Time-resolved Fluorescence Imaging Research Articles

(Invited) Comprehensive Analysis of Exciton Diffusion with Time-Resolved Fluorescence Spectroscopy, Anisotropy and Imaging

Excitons produced in materials play a crucial role in photo-energy conversion such as photovoltaics and electroluminescence devices. The temporal and spatial diffusion of excitons is a key factor for dominating the fundamental performance of optoelectronic materials. In this context, analysis of the exciton diffusion dynamics is an important issue for obtaining the rational design of the materials. Excitons diffusing in materials can be characterized by several parameters such as the energy level, orientation of the transition dipole moment and spatial distribution, and the detection of these properties is required for the comprehensive analysis. In the present study, we show time-resolved fluorescence methods for detecting the above relevant properties of excitons.First, time-resolved fluorescence spectroscopy is a basic technique for analyzing the exciton diffusion dynamics. Fluorescence intensity is detected as functions of observation wavelength and time, and the spectral intensity and shift of fluorescence bands provide information on the population of excitons and their energy level. For example, dynamic Stokes shift indicates downhill energy migration and excitons are trapped by the defective site in molecular aggregates. Second, fluorescence anisotropy can be a good indicator for tracking the exciton diffusion in amorphous materials. Fluorescence anisotropy is sensitive to orientation change in transition dipole moments (TDMs) between absorption and fluorescence. In systems where the relative orientation of adjacent molecules is different from each other, such as amorphous solids, the exciton diffusion is accompanied with the change in TDM. Thus, appropriate modeling of molecular alignment enables quantitative estimation of the exciton diffusion coefficient and diffusion length from the anisotropy signal. Finally, time-resolved imaging is a combination of time-resolved spectroscopy and super-resolution microscopy, and an emergent technique for visualizing real-space propagation of excitons in materials. The diffraction-limited excitation beam produces excitons in several hundreds of nanometers and the subsequent exciton diffusion is evaluated by the spatial width of the fluorescence spot. The temporal broadening of the fluorescence spot directly reflects the exciton distribution and we can intuitively obtain the diffusion coefficient and length. Unlike the conventional methods for bulk materials, the advantage of the time-resolved imaging is the applicability to materials with nano- and meso-scale heterogeneous structure and it enables site-selective evaluation of the exciton transport capability. In the conference site, we will also show the application of the above methods to supramolecular polymers and thermally activated delayed fluorescence materials. Figure 1

Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.

A Novel Fit-Flexible Fluorescence Soft Imager: Tri-Sensing of Intensity, Fall-Time, and Life Profile.

Time-resolved fluorescence imaging techniques, like confocal fluorescence lifetime imaging microscopy, are powerful photonic instrumentation tools of modern science with diverse applications, including: biology, medicine, and chemistry. However, complexities of the systems, both at specimen and device levels, cause difficulties in quantifying soft biomarkers. To address the problems, we first aim to understand and model the underlying photophysics of fluorescence decay curves. For this purpose, we provide a set of mathematical functions, called "life models", fittable with the real temporal recordings of histogram of photon counts. For each model, an equivalent electrical circuit, called a "life circuit", is derived for explaining the whole process. In confocal endomicroscopy, the components of excitation laser, specimen, and fluorescence-emission signal as the histogram of photon counts are modelled by a power source, network of resistor-inductor-capacitor circuitry, and multimetre, respectively. We then design a novel pixel-level temporal classification algorithm, called a "fit-flexible approach", where qualities of "intensity", "fall-time", and "life profile" are identified for each point. A model selection mechanism is used at each pixel to flexibly choose the best representative life model based on a proposed Misfit-percent metric. A two-dimensional arrangement of the quantified information detects some kind of structural information. This approach showed a potential of separating microbeads from lung tissue, distinguishing the tri-sensing from conventional methods. We alleviated by 7% the error of the Misfit-percent for recovering the histograms on real samples than the best state-of-the-art competitor. Codes are available online.

Utilizing a Paper-Based Platform for Oilfield Applications: Time-Resolved Fluorescence Imaging and Detection of Interwell Chemical Tracers.

Chemical tracers are indispensable tools for enhancing reservoir characterization and optimizing production processes in the oil and gas industry. Particularly, interwell water tracers provide key data for efficient water flood management and the improvement of production rates. However, the analysis of these water tracers within reservoir fluids is challenging, requiring laborious separation and extraction steps that often rely on complex instruments and skilled operators. Real-time analysis is especially problematic in remote areas with limited access to well-equipped laboratories. To address these challenges, we introduce a paper-based platform for the time-resolved fluorescence detection of dipicolinic acid (DPA) tracers complexed with terbium ion (Tb3+). Our innovation is driven by the need to simplify tracer analysis, make it portable, and enhance accessibility for oilfield applications. By leveraging the unique properties of cyclen-based macrocyclic ligands, we have achieved the stable and sensitive immobilization of Tb3+ on quartz microfilter paper, eliminating the need for extensive laboratory-based procedures. We achieve the stable and sensitive immobilization of Tb3+ on quartz microfilter paper by leveraging the unique properties of cyclen-based macrocyclic ligands. This innovation enables the formation of highly fluorescent, oil-blind, and optically detectable DPA-Tb3+ complexes at the paper surface. We visualize and capture these fluorescence signals using an intensified charge-coupled device camera via time gating, effectively suppressing undesirable fluorescence originating from crude oil. The quantification of DPA concentrations is achievable down to 158 ppb (9.45 × 10-7 M), as confirmed through time-resolved fluorescence microplate reader measurements. We also demonstrate the practicality of our technology by detecting DPA tracers in the presence of crude oil contamination, a common challenge encountered in oil production wells.

Tin-based perovskite films fabricated by chemical vapor deposition for photodetector application

Tin-based perovskite materials have shown great potential applications in environment-friendly optoelectronic devices. However, it is difficult to control the film growth process by solution method due to the rapid formation speed of the film. In this work, tin-based perovskite (FASnI3) films were prepared by chemical vapor deposition method. The film is mainly composed of thin nanoplates when the deposition temperature is 600 °C. The film shows good antioxidant properties by comparing the ratio between Sn4+ and Sn2+. Time-resolved fluorescence imaging measurements clearly reveal the fluorescence lifetime inhomogeneity in the film. The photodetectors based on tin-based perovskite films show good photo response properties, with a switching ratio of 808. This work provides new opportunities for non-lead perovskite thin film preparation and optoelectronic application.

Time-resolved fluorescence imaging with color-changing, “turn-on/turn-on” AIE nanoparticles

Time-resolved fluorescence imaging with color-changing, “turn-on/turn-on” AIE nanoparticles

Fluorescence molecular tomography for quantum yield and lifetime.

Fluorescence molecular tomography (FMT) is a promising modality for noninvasive imaging of internal fluorescence agents in biological tissues, especially in small animal models, with applications in diagnosis, therapy, and drug design. In this paper, we present a fluorescent reconstruction algorithm that combines time-resolved fluorescence imaging data with photon-counting microcomputed tomography (PCMCT) images to estimate the quantum yield and lifetime of fluorescent markers in a mouse model. By incorporating PCMCT images, a permissible region of interest of fluorescence yield and lifetime can be roughly estimated as prior knowledge, reducing the number of unknown variables in the inverse problem and improving the image reconstruction stability. Our numerical experiments demonstrate the accuracy and stability of the proposed reconstruction method in the presence of data noise, achieving a reconstruction error of 0.02ns for the fluorescence lifetime and an average relative error of 18% for quantum yield reconstruction.

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.

Analyte-Triggered Excited-State Intramolecular Proton Transfer- Delayed Fluorescence: A General Approach for Time-Resolved Turn-On Fluorescence Imaging.

The research of delayed fluorescence (DF) has been a hot topic in biological imaging. However, the development of analyte-triggered small molecule DF probes remains a considerable challenge. Herein a novel excited-state intramolecular proton transfer-delayed fluorescence (ESIPT-DF) approach to construct analyte-stimulated DF probes was reported. These new classes of ESIPT-DF luminophores were strategically designed and synthesized by incorporating 2-(2'-hydroxyphenyl)benzothiazole (HBT), a known ESIPT-based fluorophore, as acceptor with a series of classic donor moieties, which formed a correspondingly twisted donor-acceptor pair within each molecule. Thereinto, HBT-PXZ and HBT-PTZ exhibited significant ESIPT and DF characters with lifetimes of 5.37 and 3.65 μs in the solid state, respectively. Furthermore, a caged probe HBT-PXZ-Ga was developed by introducing a hydrophilic d-galactose group as the recognition unit specific for β-galactosidase (β-gal) and ESIPT-DF blocking agent and applied to investigate the influence of metal ions on β-gal activity on the surface of Streptococcus pneumoniae as a convenient tool. This ESIPT-DF "turn-on" approach is easily adaptable for the measurement of many different analytes using only a predictable modification on the caged group without modification of the core structure.

Analysis of In Vivo Radachlorin Accumulation through FLIM-Assisted Examination of Ex Vivo Histological Samples

We report an investigation of the in vivo accumulation of Radachlorin photosensitizer in a murine model in several types of normal and tumor tissues based on an FLIM-assisted analysis of fluorescence intensity images, time-resolved fluorescence signals, and phasor plots. Experiments were performed on ex vivo histological samples of normal and tumor tissues. It was shown that the investigation of fluorescence intensity distributions combined with that of time-resolved fluorescence images can be used for qualitative and—under some limitations—quantitative analyses of the relative uptake of this photosensitizer in tissues. The phasor plot representations of time-resolved fluorescence signals were shown to be suitable for identification of the accumulation of predominant photosensitizers in tissues.

Donor manipulation for constructing a pH sensing thermally activated delayed fluorescent probe to detect alkaliphiles

pH homeostasis is essential for alkaliphiles, given their widespread use in biotechnological applications. However, quantitative monitoring of alkaline pH in alkaliphiles remains challenging. Here, we synthesized for the first time, a thermally activated delayed fluorescent (TADF) pH probe: NI-D-OH. Our probe exhibits a good linear relationship between fluorescence intensity and pH in the neutral to alkaline range (pH 7.0–8.6), as well as long-lived TADF emission. We further show that NI-D-OH can be used to monitor intracellular pH in living organisms, and evaluate the effect of Na+ on the pH homeostasis, demonstrating the potential for alkaline pH monitoring and time-resolved fluorescence imaging.

Automated Phasor Segmentation of Fluorescence Lifetime Imaging Data for Discriminating Pigments and Binders Used in Artworks.

The non-invasive analysis of fluorescence from binders and pigments employed in mixtures in artworks is a major challenge in cultural heritage science due to the broad overlapping emission of different fluorescent species causing difficulties in the data interpretation. To improve the specificity of fluorescence measurements, we went beyond steady-state fluorescence measurements by resolving the fluorescence decay dynamics of the emitting species through time-resolved fluorescence imaging (TRFI). In particular, we acquired the fluorescence decay features of different pigments and binders using a portable and compact fibre-based imaging setup. Fluorescence time-resolved data were analysed using the phasor method followed by a Gaussian mixture model (GMM) to automatically identify the populations of fluorescent species within the fluorescence decay maps. Our results demonstrate that this approach allows distinguishing different binders when mixed with the same pigment as well as discriminating different pigments dispersed in a common binder. The results obtained could establish a framework for the analysis of a broader range of pigments and binders to be then extended to several other materials used in art production. The obtained results, together with the compactness and portability of the instrument, pave the way for future in situ applications of the technology on paintings.

Self-aggregation of the proteolytic forms of Verteporfin: An in silico and in vitro study

Verteporfin is a benzoporphyrin derivative monoacid ring A that has been historically and successfully used as a photosensitizer in clinical applications and in clinical trials including for cancer phototherapy. However, the photo-efficiency of Verteporfin is limited by self-aggregation in water, in which the various proteolytic forms present distinct self-aggregation tendencies. In this work, spectroscopic and computational studies were used to elucidate the self-aggregation behavior of the three proteolytic forms of Verteporfin. Additionally, the intracellular self-aggregation of Verteporfin was evaluated in U87MG cells line, a type of brain cancer considered incurable. The results indicated that the self-aggregation of Verteporfin in water is most likely modulated by formal charge effects following the order: neutral > monoanionic > dicationic. UV–vis electronic spectroscopy and computational dynamics analyses suggested that J-type aggregates were primarily formed. Time-resolved fluorescence and fluorescence lifetime imaging microscopy (FLIM) confirmed that although such aggregates exist in solution and inside cells, there is no evidence of preferential self-aggregation in acidic organelles, such as lysosomes. The present approach allows a better understanding of the self-aggregation of other photosensitizers, particularly the influence of different proteolytic forms and organelles on the self-aggregation of compounds that can be protonated, and the heterogeneity of photosensitizers within intracellular compartments.

Identifying the Polymorphs of Zr-Based Metal–Organic Frameworks via Time-Resolved Fluorescence Imaging

Identifying the Polymorphs of Zr-Based Metal–Organic Frameworks via Time-Resolved Fluorescence Imaging

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA’s multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA’s analysis is strongly correlated with sequencing data (Pearson’s r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

Lipid droplet formation and dynamics: tracking by time-resolved fluorescence imaging

A highly biocompatible long-lived fluorescence micelle nanoprobe AI-Cz-M was constructed for tracking the formation and dynamics of lipid droplets in living tumor cells and adipocytes in real-time and quantitatively.

Fluorescence Lifetime Imaging Probes for Cell-Based Measurements of Enzyme Activity.

Posttranslational modification (PTM) enzymes are important modulators of protein structure and function. They typically act by chemically modifying amino acids, often on side chain functional groups, to change the physiochemical landscape of the protein and thus its biophysical behavior. In particular, protein kinases are enzymes that transfer phosphate from ATP to serine, threonine, or tyrosine in protein substrates. They are key regulators of vital cellular pathways such as survival, proliferation, and apoptosis, and their dysregulation in the context of cancer has been widely investigated for the purpose of development of anticancer drugs. However, several critical questions pertaining to their physiology, such as heterogeneity of kinase signaling within and between cells, and other factors that may play into the mechanisms of drug resistance, remain unanswered. Many of the current strategies to measure kinase activity lack the scope, subcellular resolution, and real-time monitoring ability needed to obtain the type of information needed about their dynamics and localization in cells. While FRET-based biosensors are capable of dynamic single cell imaging, their applications can be limited by difficulties in multiplexing and the inherent inadequacies of steady state measurements. In this chapter, we describe our fluorescence lifetime imaging microscopy (FLIM) probe technology in which peptide kinase substrates, linked to cell-penetrating peptides and labeled with small molecule fluorophores, are used to report kinase activity through time-resolved fluorescence imaging to visualize and quantify changes to the probe's fluorescence lifetime. These can be multiplexed for more than one kinase at a time, and interpretation is not affected by differences in local intensity due to probe uptake and distribution or photobleaching. With careful choice of peptide substrate(s), fluorophore label, and imaging set-up, high specificity and spatiotemporal resolution can be achieved. Due to the mechanism by which the lifetime change occurs, this approach is compatible with other PTMs (such as acetylation, methylation), and so the considerations for kinase FLIM probe design described in this chapter should be broadly applicable for other PTMs as well.

High efficiency branched thermal activated delayed fluorescent probe based on cyanogroup for detecting Fe3+ with low limit of detection

A multipolarization branched thermal activated delayed fluorescence sensor BrACCN was designed and synthesized based on cyano-carbazole. The classical molecule structure 4CzCN was used as luminescent nucleus, which was progressively branched for photophysical optimization. On the basis of the thermal activated delayed fluorescence material 4CzCN and 4Cz-CzCN, the molecular modification was optimized to achieve long fluorescence life and high quantum yield. Through research and analysis, the fluorescence quantum yield of the branched probe was 73%, which was 1.5 times higher than that of unbranched 4CzCN. The fluorescence lifetime of the probe BrACCN was doubled up to 3 μs based on the introduction of peripheral Br atoms. Meanwhile, the probe has high selectivity and sensitivity for Fe3+ ions. In the presence of multiple ions, the probe responded only to Fe3+ with a complexing mode of 1:1 and the fluorescence was turned off. The probe has a low limit of detection (LOD) of 2.9 × 10−8 mol/L for Fe3+ ions. The probe also has a strong AIE characteristic, which is extremely beneficial for the application range of the probe. Moreover, these excellent properties of the probe will offer promising applications in live-cell confocal fluorescence imaging and time-resolved fluorescence imaging.

Spectrally and Time-Resolved Fluorescence Imaging of 22-NBD-Cholesterol in Human Peripheral Blood Mononuclear Cells in Chronic Kidney Disease Patients.

The interaction of the fluorescent probe 22-NBD-cholesterol with membranes of human peripheral blood mononuclear cells (PBMC) was tested by time- and spectrally resolved fluorescence imaging to monitor the disturbance of lipid metabolism in chronic kidney disease (CKD) and its treatment with statins. Blood samples from healthy volunteers (HV) and CKD patients, either treated or untreated with statins, were compared. Spectral imaging was done using confocal microscopy at 16 spectral channels in response to 458 nm excitation. Time-resolved imaging was achieved by time-correlated single photon counting (TCSPC) following excitation at 475 nm. The fluorescence of 22-NBD-cholesterol was mostly integrated into plasmatic membrane and/or intracellular membrane but was missing from the nuclear region. The presence of two distinct spectral forms of 22-NBD-cholesterol was uncovered, with significant variations between studied groups. In addition, two fluorescence lifetime components were unmasked, changing in CKD patients treated with statins. The gathered results indicate that 22-NBD-cholesterol may serve as a tool to study changes in the lipid metabolism of patients with CKD to monitor the effect of statin treatment.

Mapping Transport Properties of Halide Perovskites via Short-Time-Dynamics Scaling Laws and Subnanosecond-Time-Resolution Imaging

The excellent optoelectronic and transport properties of halide perovskites have led to the rapid development of perovskite-based optoelectronic devices. A fundamental understanding of charge-carrier dynamics, as well as the implementation of physical models able to accurately describe their behaviour, is essential for further improvements in the field. Here, combining advanced modeling and characterization, a method for analyzing the short time dynamics of time-resolved fluorescence imaging (TRFLIM) decays is demonstrated. A theoretical scaling law for the time derivative of transient photoluminescence decays as a function of excitation power is extracted. This scaling law, computed from classical drift-diffusion equations, defines an innovative and simple way to extract quantitative values for several transport parameters, including the external radiative-recombination coefficient. The model is notably applied on a set of images acquired with a temporal shift of 250 ps to map the top-surface recombination velocity of a triple-cation mixed-halide perovskite thin film at the microscale. The development of high-time-resolution imaging techniques coupled with a scaling method for analyzing short time dynamics provides a solid platform for the investigation of local heterogeneities in semiconductor materials and the accurate determination of the main parameters governing their carrier transport.