiple OPPs, offering a promising analytical tool for monitoring pesticide residues.
Significance. Near-infrared fluorescence (NIRF) imaging is increasingly used for perfusion assessment in clinical care and research settings. While subjective interpretation demonstrates clinical benefits, quantitative analysis is crucial for broader adoption and classification of perfusion patterns. However, strict standardization often conflicts with fast-paced clinical workflow, obstructing broad implementation.Aim and approach. This study hypothesized that normalization of fluorescence measurements could effectively correct for measurement variability, reducing the need for strict standardization in quantitative NIRF perfusion imaging. To evaluate this, a model capable of consistently simulating fluorescence perfusion patterns was employed. Experiments were conducted in an operating room using four NIRF camera systems under varying measurement conditions.Results. Normalization to maximum signal intensity provided consistent perfusion parameter values across differences in camera type, angle, rotation and settings (Δ≤1%). In cases of severe malperfusion where peak-intensity was not reached within the measurement period, normalization did not adequately correct for measurement variability (Δ increased).Conclusion. While standardization remains valuable beyond parameter accuracy, appropriate normalization substantially reduces dependence on strict measurement protocols. These findings support broader clinical adoption of quantitative NIRF imaging, extending its utility beyond specialized tertiary centers and facilitating widespread integration into routine surgical care.
