PicoQuant

Biological condensates, often formed via liquid-liquid phase separation (LLPS), are membraneless compartments organizing biochemical reactions. Recent advances have shifted the focus from identifying condensates to elucidating their dynamic biological functions, such as buffering concentrations, mediating reactions, and regulating signaling. These are critical for cellular processes and implicated in diseases like cancer and neurodegeneration. Advanced microscopy techniques, including fluorescence lifetime imaging microscopy (FLIM), FLIM-FRET, and fluorescence correlation spectroscopy (FCS), enable quantitative, real-time investigations of condensate composition, dynamics, material properties, and their responses to environmental stimuli in live cells. This perspective highlights the utility of time-resolved fluorescence and single-molecule spectroscopy techniques for shedding light on condensate functions, properties, and interactions with membranes, offering insights into cellular physiology and pathology.

Plasticity-related gene (PRG) 5 is a vertebrate specific membrane protein, that belongs to the family of lipid-phosphate phosphatases (LPPs). It is prominently expressed in neurons and is involved in cellular processes such as growth-cone guidance and spine formation. At a functional level, PRG5 induces filopodia in non-neuronal cell lines, as well as the formation of plasma membrane protrusions in primary cortical and hippocampal neurons. Overexpression of PRG5 in immature neurons leads to the induction of spine-like structures, and regulates spine density and morphology in mature neurons. Understanding spine formation is pivotal, as spine abnormalities are associated with numerous neurological disorders. Although the importance of PRG5 in neuronal function is evident, the precise mechanisms as to how exactly it induces membrane protrusions and orchestrates cellular processes remain unresolved. Here we used in vitro biochemical assays to demonstrate that in HEK293T cells a large fraction of PRG5 can be found in homo dimers and lager multimers. By using Fluorescence Lifetime Imaging (FLIM) to quantify Förster Resonance Energy Transfer (FRET), we were able to visualize and quantify the specific localization of PRG5 multimers in living HEK293T cells and in fixed immature primary hippocampal neurons. Here, we provide the first evidence that PRG5 multimers are specifically localized in non-neuronal filopodia, as well as in neuronal spine-like structures. Our findings indicate a potential functional role for PRG5 multimerization, which might be required for interaction with extracellular matrix molecules or for maintaining the stability of membrane protrusions.

Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.

Our current understanding of biomolecular condensate formation is largely based on observing the final near-equilibrium condensate state. Despite expectations from classical nucleation theory, pre-critical protein clusters were recently shown to form under subsaturation conditions in vitro; if similar long-lived clusters comprising more than a few molecules are also present in cells, our understanding of the physical basis of biological phase separation may fundamentally change. Here, we combine fluorescence microscopy with photobleaching analysis to quantify the formation of clusters of NELF proteins in living, stressed cells. We categorise small and large clusters based on their dynamics and their response to p38 kinase inhibition. We find a broad distribution of pre-condensate cluster sizes and show that NELF protein cluster formation can be explained as non-classical nucleation with a surprisingly flat free-energy landscape for a wide range of sizes and an inhibition of condensation in unstressed cells.

Background: A commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters. Results: As a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable. Conclusions: We conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

Fluorescence Resonance Energy Transfer (FRET)‐based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction‐limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super‐resolved FRET imaging based on single‐molecule localization microscopy using an early prototype of a commercial time‐resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.

Quantitative single molecule and time‐resolved fluorescence techniques offer new insights into many samples in life and materials sciences. So far, their adoption has been slow because expert knowledge was required for correct data acquisition and analysis. Now, PicoQuant have developed a new confocal microscope called Luminosa. It combines state‐of‐the‐art hardware with cutting‐edge software to deliver high‐quality data while simplifying daily operation. The software includes context‐based workflows for each technique. This article shows how it streamlines single‐molecule Förster resonance energy transfer (smFRET) experiments.

The field of quantum information processing offers secure communication protected by the laws of quantum mechanics and is on the verge of finding wider application for the information transfer of sensitive data. To improve cost-efficiency, extensive research is being carried out on the various components required for high data throughput using quantum key distribution (QKD). Aiming for an application-oriented solution, we report the realization of a multichannel QKD system for plug-and-play high-bandwidth secure communication at telecom wavelengths. We designed a rack-sized multichannel superconducting nanowire single photon detector (SNSPD) system, as well as a highly parallelized time-correlated single photon counting (TCSPC) unit. Our system is linked to an FPGA-controlled QKD evaluation setup for continuous operation, allowing us to achieve high secret key rates using a coherent-one-way protocol.

Superconducting nanowire single-photon detectors are an enabling technology for modern quantum information science and are gaining attractiveness for the most demanding photon counting tasks in other fields. Embedding such detectors in photonic integrated circuits enables additional counting capabilities through nanophotonic functionalization. Here, we show how a scalable number of waveguide-integrated superconducting nanowire single-photon detectors can be interfaced with independent fiber optic channels on the same chip. Our plug-and-play detector package is hosted inside a compact and portable closed-cycle cryostat providing cryogenic signal amplification for up to 64 channels. We demonstrate state-of-the-art multi-channel photon counting performance with average system detection efficiency of (40.5 ± 9.4)% and dark count rate of (123 ± 34) Hz for 32 individually addressable detectors at minimal noise-equivalent power of (5.1 ± 1.2) · 10 ⁻¹⁸ W/[Formula: see text]. Our detectors achieve timing jitter as low as 26 ps, which increases to (114 ± 17) ps for high-speed multi-channel operation using dedicated time-correlated single photon counting electronics. Our multi-channel single photon receiver offers exciting measurement capabilities for future quantum communication, remote sensing, and imaging applications.

Background A commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, One Pattern Analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters. Results As a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, in situ complex formation of MADS-domain proteins cannot be detected. Conclusions We conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei display strong insulation even before TADs emerge. Moreover, active transcription within a TAD leads to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affects insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

Photoinduced electron and energy transfers are ubiquitous phenomena in nature and are under investigation in physics, chemistry, and biology. Because of their fundamental relevance and multiple possible applications, they are of increasing importance in life sciences and technology. Porphyrin‐based molecular systems that can undergo efficient photoinduced electron transfer ( PET ) and/or excitation energy transfer ( EET ) processes are attracting interest currently not only for the development of solar energy conversion systems, but also for the rational design of fluorescent sensors, photocatalysts, novel drugs for photodynamic therapy, and optoelectronic devices. A detailed understanding of PET and EET, as well as the factors governing these fundamental processes in donor–acceptor systems, is of crucial importance for the development of sophisticated molecular systems for various applications. In this chapter, the fundamental principles of EET as well as PET processes are reviewed. Resonance, exchange as well as excitonic mechanisms of EET, including the influence of the bridge connecting the donor and acceptor chromophores, are discussed. Quantum mechanical and classical Marcus theories of PET are presented, allowing an estimation of the PET probability and the rate of this process as a function of the physicochemical properties of a particular molecular system as well as of its environment. Finally, several representative examples of covalently linked and self‐assembled porphyrin‐based molecular systems displaying interesting functional properties based on energy and/or electron transfer phenomena are discussed.

The transient absorption spectra of the series of diamagnetic H2TFcP, ZnTFcP, PdTFcP, and FcInTFcP compounds (TFcP(2‐)=5,10,15,20‐tetraferroceneporphyrin dianion) were investigated in polar (DMF) and non‐polar (toluene) solvents using excitation at 650 nm. The formation and the deactivation of the charge‐separated (Fc⁺‐Porphyrin−.) state were observed in all cases. The lifetime of the charge‐separated state is nearly constant for all compounds (∼20 ps) and independent of the nature of the central ion and solvent. The formation of the triplet state in all the complexes was not observed. The third, minor long‐lived (160–480 ps) component was observed in polar DMF solvent. This component was tentatively assigned to the porphyrin species that are weakly bound to the carbonyl oxygen in DMF. DFT and TDDFT calculations on the ground state, excited state, and triplet state of the target compounds were in agreement with the experimental data.

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na+ transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na+ loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na+ changes on energy depletion.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated, quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments¹. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique2,3. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g., DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE⁴), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility⁵. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative⁶ was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models7,8, and tools9,10, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists¹¹, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.