Single-particle Fluorescence Research Articles

Single-Particle Fluorescence Spectroscopy for Elucidating Charge Transfer and Catalytic Mechanisms on Nanophotocatalysts.

Photocatalysis is a cost-effective approach to producing renewable energy. A thorough comprehension of carrier separation at the micronano level is crucial for enhancing the photochemical conversion capabilities of photocatalysts. However, the heterogeneity of photocatalyst nanoparticles and complex charge migration processes limit the profound understanding of photocatalytic reaction mechanisms. By establishing the precise interrelationship between microscopic properties and photophysical behaviors of photocatalysts, single-particle fluorescence spectroscopy can elucidate the carrier separation and catalytic mechanism of the photocatalysts in situ, which provides perspectives for improving the photocatalytic efficiency. This Review primarily focuses on the basic principles and advantages of single-particle fluorescence spectroscopy and its progress in the study of plasmonic and semiconductor photocatalysis, especially emphasizing its importance in understanding the charge separation and photocatalytic reaction mechanism, which offers scientific guidance for designing efficient photocatalytic systems. Finally, we summarize and forecast the future development prospects of single-particle fluorescence spectroscopy technology, especially the insights into its technological upgrading.

Unveiling the Dynamic Mechanism of SARS-CoV-2 Entry Host Cells at the Single-Particle Level.

Understanding the dynamic features of severe acute respiratory coronavirus 2 (SARS-CoV-2) binding to the cell membrane and entry cells is crucial for comprehending viral pathogenesis and transmission and facilitating the development of effective drugs against COVID-19. Herein, we employed atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) to study the binding dynamics between the virus and cell membrane. Our findings revealed that the Omicron variant of SARS-CoV-2 virus-like particles (VLPs) exhibited a slightly stronger affinity for the angiotensin-converting enzyme-2 (ACE2) receptor compared with the Delta variant and was significantly higher than the wild-type (WT). Using a real-time force-tracing technique, we quantified the dynamic parameters for a single SARS-CoV-2 VLP entry into cells, showing that approximately 200 ms and 60 pN are required. The parameters aligned with the analysis obtained from coarse-grained molecular dynamics (CGMD) simulations. Additionally, the Omicron variant invades cells at a higher entry cell speed, smaller force, and higher probability. Furthermore, single-particle fluorescence tracking visually demonstrated clathrin-dependent endocytosis for SARS-CoV-2 entry into A549 cells. The dynamic features of endocytosis provide valuable insights into the SARS-CoV-2 entry mechanism and possible intervention strategies targeting the viral infection process.

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 μm2/s), slow (∼0.04 μm2/s), and fast (∼0.14 μm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

Donor-acceptor covalent organic framework/ZnIn2S4 core-shell structure S-scheme heterostructure for efficient photocatalytic hydrogen evolution

Covalent organic frameworks (COFs) show promising prospect as the photocatalysts with advantages of exceptional light adsorption capability, large specific surface area, and adjustable band structure. However, COFs usually suffer from severe recombination of photogenerated carriers. Therefore, there is an urgent demand to design effective COF-based heterostructures to enhance the separation of carriers. In this work, a porphyrin-based COF with electron donor-acceptor structure is synthesized via condensation polymerization by using 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPPP) and N,N,N',N'-tetra(4-formylphenyl)benzidin (NFPBD) as the donor and acceptor, respectively. Subsequently, ZnIn2S4 (ZIS) are successfully in-situ grown on the surface of porphyrin-based COF, forming a novel core-shell structure. The in-situ synthesized ZIS with positive charges can be easily adsorbed on the negatively charged sites of the COF’s surface via the electrostatic interaction. This organic/inorganic hybrid COF-ZIS heterostructure exhibits a superior photocatalytic hydrogen evolution (PHE) rate of 695 μmol g−1 h−1, approximately three times higher than that of ZIS. The construction of COF-ZIS heterostructure plays an important role in enhancing the separation and transport of photogenerated carriers, which provides more electrons at the surface of ZIS to take part in proton reduction. Electron paramagnetic resonance spectra confirm the charge carriers transfer mode in the COF-ZIS heterostructure via an S-scheme mechanism. Moreover, upon loading Pt as the cocatalyst, the heterostructure achieves an effective PHE rate of 2711 μmol g−1 h−1 along with an exceptional stability. Additionally, the COF-ZIS heterostructure reaches up to 2.45% of apparent quantum efficiency at 400 nm. Notably, the average lifetime of the COF-ZIS heterostructure increases by 43.2% and 98.9% compared to that of ZIS and the COF individually, as observed through single-particle fluorescence spectroscopy. This work gives valuable inspiration into the building of donor-acceptor COF-based S-scheme heterostructures to achieve highly effective green energy conversion by aligning band structures.

The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact.

Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

A fluorescence approach for an online measurement technique of atmospheric microplastics.

Microplastic particles in the atmosphere are regularly detected in urban areas as well as in very remote locations. Yet the sources, chemical transformation, transport, and abundance of airborne microplastics still remain largely unexplained. Therefore, their impact on health, weather and climate related processes lacks comprehensive understanding. Single particle detection presents a substantial challenge due to its time-consuming process and is conducted solely offline. To get more information about the distribution, fluxes and sources of microplastics in the atmosphere, a reliable and fast online measurement technique is of utmost importance. Here we demonstrate the use of the autofluorescence of microplastic particles for their online detection with a high sensitivity towards different widely used polymers. We deploy online, single particle fluorescence spectroscopy with a Wideband Integrated Bioaerosol Sensor WIBS 5/NEO (Droplet Measurement Technologies, USA), which enables single particle fluorescence measurements at two excitation wavelengths (280 nm and 370 nm) and in two emission windows (310-400 nm and 420-650 nm). We investigated shredded (<100 μm) everyday plastic products (drinking bottles and yogurt cups) and pure powders of polyethylene terephthalate (PET), polyethylene and polypropylene. For the broad range of typical plastic products analyzed, we detected fluorescence on a single particle level using the WIBS. The online detection can identify particles smaller than 2 μm. In the case of microplastic particles from a PET bottle, 1.2 μm sized particles can be detected with 95% efficiency. Comparison with biological aerosols reveals that microplastics can be distinguished from two abundant pollen species and investigation of the complete fluorescence excitation emission maps of all samples shows that online identification of microplastics might be possible with fluorescence techniques if multiple channels are available.

Regionally sourced bioaerosols drive high-temperature ice nucleating particles in the Arctic

Primary biological aerosol particles (PBAP) play an important role in the climate system, facilitating the formation of ice within clouds, consequently PBAP may be important in understanding the rapidly changing Arctic. Within this work, we use single-particle fluorescence spectroscopy to identify and quantify PBAP at an Arctic mountain site, with transmission electronic microscopy analysis supporting the presence of PBAP. We find that PBAP concentrations range between 10−3–10−1 L−1 and peak in summer. Evidences suggest that the terrestrial Arctic biosphere is an important regional source of PBAP, given the high correlation to air temperature, surface albedo, surface vegetation and PBAP tracers. PBAP clearly correlate with high-temperature ice nucleating particles (INP) (>-15 °C), of which a high a fraction (>90%) are proteinaceous in summer, implying biological origin. These findings will contribute to an improved understanding of sources and characteristics of Arctic PBAP and their links to INP.

Illuminating bunyavirus entry into host cells with fluorescence.

Bunyavirales constitute the largest order of enveloped RNA viruses, many members of which cause severe diseases in humans and domestic animals. In recent decades, innovative fluorescence-based methods have paved the way to visualize and track single fluorescent bunyaviral particles in fixed and live cells. This technological breakthrough has enabled imaging of the early stages of infection and the quantification of every step in the bunyavirus cell entry process. Here, we describe the latest procedures for rendering bunyaviral particles fluorescent and discuss the advantages and disadvantages of each approach in light of the most recent advances in fluorescence detection and monitoring of bunyavirus entry. In this mini-review, we also illustrate how fluorescent viral particles are a powerful tool for deciphering the cellular entry process of bunyaviruses, the vast majority of which have not yet been analyzed.

Combined SPT and FCS methods reveal a mechanism of RNAP II oversampling in cell nuclei

Gene expression orchestration is a key question in fundamental and applied research. Different models for transcription regulation were proposed, yet the dynamic regulation of RNA polymerase II (RNAP II) activity remains a matter of debate. To improve our knowledge of this topic, we investigated RNAP II motility in eukaryotic cells by combining single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) techniques, to take advantage of their different sensitivities in order to analyze together slow and fast molecular movements. Thanks to calibrated samples, we developed a benchmark for quantitative analysis of molecular dynamics, to eliminate the main potential instrumental biases. We applied this workflow to study the diffusion of RPB1, the catalytic subunit of RNAP II. By a cross-analysis of FCS and SPT, we could highlight different RPB1 motility states and identifyed a stationary state, a slow diffusion state, and two different modes of subdiffusion. Interestingly, our analysis also unveiled the oversampling by RPB1 of nuclear subdomains. Based on these data, we propose a novel model of spatio-temporal transcription regulation. Altogether, our results highlight the importance of combining microscopy approaches at different time scales to get a full insight into the real complexity of molecular kinetics in cells.

Size-dependent miscibility controls the kinetics of anion exchange in cesium lead halide nanocrystals.

Anion exchange is a facile, post-synthetic method to tune the emission wavelength of colloidal cesium lead halide (CsPbX3, X = Cl, Br, I) perovskite nanocrystals. While colloidal nanocrystals can exhibit size-dependent phase stability and chemical reactivity, the role of size in the mechanism of anion exchange in CsPbX3 nanocrystals has not been elucidated. We used single-particle fluorescence microscopy to monitor the transformation of individual CsPbBr3 nanocrystals to CsPbI3. By systematically varying the size of the nanocrystals and the concentration of substitutional iodide, we observed that smaller nanocrystals exhibit longer transition times in their fluorescence trajectories, while larger nanocrystals undergo a more abrupt transition during anion exchange. Monte Carlo simulations were used to rationalize the size-dependent reactivity, in which we varied how each exchange event affects the probability for further exchange. Greater cooperativity for simulated ion exchange leads to shorter transition times to complete the exchange. We propose that size-dependent miscibility between CsPbBr3 and CsPbI3 at the nanoscale controls the reaction kinetics. Smaller nanocrystals maintain a homogeneous composition during anion exchange. As the nanocrystal size increases, variations in the octahedral tilting patterns of the perovskite crystals lead to different structures for CsPbBr3 and CsPbI3. Thus, an iodide-rich region must first nucleate within larger CsPbBr3 nanocrystals, which is followed by rapid transformation to CsPbI3. While higher concentrations of substitutional anions can suppress this size-dependent reactivity, the inherent differences in reactivity between nanocrystals of different sizes are important to consider when scaling up this reaction for applications in solid-state lighting and biological imaging.

Highly enhanced photoluminescence and suppressed blinking of N-doped carbon dots by targeted passivation of amine group for mechanistic insights and Vis-NIR excitation bioimaging application

Carbon dots (CDs) have attracted extensive attention owing to their attractive optical properties, small size, low cost, and good biocompatibility. However, there are still challenges in unequivocally unraveling the photoluminescence (PL) origin and the structure-function relationship associated with the surface groups of CDs. Herein, we have successfully enhanced the PL efficiency of GA/N-CDs by targeted passivation of the amine groups of N-doped CDs (N-CDs) with glutaraldehyde (GA), to facilitate their bioimaging applications under one-photon and two-photon excitations. In particular, we have systematically investigated the regulation effects of this surface modification on the optical properties, exciton dynamics, and blinking behavior using steady-state and time-resolved spectroscopies, and single-particle fluorescence imaging microscopes. The effective passivation of the amine surface group of N-CDs not only regulates the energy levels and properties of surface states, but importantly activates an additional emissive center via an aldehyde-amine condensation reaction. Meanwhile, the hydrogen bonding interactions between GA and the related surface groups of N-CDs further restrain the non-radiative exciton relaxations. This synergetic effect greatly enhances the PL efficiency of two separate emission centers and the photostability of GA/N-CDs. Moreover, the photoblinking of N-CDs is significantly suppressed and the two-photon excitation PL property is also markedly increased after the surface modification, endowing GA/N-CDs the outstanding performances for fluorescence bioimaging under Vis-NIR excitations. Our findings contribute to mechanistic insights into the correlation between photophysical properties and surface states/groups, as well as demonstrate an effective post-modification strategy to improve the PL efficiency and stability of carbon-based dots.

SARS-CoV-2 Binding to Terminal Sialic Acid of Gangliosides Embedded in Lipid Membranes.

Multiple recent reports indicate that the S protein of SARS-CoV-2 specifically interacts with membrane receptors and attachment factors other than ACE2. They likely have an active role in cellular attachment and entry of the virus. In this article, we examined the binding of SARS-CoV-2 particles to gangliosides embedded in supported lipid bilayers (SLBs), mimicking the cell membrane-like environment. We show that the virus specifically binds to sialylated (sialic acid (SIA)) gangliosides, i.e., GD1a, GM3, and GM1, as determined from the acquired single-particle fluorescence images using a time-lapse total internal reflection fluorescence (TIRF) microscope. The data of virus binding events, the apparent binding rate constant, and the maximum virus coverage on the ganglioside-rich SLBs show that the virus particles have a higher binding affinity toward the GD1a and GM3 compared to the GM1 ganglioside. Enzymatic hydrolysis of the SIA-Gal bond of the gangliosides confirms that the SIA sugar unit of GD1a and GM3 is essential for virus attachment to the SLBs and even the cell surface sialic acid is critical for the cellular attachment of the virus. The structural difference between GM3/GD1a and GM1 is the presence of SIA at the main or branched chain. We conclude that the number of SIA per ganglioside can weakly influence the initial binding rate of SARS-CoV-2 particles, whereas the terminal or more exposed SIA is critical for the virus binding to the gangliosides in SLBs.

Three-dimensional single particle tracking using 4π self-interference of temporally phase-shifted fluorescence

Single particle tracking in three dimensions is an indispensable tool for studying dynamic processes in various disciplines, including material sciences, physics, and biology, but often shows anisotropic three-dimensional spatial localization precision, which restricts the tracking precision, and/or a limited number of particles that can be tracked simultaneously over extended volumes. Here we developed an interferometric, three-dimensional fluorescence single particle tracking method based on conventional widefield excitation and temporal phase-shift interference of the emitted, high-aperture-angle, fluorescence wavefronts in a greatly simplified, free-running, triangle interferometer that enables tracking of multiple particles at the same time with <10-nm spatial localization precision in all three dimensions over extended volumes (~35 × 35 × 2 μm3) at video rate (25 Hz). We applied our method to characterize the microenvironment of living cells and up to ~40 μm deep in soft materials.

Simultaneous single-particle tracking and fluorescence lifetime measurement with a single-photon detector array.

Fluorescence single-particle tracking (SPT) techniques are a fundamental tool for providing information on the complex functions and interactions of individual particles in biological environments. The main concern about following single particles resides mostly in how to make registered photons informative and the solution is usually optimized only to better extrapolate either the temporal or the spatial information. This tradeoff ultimately reflects into the contraposition of wide-field camera-based techniques and real-time SMT, with the latter representing the state of the art, but at the cost of high technical complexity. Furthermore, fluorescence lifetime information, despite being accessible in many cases, has yet to be measured consistently. Within the context of the BrightEyes ERC-2018-COG project, we present a real-time three-dimensional SPT implementation based on a confocal laser-scanning microscope featuring a 5×5 single-photon avalanche photodiode (SPAD) array detector capable of overcoming the above limitations due to an hybrid approach. Briefly, each sensitive element of the detector acts as a confocal pinhole, and the recorded intensity distribution mirrors the position of the particle with respect to the center of the excitation volume. The estimation is then used as input for a real-time feedback tracking system. Notably, each pixel is an independent photon counting detector, allowing for concurrent measurement of the fluorescence lifetime. The system is capable of experimentally tracking single particles with localization precision up to 30 nm with 100 photons and microsecond time resolution. The same photon stream is used to perform fluorescence lifetime measurements. The technique has been validated by tracking 20 nm fluorescent beads moved along a predetermined path and by measuring the diffusion coefficient when freely diffusing in water-glycerol solutions. As application, the movement of lysosomes in living cells has been investigated while assessing the lifetime of the GFP marker.

Single-Particle Analysis of the Interaction Between Molecules and Protein Aggregated Species by Dual-Color Time-Resolved Fluorescence Spectroscopy.

Amyloid protein aggregation is widely involved in a number of neurodegenerative diseases for which novel therapeutic and diagnostic strategies are still needed. Owing to the complex and heterogeneous nature of the aggregated species responsible for toxicity in these disorders, a detailed characterization of the interaction of molecules of interest with the amyloid aggregates is a challenging endeavor. Here, we present the experimental and analytical steps of a protocol which combines dual-color fluorescence cross-correlation spectroscopy and dual-color single-particle fluorescence spectroscopy to quantify the binding affinity and stoichiometryof an inhibitor of α-synuclein amyloid aggregation. This approach allows studying the interaction in detail and through two independent analytical methods, thus yielding a remarkably robust tool that could be extended to investigating the interaction of molecules of interest to other pathogenic protein aggregates as well as multi-ligand/multi-receptor complexes.

Changes in the Fluorescence of Biological Particles Exposed to Environmental Conditions in the National Capitol Region

A variety of methods have been used to study atmospheric bioaerosols. A common technique employed for the detection and measurement of bioaerosols is the measurement of the autofluorescence of biological particles when excited by ultraviolet light. We examined the changes in the fluorescence spectra of bioaerosols when exposed to ambient outdoor conditions for periods of several hours. The bioaerosols in this study were contained in a Captive Aerosol Growth and Evolution (CAGE) chamber that employed two rotating drums constructed with an exterior FEP Teflon film to allow sunlight to penetrate and an inner ePTFE membrane to allow ambient trace gasses to permeate the drums. The bioaerosols were periodically measured with a TSI UV-APS (excited at 355 nm) and a single-particle fluorescence spectrometer (excited at 351 and 263 nm). The data indicate changes in both fluorescence spectral profile and intensity from Bacillus thuringiensis var. kurstaki spores and MS2 bacteriophage particles during the experiments. The changes observed in these particles appear to be due to a combination of the environmental conditions rather than attributable to any single factor. The results of this study indicate that bioaerosols are significantly altered by atmospheric aging processes and that these changes may affect measurements by ultra-violet light induced fluorescence (UV-LIF) or other spectroscopic techniques.

The Impact of Hidden Structure on Aggregate Disassembly by Molecular Chaperones.

Protein aggregation, or the uncontrolled self-assembly of partially folded proteins, is an ever-present danger for living organisms. Unimpeded, protein aggregation can result in severe cellular dysfunction and disease. A group of proteins known as molecular chaperones is responsible for dismantling protein aggregates. However, how protein aggregates are recognized and disassembled remains poorly understood. Here we employ a single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS), in combination with two structurally distinct aggregate types grown from the same starting protein, to examine the mechanism of chaperone-mediated protein disaggregation. Using the core bi-chaperone disaggregase system from Escherichia coli as a model, we demonstrate that, in contrast to prevailing models, the overall size of an aggregate particle has, at most, a minor influence on the progression of aggregate disassembly. Rather, we show that changes in internal structure, which have no observable impact on aggregate particle size or molecular chaperone binding, can dramatically limit the ability of the bi-chaperone system to take aggregates apart. In addition, these structural alterations progress with surprising speed, rendering aggregates resistant to disassembly within minutes. Thus, while protein aggregate structure is generally poorly defined and is often obscured by heterogeneous and complex particle distributions, it can have a determinative impact on the ability of cellular quality control systems to process protein aggregates.

Single-particle fluorescence tracking combined with TrackMate assay reveals highly heterogeneous and discontinuous lysosomal transport in freely orientated axons.

Axonal transport plays a significant role in the establishment of neuronal polarity, axon growth, and synapse formation during neuronal development. The axon of a naturally growing neuron is a highly complex and multifurcated structure with a large number of bends and branches. Nowadays, the study of dynamic axonal transport in morphologically complex neurons is greatly limited by the technological barrier. Here, a sparse gene transfection strategy was developed to locate fluorescent mCherry in the lysosome of primary neurons, thus enabling us to track the lysosome-based axonal transport with a single-particle resolution. Thereby, several axonal transport models were observed, including the forward or backward transport model, stop-and-go model, repeated back-and-forth transport model, and cross-branch transport model. Then, the accurate single-particle velocity quantification by TrackMate revealed a highly heterogeneous and discontinuous transportation process of lysosome-based axonal transport in freely orientated axons. And, multiple physical factors, such as the axonal structure and the size of particles, were disclosed to affect the velocity of particle transporting in freely orientated axons. The combined single-particle fluorescence tracking and TrackMate assay can be served as a facile tool for evaluating axonal transport in neuronal development and axonal transport-related diseases.

Characterization of sub-pollen particles in size-resolved atmospheric aerosol using chemical tracers.

Pollen grains may contain allergens that exacerbate allergic respiratory diseases like asthma and rhinitis. In the presence of water, pollen grains (10–100 μm) can rupture to produce sub-pollen particles (SPP) with diameters <2.5 μm, which in comparison to intact pollen grains, have longer atmospheric lifetimes and greater penetration to the lower lung. The current study examines SPP, fungal spores, and bacteria in size-resolved atmospheric particulate matter (PM) using chemical and biological tracers. During springtime tree pollen season in Iowa City, Iowa, fine particle (PM2.5) concentrations of fructose (a pollen chemical tracer) increased on rainy sampling periods, especially during severe thunderstorms, and peaked when a tornado struck nearby. Submicron fluorescent particles, measured by single-particle fluorescence spectroscopy, were also enhanced during rain events, particularly thunderstorms in agreement with the chemical tracer measurements. PM2.5 sucrose (a pollen chemical tracer) concentrations were higher in early spring when nighttime temperatures were closer to freezing, while fructose concentrations were higher in late spring with warmer temperatures, consistent with chemical tracers being sensitive to seasonal temperature influences. The first co-located measurements of fructose and Bet v 1 (birch pollen allergen), indicated that SPP ranged in diameter from <0.25 to 2.5 μm during rainy sampling periods and that allergens and carbohydrates exhibited distinct size distributions. Meanwhile, mannitol (a fungal spore tracer) peaked on warm, dry days following rain and was primarily in supermicron particles (>1.0 μm), which is consistent with intact fungal spore diameters (1–30 μm). Bacterial endotoxins in PM also increased during extreme weather events, primarily in supermicron particles. While the concentrations of fructose, mannitol, and endotoxin all increased in PM2.5 μm during thunderstorms, the greatest relative increase in concentration was observed for fructose. Together, these observations suggest that SPP containing starch granules and allergens (Bet v 1) were released during rainy sampling periods. This study advances the use of chemical tracers to track SPP and other bioaerosols in the atmosphere, by providing new insight to their size distribution and response to extreme weather conditions.