A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Recycling of the actin monomer pool limits the lifetime of network turnover.

Intracellular organization is largely mediated by actin turnover. Cellular actin networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called "dynamic steady state," allows cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled by an actin comet in a microwell containing a limited amount of components. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly, and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated protein (CAP) is the key step in allowing the reuse of monomers for multiple assembly cycles. ATP supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis.

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

Light-field tomographic fluorescence lifetime imaging microscopy.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging technique that enables the visualization of biological samples at the molecular level by measuring the fluorescence decay rate of fluorescent probes. This provides critical information about molecular interactions, environmental changes, and localization within biological systems. However, creating high-resolution lifetime maps using conventional FLIM systems can be challenging, as it often requires extensive scanning that can significantly lengthen acquisition times. This issue is further compounded in three-dimensional (3D) imaging because it demands additional scanning along the depth axis. To tackle this challenge, we developed a computational imaging technique called light-field tomographic FLIM (LIFT-FLIM). Our approach allows for the acquisition of volumetric fluorescence lifetime images in a highly data-efficient manner, significantly reducing the number of scanning steps required compared to conventional point-scanning or line-scanning FLIM imagers. Moreover, LIFT-FLIM enables the measurement of high-dimensional data using low-dimensional detectors, which are typically low cost and feature a higher temporal bandwidth. We demonstrated LIFT-FLIM using a linear single-photon avalanche diode array on various biological systems, showcasing unparalleled single-photon detection sensitivity. Additionally, we expanded the functionality of our method to spectral FLIM and demonstrated its application in high-content multiplexed imaging of lung organoids. LIFT-FLIM has the potential to open up broad avenues in both basic and translational biomedical research.

A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

Histone FRET reports the spatial heterogeneity in nanoscale chromatin architecture that is imparted by the epigenetic landscape at the level of single foci in an intact cell nucleus.

Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.

Finding the partner: FRET and beyond.

Given the importance of aberrant protein-protein interactions (PPIs) in disease, the recent drug discovery focuses on targeting the altered PPIs to treat the disease. In this context, identifying the atypical PPIs underlying the disease is critical for the development of diagnostics and therapeutics. Various biochemical, biophysical, and genetic methods have been reported to study PPIs. Here, we are giving a short account of those techniques with more emphasis on Förster resonance energy transfer (FRET), which can be used to monitor macromolecular interactions in live cells. Besides the basics of FRET, we explain the modifications of its application, like Single molecule FRET (smFRET), Fluorescence Lifetime Imaging Microscopy-FRET (FLIM-FRET), and photoswitching FRET. While smFRET is extensively used for evaluating the biology of nucleic acids and also to develop diagnostics, FLIM-FRET is widely exploited to study the PPIs underlying neurological disorders and cancer. Photoswitching FRET is a relatively newer technique and it has tremendous potential to unravel the significance of different PPIs. Besides these modifications, there are several advancements in the field by introducing new fluorophores. Identification of lanthanide chelates, quantum dots, and other nanoparticle fluorophores has revolutionized the applications of FRET in diagnostics and basic biology. Yet, these methods can be employed to study the interactions of only two molecules. Since the majority of the PPIs are multimeric complexes, we still need to improve our technologies to study these interactions in live cells in real-time.

Comprehensive review of indicators and techniques for optical mapping of intracellular calcium ions.

Calcium ions (Ca2+) play crucial roles in almost every cellular process, making the detection of changes in intracellular Ca2+ essential to understanding cell function. The fluorescence indicator method has garnered widespread application due to its exceptional sensitivity, rapid analysis, cost-effectiveness, and user-friendly nature. It has successfully delineated the spatial and temporal dynamics of Ca2+ signaling across diverse cell types. However, it is vital to understand that different indicators have varying levels of accuracy, sensitivity, and stability, making choosing the right inspection method crucial. As optical detection technologies advance, they continually broaden the horizons of scientific inquiry. This primer offers a systematic synthesis of the current fluorescence indicators and optical imaging modalities utilized for the detection of intracellular Ca2+. It elucidates their practical applications and inherent limitations, serving as an essential reference for researchers seeking to identify the most suitable detection methodologies for their calcium-centric investigations.

Changes of Development from Childhood to Late Adulthood in Rats Tracked by Urinary Proteome.

To date, studies of development have mainly focused on the embryonic stage and a short time thereafter. There has been little research on the whole life of an individual from childhood to aging and death. For the first time, we used noninvasive urinary proteome technology to track changes in several important developmental time points in a group of rats, covering 10 time points from childhood, adolescence, young adulthood, middle adulthood, and near-death in old age. Similar to previous studies on puberty, proteins were detected and they are involved in sexual or reproductive maturation, mature spermatozoa in seminiferous tubules (first seen), gonadal hormones, decline of estradiol, brain growth, and central nervous system myelination, and our differential protein enrichment pathways also included reproductive system development, tube development, response to hormone, response to estradiol, brain development, and neuron development. Similar to previous studies in young adults, proteins were detected and they are involved in musculoskeletal maturity, peak bone mass, development of the immune system, and growth and physical development, and our differential proteins enrichment pathways also included skeletal system development, bone regeneration, system development, immune system processes, myeloid leukocyte differentiation, and developmental growth. Studies on aging-related changes in neurons and neurogenesis have been reported, and we also found relevant pathways in aged rats, such as regulation of neuronal synaptic plasticity and positive regulation of long-term neuronal synaptic plasticity. However, at all time points throughout life, there were many biological pathways revealed by differential urinary protein enrichment involving multiple organs, tissues, systems, etc. that have not been mentioned in existing studies. This study shows comprehensive and detailed changes in rat lifetime development through the urinary proteome, helping to fill the gap in development research. Moreover, it provides a new approach to monitoring changes in human health and diseases of aging using the urinary proteome.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Organic molecular dynamics and charge-carrier lifetime in lead iodide perovskite MAPbI3.

The long charge carrier lifetime of the hybrid organic-inorganic perovskites (HOIPs) is the key for their remarkable performance as a solar cell material. The microscopic mechanism for the long lifetime is still in debate. Here, by using a muon spin relaxation technique that probes the fluctuation of local magnetic fields, we show that the muon depolarization rate (Δ) of a prototype HOIP methylammonium lead iodide (MAPbI3) shows a sharp decrease with increasing temperature in two steps above 120 K and 190 K across the structural transition from orthorhombic to tetragonal structure at 162 K. Our analysis shows that the reduction of Δ is quantitatively in agreement with the expected behavior due to the rapid development of methyl ammonium (MA) jumping rotation around the C 3 and C 4 symmetry axes. Our results provide direct evidence for the intimate relation between the rotation of the electric dipoles of MA molecules and the charge carrier lifetime in HOIPs.

Hybrid measurement of respiratory aerosol reveals a dominant coarse fraction resulting from speech that remains airborne for minutes.

Accurate measurements of the size and quantity of aerosols generated by various human activities in different environments are required for efficacious mitigation strategies and accurate modeling of respiratory disease transmission. Previous studies of speech droplets, using standard aerosol instrumentation, reported very few particles larger than 5 µm. This starkly contrasts with the abundance of such particles seen in both historical slide deposition measurements and more recent light scattering observations. We have reconciled this discrepancy by developing an alternative experimental approach that addresses complications arising from nucleated condensation. Measurements reveal that a large volume fraction of speech-generated aerosol has diameters in the 5- to 20-µm range, making them sufficiently small to remain airborne for minutes, not hours. This coarse aerosol is too large to penetrate the lower respiratory tract directly, and its relevance to disease transmission is consistent with the vast majority of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiating in the upper respiratory tract. Our measurements suggest that in the absence of symptoms such as coughing or sneezing, the importance of speech-generated aerosol in the transmission of respiratory diseases is far greater than generally recognized.

Single-molecule fluorescence imaging and deep learning reveal highly heterogeneous aggregation of amyloid-ß 42.

Polymorphism in the structure of amyloid fibrils suggests the existence of many different assembly pathways. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity, but in practice it is extremely difficult to probe individual aggregation pathways in a mixture. Here, we present development of a method combining single-molecule fluorescence lifetime imaging and deep learning for monitoring individual fibril formation in real time and their high-throughput analysis. A deep neural network (FNet) separates an image of highly overlapping fibrils into single fibril images, which allows for tracking the growth and changes in characteristics of individual fibrils. Using this method, we investigated aggregation of the 42-residue amyloid-ß peptide (Aß42). We demonstrate that highly heterogeneous fibril formation can be quantitatively characterized in terms of the number of cross-ß subunits, elongation speed, growth polarity, and conformation of fibrils. Tracking individual fibril formation and growth also leads to the discovery of a general nucleation mechanism (termed heterogeneous secondary nucleation), where a fibril is formed on the surface of an oligomer with a different structure. Our development will be broadly applicable to characterization of heterogeneous aggregation processes of other proteins.

Bright Nonblinking Photoluminescence with Blinking Lifetime from a Nanocavity-Coupled Quantum Dot.

Colloidal quantum dots (QDs) are excellent luminescent nanomaterials for many optoelectronic applications. However, photoluminescence blinking has limited their practical use. Coupling QDs to plasmonic nanostructures shows potential in suppressing blinking. However, the underlying mechanism remains unclear and debated, hampering the development of bright nonblinking dots. Here, by deterministically coupling a QD to a plasmonic nanocavity, we clarify the mechanism and demonstrate unprecedented single-QD brightness. In particular, we report for the first time that a blinking QD could obtain nonblinking photoluminescence with a blinking lifetime through coupling to the nanocavity. We show that the plasmon-enhanced radiative decay outcompetes the nonradiative Auger process, enabling similar quantum yields for charged and neutral excitons in the same dot. Meanwhile, we demonstrate a record photon detection rate of 17 MHz from a colloidal QD, indicating an experimental photon generation rate of more than 500 MHz. These findings pave the way for ultrabright nonblinking QDs, benefiting diverse QD-based applications.

Vertical Dipole Dominates Charge Carrier Lifetime in Monolayer Janus MoSSe.

Two-dimensional semiconductor materials with vertical dipoles are promising photocatalysts as vertical dipoles not only promote the electron-hole separation but also enhance the carrier redox ability. However, the influence of vertical dipoles on carrier recombination in such materials, especially the competing relationship between vertical dipoles and band gaps, is not yet clear. Herein, first-principles calculations and nonadiabatic molecular dynamics simulations were combined to clarify the influence of band gap and vertical dipole on the carrier lifetime in Janus MoSSe monolayer. By comparing with the results of MoS2 and MoSe2 as well as exploring the carrier lifetime of MoSSe under strain regulation, it has been demonstrated that the vertical dipole, rather than the band gap, is the dominant factor affecting the carrier lifetime. Strikingly, a linear relationship between the carrier lifetime and vertical dipole is revealed. These findings have important implications for the design of high-performance photocatalysts and optoelectronic devices.

Enhanced Nonradiative Charge Recombination in Microfiber-Based Bismuthene.

After the first report of a graphene-based passive mode-locking ultrafast fiber laser, two-dimensional materials as efficient saturable absorbers offer a new horizon in ultrafast fiber laser. However, the interactions on atomic scale between these two-dimensional materials and fiber and the fiber effect on the carrier dynamics have not been realized. To figure out the exact role of fiber and the carrier dynamics affected by the fiber substrate related to ultrafast photonics, bismuthene, a newly reported 2D quantum material used in a passive mode-locking fiber laser, deposited on α-quartz has been investigated. We surprisingly found that the α-quartz substrate can strongly accelerate the nonradiative electron-hole recombination of bismuthene in theory, and the transient absorption spectra of bismuthene on normal glass and α-quartz further verify the substrate effect on carrier dynamics of bismuthene. The discovery provides new thinking about substrate effect to regulate the performance of ultrafast mode-locking fiber lasers as well as ultrafast photonics.

Diffusion-Dominated Luminescence Dynamics of CsPbBr3 Studied Using Cathodoluminescence and Microphotoluminescence Spectroscopy.

Time-resolved or time-correlation measurements using cathodoluminescence (CL) reveal the electronic and optical properties of semiconductors, such as their carrier lifetimes, at the nanoscale. However, halide perovskites, which are promising optoelectronic materials, exhibit significantly different decay dynamics in their CL and photoluminescence (PL). We conducted time-correlation CL measurements of CsPbBr3 using Hanbury Brown-Twiss interferometry and compared them with time-resolved PL. The measured CL decay time was on the order of subnanoseconds and was faster than PL decay at an excited carrier density of 2.1 × 1018 cm-3. Our experiment and analytical model revealed the CL dynamics induced by individual electron incidences, which are characterized by highly localized carrier generation followed by a rapid decrease in carrier density due to diffusion. This carrier diffusion can play a dominant role in the CL decay time for undoped semiconductors, in general, when the diffusion dynamics are faster than the carrier recombination.

Thermally Prolonged NIR-II Luminescence Lifetimes by Cross-Relaxation.

Temperature regulates nonradiative processes in luminescent materials, fundamental to luminescence nanothermometry. However, elevated temperatures often suppress the radiative process, limiting the sensitivity of thermometers. Here, we introduce an approach to populating the excited state of lanthanides at elevated temperatures, resulting in a sizable lifetime lengthening and intensity increase of the near-infrared (NIR)-II emission. The key is to create a five-energy-level system and use a pair of lanthanides to leverage the cross-relaxation process. We observed the lifetime of NIR-II emission of Er3+ has been remarkably increased from 3.85 to 7.54 ms by codoping only 0.5 mol % Ce3+ at 20 °C and further increased to 7.80 ms when increasing the temperature to 40 °C. Moreover, this concept is universal across four ion pairs and remains stable within aqueous nanoparticles. Our findings emphasize the need to design energy transfer systems that overcome the constraint of thermal quenching, enabling efficient imaging and sensing.

A fluorescence lifetime-based novel method for accurate lipid quantification of BODIPY vital-stained C. elegans.

Lipid droplets (LDs) are organelles associated with lipid storage and energy metabolism, thus, their morphology and quantity are of significant research interest. While commercially available BODIPY dye effectively labels LDs in various cell types, it also labels lysosome-related organelles (LROs) in C. elegans, leading to non-specific LD quantification. Here, we report that the fluorescent signals of BODIPY exhibit distinct fluorescence lifetime patterns for LROs and LDs, which can be captured, visualized, and filtered by fluorescence lifetime imaging microscopy. Furthermore, we proposed and validated a method based on fluorescence lifetime that can improve the accuracy of fat storage quantification in BODIPY vital-staining worms, which holds broad applications, including rapid and accurate LD quantification in forward genetic screening. Additionally, our method enables observing dynamic LD-LRO interactions in living worms, a unique capability of BODIPY vital staining. Our findings highlight distinct BODIPY fluorescence lifetime characteristics of LDs and LROs, providing a valuable tool for future research on LDs, LROs, or their interactions.

Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.