Fluorescence Lifetime Measurements Research Articles

Structural Studies of a Stapled Peptide with Native Ion Mobility-Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase.

Native mass spectrometry has emerged as an important tool for gas-phase structural biology. However, the conformations that a biomolecular ion adopts in the gas phase can differ from those found in solution. Herein, we report a synergistic, native ion mobility-mass spectrometry (IM-MS) and transition metal ion Förster resonance energy transfer (tmFRET)-based approach to probe the gas-phase ion structures of a nonstapled peptide (nsp; Ac-CAARAAHAAAHARARA-NH2) and a stapled peptide (sp; Ac-CXARAXHAAAHARARA-NH2). The stapled peptide contains a single hydrocarbon chain connecting the peptide backbone in the i and i + 4 positions via a Grubbs ring-closure metathesis. Fluorescence lifetime measurements indicated that the Cu-bound complexes of carboxyrhodamine 6g (crh6g)-labeled stapled peptide (sp-crh6g) had a shorter donor-acceptor distance (rDA) than the labeled nonstapled peptide (nsp-crh6g). Experimental collision cross-section (CCS) values were then determined by native IM-MS, which could separate the conformations of Cu-bound complexes of nsp-crh6g and sp-crh6g. Finally, the experimental CCS (i.e., shape) and rDA (i.e., distance) values were used as constraints for computational studies, which unambiguously revealed how a staple reduces the elongation of the peptide ions in the gas phase. This study demonstrates the superiority of combining native IM-MS, tmFRET, and computational studies to investigate the structure of biomolecular ions.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2β2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

AlGaN Ultraviolet Micro-LEDs

AlGaN-based UV LEDs have proven to have broad applications in many fields including sterilization, disinfection, purification, and phototherapy, but the performance still needs to be further improved. Benefiting from advantages such as better current spreading, lower self-heating effect and higher light extraction efficiency achieved by the reduced LED size, UV micro-LEDs are expected to improve quantum efficiency and thus can expand more potential applications. In this article, performance enhancement techniques and applications of UV micro-LEDs will be reviewed, providing an outlook for further development of AlGaN-based UV-LEDs. The micro-LED format offers possibilities to address fundamental issues that UV LEDs confront in general, but also demonstrates specific characteristics and performance advantages opening up new areas of application including high-speed optical communication, time-resolved fluorescence lifetime measurement, optical pumping, direct writing and charge management.

Focus on software, data acquisition, and instrumentation

Focus on software, data acquisition, and instrumentation

A Dynamically Responsive Chemosensor That Can be Modulated by an Effector: Amplification Sensing by Positive Heterotropic Allosterism

Abstract Induced-fit chemosensors that can control their molecular recognition behavior using external stimuli such as temperature, light, pH, and chemical additives, have attracted much attention from several chemists. In this study, we report a bisporphyrin–bisthiourea–binaphthyl conjugated chemosensor that can be modulated using a chiral dianion. The optical properties of the chemosensor measured using fluorescence and UV/vis absorption spectroscopies, as well as fluorescence lifetime measurements, indicated that the unique conjugation enabled S2 fluorescence. The binding constants of the chemosensor for amino acids were amplified a thousand-fold compared to that of the reference compound due to positive heterotropic allosterism. The present study provides new perspectives for signal amplification of chemosensors by allosterism.

Excited state lifetime modulation in semiconductor nanocrystals for super-resolution imaging

We report on proof of principle measurements of a concept for a super-resolution imaging method that is based on excitation field density-dependent lifetime modulation of semiconductor nanocrystals. The prerequisite of the technique is access to semiconductor nanocrystals with emission lifetimes that depend on the excitation intensity. Experimentally, the method requires a confocal microscope with fluorescence-lifetime measurement capability that makes it easily accessible to a broad optical imaging community. We demonstrate with single particle imaging that the method allows one to achieve a spatial resolution of the order of several tens of nanometers at moderate fluorescence excitation intensity.

Photosensitized protein damage by water-soluble phthalocyanine zinc(II) and gallium(III) complexes through electron transfer and singlet oxygen production

The cationic phthalocyanines (ZnPyPc and GaPyPc) photosensitized the oxidation of human serum albumin (HSA). The scavenger experiment using sodium azide suggested the contribution of 1O2. However, the HSA photodamage was observed in the presence of sodium azide. Because the redox potential of these phthalocyanines were relatively small and the Gibbs energy of the electron transfer from tryptophan to singlet excited states of these phthalocyanines is negative, electron-transfer oxidation is possible in terms of thermodynamics. Fluorescence lifetime measurement supported the electron-transfer mechanism. This study showed that electron transfer–supported oxidation plays an important role in the protein photodamage by cationic phthalocyanines.

Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue.

Fluorescence lifetime (FLT) multiplexing and multispectral imaging (MSI) are both frequently employed for in vitro and ex vivo biological studies. In vivo applications of MSI for deep seated fluorophores require consideration of diffusive light propagation in biological tissue. We have previously shown that a well-known redshift of fluorescence spectra in diffusive medium induces a fluorophore cross-talk, which cannot be accounted for even with known optical properties of the medium. In contrast, FLT measurements remain largely unaffected by light propagation in tissue, enabling zero cross-talk and accurate relative quantification. While a fully quantitative estimation of fluorophore concentrations requires depth resolved tomographic imaging, this is often not possible due to the difficulty of estimating tissue optical properties and modelling light propagation in complex tissue geometries. Here, we experimentally investigate the performance of planar (non-tomographic) MSI and FLT multiplexing for the quantitative recovery of multiple near-infrared fluorophores embedded in 4-8 mm thick tissue. We show that FLT multiplexing provides a superior quantification accuracy (error < 10%) compared to MSI (error = 20-107%) in tissue. The error rates for MSI increased with tissue thickness and can be directly attributed to the spectral redshift induced cross-talk between emission spectra. Our data indicate that planar FLT multiplexing can provide high quantification accuracy in thick biological tissue without a need for optical property estimation, thereby offering an important validation tool for rapid quantification of fluorophore concentrations in bulk tissue.

Protein Mobility Measurements through Oxidative Green-to-Red Photoconversion of EGFP.

Protein dynamics plays a key role in live cell functioning, stimulating the development of new experimental techniques for studying protein transport phenomena. Here, we introduce a relaxation method that is based on the rapid formation of a nonequilibrium concentration profile of the enhanced green fluorescent protein (EGFP) across a sample by its oxidative green-to-red photoconversion. Following the blue-light irradiation of a part of a sample containing EGFP and an oxidant, the diffusion-controlled response of a system is monitored. Changes in the concentration of the initial green-emitting and oxidized red-emitting forms are simultaneously tracked by fluorescence lifetime measurements using the time-correlated single photon counting. We show that the diffusion coefficient of EGFP in water, determined by this method, is in good agreement with previously published data. This approach opens a way for the studies of intracellular viscosity changes combined with sensing of elevated levels of reactive oxygen species.

Fluorescence signatures of SARS-CoV-2 spike S1 proteins and a human ACE-2: excitation-emission maps and fluorescence lifetimes.

.Significance: Fast and reliable detection of infectious SARS-CoV-2 virus loads is an important issue. Fluorescence spectroscopy is a sensitive tool to do so in clean environments. This presumes a comprehensive knowledge of fluorescence data.Aim: We aim at providing fully featured information on wavelength and time-dependent data of the fluorescence of the SARS-CoV-2 spike protein S1 subunit, its receptor-binding domain (RBD), and the human angiotensin-converting enzyme 2, especially with respect to possible optical detection schemes.Approach: Spectrally resolved excitation-emission maps of the involved proteins and measurements of fluorescence lifetimes were recorded for excitations from 220 to 295 nm. The fluorescence decay times were extracted by using a biexponential kinetic approach. The binding process in the SARS-CoV-2 RBD was likewise examined for spectroscopic changes.Results: Distinct spectral features for each protein are pointed out in relevant spectra extracted from the excitation-emission maps. We also identify minor spectroscopic changes under the binding process. The decay times in the biexponential model are found to be and .Conclusions: Specific material data serve as an important background information for the design of optical detection and testing methods for SARS-CoV-2 loaded media.

Abnormal push-pull benzo[4,5]imidazo[1,2-a][1,2,3]triazolo[4,5-e]pyrimidine fluorophores in planarized intramolecular charge transfer (PLICT) state: Synthesis, photophysical studies and theoretical calculations

The combination of excellent luminescence with high solvent polarity effect and aggregation induced emission (AIE) is an ideal combination for creating fluorophores/probes with high microenvironmental sensitivity. However, many push-pull chromophores of the D−A type in common intramolecular charge transfer (ICT) state with a significant solvatochromic effect and AIE activity, have poor luminescent properties. Herein, to overcome this problem by using reactions of nucleophilic aromatic hydrogen substitution (SNH), we have designed a series of novel 4-heteroaryl-substituted 2-aryl-2H-benzo[4,5]imidazo[1,2-a][1,2,3]triazolo[4,5-e]pyrimidine fluorophores possessing a planarized intramolecular charge transfer (PLICT) state. All these fluorophores exhibited high luminescence quantum yields (up to 60%) and large Stokes shift values of up to 7459 cm−1. Among them, the fluorophore 4h was found to exhibit the most pronounced positive solvatochromic effect and the probe 4f exhibited the most pronounced aggregation induced emission characteristics. This AIE behavior was further confirmed by means of time-resolved fluorescence lifetime measurements as well as DFT-assisted geometry optimization studies. In the presence of trifluoroacetic acid (TFA) compound 4h exhibited a well-pronounced acidochromism via visible color change from yellow-green to orange which returned to the original yellow-green solution after the addition of triethylamine (TEA). The Stern-Volmer constant for the probe 4h towards TFA was 38 M−1. Finally, for the compounds 4f, g, h theoretical calculations in the ground and excited states in different solvents were carried out to confirm the PLICT process. Based on all above the herein reported PLICT fluorophores 4a-h can be successfully applied as biological probes and optical switches.

Time-resolved fluorescence and anisotropy studies of red pigments present in acrylic formulations

Red organic pigments are frequently found in modern paintings and murals based on acrylic formulations. The detection of these molecules is valuable to guide investigations about cultural heritage and for restoration efforts. These studies usually employ microscopic amounts of materials that are obtained through swabbing or micro-sampling. In this contribution we describe the time-resolved emission properties of a set of red pigments with the objective of characterizing their excited state properties and developing strategies to identify their presence through fluorescence lifetime measurements, even in concentrations of the order of 10−9 M. As we show, using different solvent systems, the emission decay measurements can be setup to be a robust identification technique that avoids problems with evaporation or partial solubility. We also show that the sensitivity of these determinations is improved using a confocal type of setup with a high numerical aperture lens to ensure a high photon capture. This setup also allows for the samples to be prepared in microliter level volumes which implies a relatively high concentration of the pigments. In addition, we show that the lifetime measurements can be complemented with determinations of the emission anisotropy decays with the same experimental setup, which provides an additional property specific to each pigment, permitting an accurate differentiation between fluorophores.

Revealing superradiant emission in the single-to-bulk transition of quantum emitters in nanodiamond agglomerates

Individual quantum emitters form a fundamental building block for emerging quantum technologies. Collective effects of emitters, such as superradiance, might improve the performance of applications even further. When scaling materials to larger sizes, however, the optical density of states is modified by the surrounding material, and the collective coupling in small domains might be covered by transitions to bulk properties due to the presence of multiple collectively emitting domains, which inhomogeneously add. Here, we probe the optical properties of nitrogen vacancy centers in agglomerates of nanodiamonds. We quantify the transition from individual emitters to bulk emission by fluorescence lifetime measurements, and find a transition to occur on a length scale of wavelengths around the emitter. While our lifetime measurements are consistent with superradiant decay, the second-order correlation function, which is a standard measure to reveal collective properties, fails to probe collective effects for our case of an ensemble of collectively contributing domains to the emission. Therefore, we propose and apply a new measure to trace collective effects based on the intensity fluctuations of the emitted light. Our work points toward systematically studying collective effects in a scalable solid-state quantum system, and using them for quantum optical applications in agglomerates of highly-doped nanodiamonds.

Use of nanosecond excitation pulses in fluorescence lifetime measurement via phasor analysis.

We investigated the possibility of using long excitation pulses in fluorescence lifetime imaging microscopy (FLIM) using phasor analysis. It has long been believed that the pulse width of an excitation laser must be shorter than the lifetime of a fluorophore in a time-domain FLIM system. Even though phasor analysis can effectively minimize the pulse effect by using deconvolution, the precision of a measured lifetime can be degraded seriously. Here, we provide a fundamental theory on pulse-width-dependent measurement precisions in lifetime measurement in the phasor plane. Our theory predicts that high-precision lifetimes can be obtained even with a laser whose pulse width is four times larger than the lifetime of a fluorophore. We have experimentally demonstrated this by measuring the lifetimes of fluorescence probes with 2.57 ns and 3.75 ns lifetimes by using various pulse widths (0.52-38 ns) and modulation frequencies (10-200 MHz). We believe our results open a new possibility of using long pulse-width lasers for high-precision FLIM.

Composition and concentration-dependent photoluminescence of nitrogen-doped carbon dots

Photoluminescent carbon dots have received significant research interest in recent years owing to their extraordinary optical properties, biocompatibility, and versatile functionalities. Nitrogen-doping is a widely used strategy for enhancing the photo-electronic functionalities of carbon dots. However, there is a lack of systematic study on the composition and concentration-dependency emission behaviour of N-doped carbon dots in the literature. In this study, multicolour carbon dots (CDs) having different degree of nitrogen doping were synthesized by varying the molar ratio of citric acid to urea in the precursor via hydrothermal treatment. The effects of nitrogen doping on chemical, optical, and electronic properties of CDs were characterized using various techniques including transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FTIR); fluorescence and absorption spectroscopy; fluorescence lifetime and Hall effect measurements. Three main emissive centres were recognized in concentration-dependent fluorescence study of N-CDs which can be ascribed to molecular type of fluorescence, core emission, and mid-gap nitrogen states on the edge/surface of CDs. A plausible mechanism in relation to the obtained results is proposed. This work provides insights on the opto-electro-tunability of CDs via N-doping.

In vivo two-photon fluorescence lifetime imaging microendoscopy based on fiber-bundle

Fluorescence lifetime imaging microendoscopy (FLIME) has been reported to investigate the physicochemical microenvironment in biological tissue. In this work, we designed a two-photon (TP) FLIME system based on a fiber-bundle glued with an achromatic mini-objective with 1.4-mm diameter, which was coupled to a standard TP microscope containing a dispersion precompensation module in the laser source. With 840 nm excitation, the field of view and lateral resolution of our system are 390 µm and 1.55 µm, respectively. To examine the capability of imaging in vivo, we obtained Z-stack (0-130 µm) TP-FLIME images from the intestine's surface of a mouse injected with squaraine dye. Further, we utilized the TP-FLIME system to image the kidney, liver, and xenografted tumor at 100-µm depth in vivo with cellular resolution, which features the distribution of cells and tissue structures with better contrast than intensity images. These results demonstrated that the proposed system is capable of measuring fluorescence lifetime in situ and provides a powerful tool to research the deep tissue microenvironment naturally.

A new mitochondrial probe combining pyrene and a triphenylphosphonium salt for cellular oxygen and free radical detection via fluorescence lifetime measurements

To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, we connected pyrene derivatives (PB) to a triphenylphosphonium salt (TPP+) as a mitochondrial vector forming PB-TPP+ probes. Two pyrene isomers with the n-butyltriphenylphosphonium moieties attached at their 1- or 2- positions were synthesized and characterized. Using the long fluorescence lifetime of pyrene, it was possible to monitor the variation of cellular free radicals and oxygen and to follow the reversibility of both quenchers in real-time. We compared the behavior of these new probes to the previously published pyrene-probes, functionalized by a mitochondrial-penetrating peptide, allowing their transfer to the mitochondria (Mito-PB) or to the cytosolic membrane for pyrene butyric acid (PBA). The high cellular uptake of the new probes allows cells to be loaded with an initial concentration 40 times lower than that for Mito-PB probes, without inducing perturbations in cell growth. The variation in free radicals and oxygen levels was monitored within cells under different stress conditions through the fluorescence lifetime of the new TPP+-based probes giving comparable results to those obtained for MPP-based probes. However, at a loading concentration as low as 25 nM, our technique allows the detection of increased production of free radicals in the mitochondria in the presence of the TPP+ vector, a warning to the user of this well-known vector.

Spectroscopic study of Er-doped Ga2Ge5S13 glass for mid-IR laser applications

We present mid-IR spectroscopic characterization of the low-phonon chalcogenide glass, Ga2Ge5S13 (GGS) doped with Er3+ ions. Under the excitation at ∼800 nm, Er3+:GGS exhibited broad mid-IR emission bands centered at ∼2.7, ∼3.5, and ∼4.5 µm at room temperature. The emission lifetime of the 4I9/2 level of Er3+ ions in GGS glass was found to be millisecond-long at room temperature. The measured fluorescence lifetimes were nearly independent of temperature, indicating negligibly small nonradiative decay rate for the 4I9/2 state, as can be expected for a low-maximum-phonon energy host. The transition line-strengths, radiative lifetimes, fluorescence branching ratios were calculated by using the Judd-Ofelt method. The peak stimulated emission cross-section of the 4I9/2 → 4I11/2 transition of Er3+ ion was determined to be ∼0.10×10−20 cm2 at room temperature.

Synthesis and Solvent Dependent Fluorescence of Some Piperidine-Substituted Naphthalimide Derivatives and Consequences for Water Sensing.

Novel fluorescent strigolactone derivatives that contain the piperidine-substituted 1,8-naphthalimide ring system connected through an ether link to a bioactive 3-methyl-furan-2-one unit were synthesized and their spectroscopic properties investigated. The solvatochromic behavior of these piperidine-naphthalimides was monitored in solvents of different polarity using the electronic absorption and fluorescence spectra. These compounds exhibited a strong positive solvatochromism taking into account the change of solvent polarity, and the response mechanism was analyzed by fluorescence lifetime measurements. According to Catalan and [f(n), f(ε), β, α] solvent scales, the dipolarity and polarizability are relevant to describe the solute–solvent interactions. The emission chemosensing activity was discussed in order to determine the water content in organic environments. The emission intensity of these compounds decreased rapidly in dioxane, increasing water level up to 10%. Measuring of quantum yield indicated that the highest values of quantum efficiency were obtained in nonpolar solvents, while in polar solvents these derivatives revealed the lowest quantum yield. The fluorescence decay can be described by a monoexponential model for low water levels, and for higher water contents a biexponential model was valid.

Investigation of Thermally Activated Delayed Fluorescence in Donor–Acceptor Organic Emitters with Time-Resolved Absorption Spectroscopy

In this contribution, we utilize multiple time-resolved and nonlinear optical measurements and quantum chemical simulations to investigate the excited-state dynamics of organic chromophores with thermally activated delayed fluorescent (TADF) characteristics. We are most interested in probing the influence of a phenylene linker on the photophysical properties of emitters composed of carbazole-based donors linked to either a phthalonitrile (PN) or diphenyltriazine (Trz) acceptor. The PN-acceptor compounds display a near double-fluorescence quantum yield (ΦF) enhancement in oxygen-free conditions. The fluorescent lifetime measurements indicate that the Trz-acceptor compounds are more efficient fluorescence emitters and quickly go from a delocalized to localized state. They also reveal that only the PN-acceptor compounds display a long-lived emissive lifetime component associated with TADF activity. Analysis of the nanosecond transient absorption spectra and kinetics reveals long-lived excited-state absorption (ESA) bands associated with triplet states for the PN-acceptor compounds. No ESA bands were observed for the Trz-acceptor compounds, despite observing a quantum yield enhancement for the Trz-acceptor compounds after oxygen purging. From the transient absorption measurements, it was determined that the PN-acceptor compounds have reverse intersystem crossing rates (krISC) that are able to compete with other triplet decay pathways. From quantum chemical calculations, it is proposed that inclusion of the phenylene linker prevents sufficient highest occupied molecular orbital/lowest unoccupied molecular orbital separation and suppresses TADF activity and that directly linking multiple donors to the acceptor will aid in achieving TADF activity.