Changes In Fluorescence Lifetime Research Articles

Luminescence enhancement of Y2O3 thin films based on LSPR effect of Ag nanolayers

Based on the local surface plasmon resonance effect of Ag nanolayer, the luminescence enhancement in the visible light range of multilayer thin film has been investigated combined with metal/spacer molecule/photoluminescence material structure. The luminescence enhancement effect of multilayer thin films can be improved by adjusting the spin-coating number of Ag nanolayer and SiO2 spacer in the multilayer structure. At the same time, the mechanism of enhanced luminescence has been analyzed according to the change in fluorescence lifetime. The effect of the coupling between Ag nanolayer and luminescence layer on the radiative transition rate and fluorescence quantum yield have been discussed. The up-conversion luminescence intensity of the noble metal-modified multilayer thin film can be further improved by combining the multilayer structure with sensitized layer.

A Toxicogenic Interaction between Intracellular Amyloid-β and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid β (Aβ) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aβ oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aβ-ApoE interaction suppresses these changes and concomitantly reduces Aβ toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aβ oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Fast and slow: Recording neuromodulator dynamics across both transient and chronic time scales.

Neuromodulators transform animal behaviors. Recent research has demonstrated the importance of both sustained and transient change in neuromodulators, likely due to tonic and phasic neuromodulator release. However, no method could simultaneously record both types of dynamics. Fluorescence lifetime of optical reporters could offer a solution because it allows high temporal resolution and is impervious to sensor expression differences across chronic periods. Nevertheless, no fluorescence lifetime change across the entire classes of neuromodulator sensors was previously known. Unexpectedly, we find that several intensity-based neuromodulator sensors also exhibit fluorescence lifetime responses. Furthermore, we show that lifetime measures in vivo neuromodulator dynamics both with high temporal resolution and with consistency across animals and time. Thus, we report a method that can simultaneously measure neuromodulator change over transient and chronic time scales, promising to reveal the roles of multi-time scale neuromodulator dynamics in diseases, in response to therapies, and across development and aging.

Spectroscopic Identification of Charge Transfer of Thiolated Molecules on Gold Nanoparticles via Gold Nanoclusters.

Investigation of charge transfer needs analytical tools that could reveal this phenomenon, and enables understanding of its effect at the molecular level. Here, we show how the combination of using gold nanoclusters (AuNCs) and different spectroscopic techniques could be employed to investigate the charge transfer of thiolated molecules on gold nanoparticles (AuNP@Mol). It was found that the charge transfer effect in the thiolated molecule could be affected by AuNCs, evidenced by the amplification of surface-enhanced Raman scattering (SERS) signal of the molecule and changes in fluorescence lifetime of AuNCs. Density functional theory (DFT) calculations further revealed that AuNCs could amplify the charge transfer process at the molecular level by pumping electrons to the surface of AuNPs. Finite element method (FEM) simulations also showed that the electromagnetic enhancement mechanism along with chemical enhancement determines the SERS improvement in the thiolated molecule. This study provides a mechanistic insight into the investigation of charge transfer at the molecular level between organic and inorganic compounds, which is of great importance in designing new nanocomposite systems. Additionally, this work demonstrates the potential of SERS as a powerful analytical tool that could be used in nanochemistry, material science, energy, and biomedical fields.

Three-Four Photon Transition Mn(II) Complex Monitoring Lysosome-Related ATP in Real Time via Fluorescence Lifetime Imaging.

Currently, in situ monitoring of the adenosine triphosphate (ATP) level in lysosomes is critical to understand their involvement in various biological processes, but it remains difficult due to the interferences of limited targeting and low resolution of fluorescent probes. Herein, we report a classic Mn(II) probe (FX2-MnCl2) with near-infrared (NIR) nonlinear (NLO) properties, accompanied by three-four photon transition and fivefold fluorescence enhancement in the presence of ATP. FX2-MnCl2 combines with ATP through dual recognition sites of diethoxy and manganese ions to reflect slightly fluorescence lifetime change. Through the synergy of multiphoton fluorescence imaging (MP-FI) and multiphoton fluorescence lifetime imaging microscopy (MP-FLIM), it is further demonstrated that FX2-MnCl2 displays lysosome-specific targeting behavior, which can monitor lysosome-related ATP migration under NIR laser light. This work provides a novel multiphoton transformation fluorescence complex, which might be a potential candidate as a simple and straightforward biomarker of lysosome ATP in vitro for clinical diagnosis.

Fibre-based fluorescence-lifetime imaging microscopy: a real-time biopsy guidance tool for suspected lung cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.

Phasor-FLIM analysis of cellulose paper ageing mechanism with carbotrace 680 dye

Ageing of paper is a complex process of great relevance for application purposes because of its widespread use as support for information storage in books and documents, and as common low-cost and green packaging material, to name a few. A key factor in paper ageing is the oxidation of cellulose, a macromolecule of natural origin that constitutes the main chemical component of paper. Such a complex process results in changes in the cellulose polymeric chains in chemical and structural properties. The scope of this work is to explore the effects of oxidation of cellulose as one of the principal mechanisms of ageing of paper using a fluorescence-based approach. To this aim, fluorescence-lifetime imaging microscopy (FLIM) measurements on pure cellulose samples stained using Carbotrace 680 dye were performed, and data were analyzed by phasor approach. The comparison with results from conventional techniques allowed to map paper microstructure as a function of the sample oxidation degree correlating the fluorescence-lifetime changes to cellulose oxidation. A two-step oxidation kinetics that produced specific modification in paper organization was highlighted indicating that FLIM measurements using Carbotrace 680 dye may provide a simple tool to obtain information on the oxidation process also adding spatial information at sub-micrometric scale.

Evaluating Riboglow-FLIM probes for RNA sensing.

We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. In vitro measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.

PDT-Induced Variations of Radachlorin Fluorescence Lifetime in Living Cells In Vitro

Variations in the fluorescence lifetimes of Radachlorin photosensitizers in HeLa and A549 cells, caused by photodynamic treatment, were studied using fluorescence lifetime imaging microscopy (FLIM). An analysis of FLIM images of the cells demonstrated a substantial decrease in the mean Radachlorin fluorescence lifetime and intensity as a result of UV irradiation of the photosensitized cells at different doses, with higher doses causing a more pronounced decrease in the mean fluorescence lifetime in cells. The post-treatment decrease in Radachlorin fluorescence intensity was accompanied by the appearance of an additional rapidly decaying fluorescence component and a nonlinear decrease in the weighted fluorescence lifetime obtained from double-exponential fits of time-resolved fluorescence signals. Experiments performed in the aqueous solutions of the photosensitizer revealed similar irreversible changes in the Radachlorin fluorescence lifetime and intensity. Therefore, the observed phenomena occurred most likely due to the photodegradation of the photosensitizer molecules and can be applied for dosimetry and monitoring of irradiation doses in different areas of malignant tissues in the course of photodynamic treatment.

Steady-State and Time-Resolved Fluorescence Study of Selected Tryptophan-Containing Peptides in an AOT Reverse Micelle Environment.

The aim of this study was to demonstrate the utility of time-resolved fluorescence spectroscopy in the detection of subtle changes in the local microenvironment of a tryptophan chromophore in a confined and crowded medium of AOT reverse micelles, which mimic biological membranes and cell compartmentalization. For this purpose, fluorescence properties of L-tryptophan and several newly synthesized tryptophan-containing peptides in buffer and in an AOT reverse micelle medium were determined. It was shown that insertion of tryptophan and its short di- and tripeptides inside micelles led to evident changes in both the steady-state emission spectra and in fluorescence decay kinetics. The observed differences in spectral characteristics, such as a blue shift in the emission maxima, changes in the average fluorescence lifetime, and the appearance of environmental-dependent fluorescent species, showed the utility of time-resolved fluorescence spectroscopy as a sensitive tool for detecting subtle conformational modifications in tryptophan and its peptides induced by changes in polarity, viscosity, and specific interactions between chromophores and water molecules/polar groups/ions that occur inside reverse micelles.

Spectroscopy evidence of interaction of 1,5 disubstituted piperidino-amido anthraquinone derivative with human telomeric G-quadruplex DNA: Basis of anticancer action

The binding of ligands to G-quadruplex (G4) structures at the ends of human telomeric DNA induces thermal stabilization, which interferes with telomere maintenance by disrupting the association of telomeres with the telomerase enzyme—an important marker for cancer. Understanding the binding mode of the ligand-G quadruplex DNA complex is imperative for evaluating the relative efficacy and specificity of their interaction. We focused on the interaction of 1,5-Bis(3-piperidino propionamido) anthracene-9,10-dione with human telomeric DNA sequences using surface plasmon resonance, absorbance, fluorescence (steady-state and lifetime), and circular dichroism spectroscopy techniques. The ligand binding with HTel22 (Kb = 8.4 × 105 M−1) induced cell toxicity with an IC50 value of ∼ 8.64 µM in MCF-7 cancer cell lines. Significant hypochromism (59 %), fluorescence quenching (97 %), no change in fluorescence lifetime and absence of induced Circular Dichroism (CD) band upon addition of G4 DNA to ligand, suggest a groove/external binding mode. CD spectral changes reflect rearrangement in parallel and antiparallel strands to accommodate the ligand. Docking results reveal specific short contacts of the ligand's side chain, including 9CO, 13N, 14NH, and 11CO, with the grooves of the G4 DNA, without making any contact with the loops, favoring an energetically more favorable conformation. Thermal denaturation profiles, obtained by Differential Scanning Calorimetry and CD, show the stabilization of wHTel26 and HTel22 G4 DNA in K+ and Na+ rich solutions by 21.2 and 17.6 °C, respectively, which may restrict the access of telomerase to telomeres. The findings indicate the potential of modifying anthraquinone substituent groups for therapeutic applications.

Photophysical properties of Radachlorin photosensitizer in solutions of different pH, viscosity and polarity

We present a thorough experimental investigation of fluorescence properties of Radachlorin photosensitizer in solutions of different acidity, viscosity and polarity. Experiments were performed using time-resolved fluorescence lifetime imaging and time-resolved analysis of polarized fluorescence. Variations of solution acidity resulted in considerable changes of Radachlorin fluorescence quantum yield and lifetime in the pH range from 4 to 7, but did not affect the rotational diffusion time, and almost did not change the quantum yield and characteristic times of singlet oxygen phosphorescence. Variations of solution polarity and viscosity were achieved by changing ethanol or methanol fraction in aqueous solution. The decrease of solution polarity resulted in nonlinear rise of Radachlorin fluorescence quantum yield and lifetime up to alcohol concentration of 50%–65%, as well as in considerable rise of singlet oxygen quantum yield and significant changes in characteristic times of its phosphorescence. Variations of solution viscosity resulted in changes of rotational diffusion time of Radachlorin molecules, which appeared to be in perfect correlation with methanol solution viscosity. Good correspondence with ethanol solution viscosity was observed only up to 50% alcohol fraction. Deviations of rotational diffusion time of Radachlorin molecules from direct proportionality with solution viscosity at higher ethanol concentrations were suggested to be due to different solvation conditions. The data obtained can give important reference points for analysis of microenvironment of Radachlorin molecules, their intracellular localization and performance in singlet oxygen generation.

Time-Resolved Fluorescence Spectroscopy of Blood, Plasma and Albumin as a Potential Diagnostic Tool for Acute Inflammation in COVID-19 Pneumonia Patients.

Fluorescence lifetime measurements of blood or plasma offer valuable insights into the microenvironment and molecular interactions of fluorophores, particularly concerning albumin. Neutrophil- and hypoxia-induced oxidative stress in COVID-19 pneumonia patients leads to hyperinflammation, various oxidative modifications of blood proteins, and potential alterations in the fluorescence lifetime of tryptophan-containing proteins, especially albumin. The objective of this study was to investigate the efficacy of time-resolved fluorescence spectroscopy of blood and plasma as a prompt diagnostic tool for the early diagnosis and severity assessment of COVID-19-associated pneumonia. This study examined a cohort of sixty COVID-19 patients with respiratory symptoms. To investigate whether oxidative stress is the underlying cause of the change in fluorescence lifetime, human serum albumin was treated with chloramine T. The time-resolved spectrometer Life Spec II (Edinburgh Instruments Ltd., Livingston, UK), equipped with a sub-nanosecond pulsed 280 nm diode, was used to measure the fluorescence lifetime of blood and plasma. The findings revealed a significant reduction in the fluorescence lifetime of blood (diluted 200 times) and plasma (diluted 20 times) at 360 nm in COVID-19 pneumonia patients compared with their respective values recorded six months post-infection and those of healthy individuals. Significant negative correlations were observed between the mean fluorescence lifetime of blood and plasma at 360 nm and several severity biomarkers and advanced oxidation protein products, while a positive correlation was found with albumin and the albumin-globulin ratio. The time-resolved fluorescence spectroscopy method demonstrates the potential to be used as a preliminary screening technique for identifying patients who are at risk of developing severe complications. Furthermore, the small amount of blood required for the measurements has the potential to enable a rapid fingerstick blood test.

Efficient single-cell oxygen consumption rate characterization based on frequency domain fluorescence lifetime imaging microscopy measurement and microfluidic platform.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

LUMINESCENT PROPERTIES OF Nd(III) COMPLEXES WITH ETHY­LENE­DIAMINE-N,N'-DISUCCINIC AND N,N-BIS(PHOSPHONO­METHYL)-2-AMINOPROPIONIC ACIDS

An analysis of the fluorescent characteristics of ethylenediamine-N,N'-disuccinic and N,N-bis(phosphonomethyl)-2-aminopropionic acids was carried out depending on the pH of the solutions. It was established that the change in fluorescence intensity and lifetime is associated with the formation of variously protonated forms of acids in which stable H-cycles are formed with the participation of hydrogen bonds. The energies of the singlet and triplet levels of the ligands were experimentally determined, the values of which are higher than the energy of the radiative level of the Nd(III) ion, which indicates the possibility of intramolecular transfer of the excitation energy to the resonance level of the lanthanide ion. It was established that both homo- and heteronuclear complexes of Nd(III) exhibit 4f-luminescence in the near-IR region. It was found that for phosphorus-containing complexes there is an increase in luminescence intensity and relative quantum yields in comparison with aminocarboxylate analogs. In heterometallic complexes based on aminopolycarboxylic acids, the intramolecular transfer of energy from the excited level of Co(II) to the resonance level of the f-metal leads to sensitization of the 4f-luminescence of the neodymium ion.

Increased intracellular crowding during hyperosmotic stress

Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced green fluorescent protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster resonance energy transfer sensor (GimRET). This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. Our approach can therefore be used to reveal in live cells short-lived transient states through which MMC builds over time, which could not be observed when measuring changes in other physical properties that occur at slower time scales.

Impact of cigarette smoking on fluorescence lifetime of ocular fundus

Cigarette smoking is known to adversely affect cellular metabolism and is a risk factor for various retinal diseases. Fluorescence lifetime imaging ophthalmoscopy (FLIO) has the potential to detect metabolic changes in the ocular fundus. Aim of this study was to analyze the influence of cigarette smoking on fluorescence lifetime (FLT) of healthy eyes using FLIO. Twenty-six non-smokers and 28 smokers aged between 20 and 37 years without systemic and ocular diseases were investigated by FLIO (excitation: 473 nm, emission: short spectral channel (SSC) 498–560 nm, long spectral channel (LSC) 560–720 nm). The FLT at the ETDRS grid regions were analyzed and compared. In SSC, the mean FLT (τm) of smokers was significantly longer in the ETDRS inner ring region, whereas the τm in LSC was significantly shorter in the outer ring. For the long component (τ2), smokers with pack year < 7.11 showed significantly shorter τ2 in SSC than non-smokers and the smokers with pack year ≥ 7.11. There were no significant differences in retinal thickness. The lack of obvious structural differences implies that the observed FLT changes are likely related to smoking-induced metabolic changes. These results suggest that FLIO may be useful in assessing retinal conditions related to lifestyle and systemic metabolic status.

Abstract 2389: In vivo multiphoton autofluorescence imaging is sensitive to CD8 T cell and tumor cell metabolic changes during immunotherapy in a murine melanoma model

Abstract Introduction: In vivo multiphoton autofluorescence microscopy provides live, label free, single cell imaging of metabolic changes. These metabolic changes are quantified via the metabolic coenzymes NAD(P)H and FAD which are autofluorescent molecules endogenous to all cells. Metabolic reprogramming of tumor and immune cells is closely associated with cancer progression and cell phenotype. We aim to study metabolic changes during administration of an effective, triple-combination immunotherapy within murine melanoma tumors. This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2, provided by Anyxis Immuno-Oncology GmbH of Vienna, Austria), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors. Previous work has shown that a T cell response is critical to the efficacy of this therapy, so we created an mCherry-labeled T cell mouse model to study this response. Methods: We implanted syngeneic B78 (GD2+) melanoma cells into the flanks of mCherry-labeled CD8 T cell reporter mice (C57Bl/6 background) to induce tumors. Under anesthesia, skin flap surgery was performed and tumors were imaged at several time points during therapy. Multiphoton imaging was performed to collect NAD(P)H, FAD, mCherry, and collagen signal with a 40X objective. Fluorescence lifetime data was collected using time correlated single photon counting electronics. Tissues were harvested and analyzed via flow cytometry and multiplex immunofluorescence to corroborate intravital imaging findings and characterize the immune infiltrate. Results: Here we demonstrate that our in vivo imaging is sensitive to metabolic changes in both B78 melanoma and CD8 T cells from immunotherapy treated versus control tumors. These metabolic differences include changes in protein binding, redox balance, and fluorescence lifetime. We also show remodeling of collagen, a major component of the extracellular matrix, during immunotherapy that may be explained by an observed decrease in macrophages. Flow cytometry and multiplex immunofluorescence illustrate changes in the immune infiltrate composition, activation, cytotoxicity, and spatial distribution during therapy. We are also currently investigating metabolic changes in an MC38 mouse model - a hot tumor model - that we anticipate will provide excellent metabolic contrast to our B78 immunologically cold tumor model. Conclusions: These results show that in vivo metabolic imaging enables single cell quantification of metabolic changes in tumor and immune cells during therapy. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. With continued efforts, this imaging platform may be leveraged to develop new combinations of immunotherapies. Citation Format: Alexa R. Heaton, Anna Hoefges, Peter R. Rehani, Nathaniel J. Burkard, Arika S. Feils, Dan V. Spiegelman, Noah W. Tsarovsky, Alina A. Hampton, Amy K. Erbe Gurel, Alexander L. Rakhmilevich, Paul M. Sondel, Melissa C. Skala. In vivo multiphoton autofluorescence imaging is sensitive to CD8 T cell and tumor cell metabolic changes during immunotherapy in a murine melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2389.

Towards precise optical measurements of steady state of and small changes in resting membrane potentials.

While great progress has been achieved in developing optical methods for measuring fast changes in membrane potential (like action potentials) in excitable cells, less progress has been made in precise (and calibrated) measurements of steady state resting membrane potentials (RMPs) and small changes in RMPs (in excitable or non-excitable cells). In excitable cells, small changes in RMPs are associated with multiple physiological processes such as sub-threshold events in neuronal signaling and in synaptic plasticity. They also play an important role in cell differentiation and proliferation of cardiomyocytes. In non-excitable cells, such as bacterial colonies, RMP changes play important roles in intercellular communication, coordination, metabolism, and stress response. Accurate and precise recordings of minute RMP changes require noise-immune optical tools. We have been developing an RMP (calibrated) recording approach that is capable of measuring small changes RMPs. Our approach relies on 3 components: (i) a novel optical transducer (voltage sensing dye) that utilizes intrinsic photoinduced electron transfer (PeT) mechanism to sense RMP by fluorescence lifetime changes; (ii) a novel time-gated SPAD array imager that allows widefield fluorescence lifetime imaging (FLIM); (iii) a quantitative phasor analysis of the recoded FLIM data. This package provides high-throughput, quantification of pixel-wise lifetime information. Two aspects of our approach - the novel VSD and the data analysis - have been tested in excitable cardiac muscle cells (NVRM) and in non-excitable bacterial cells (B. subtilis). B. subtilis RMPs have been estimated for normal cultures and for cultures under perturbing chemical conditions. Drug induced changes of NVRM RMPs have been also estimated. Implementation of our 3-pronged approach towards studying electrical signaling/coupling networks in bacterial biofilms and in cardiomyocytes will be discussed.

Conjugated hypercrosslinked polymers imprinted with 3,5-dinitrosalicylic acid for the fluorescent determination of α-amylase activity

The discovery of a series of coupling reactions between various building blocks has driven the development of porous organic polymers, but the common usage of expensive and air-sensitive organometallic catalysts and complex procedures in harsh syntheses has limited their expansion. A microporous hypercrosslinked polymer (HCP) was synthesized by polymerizing a naphthalene monomer and a 1,4-dimethoxybenzene crosslinker using Friedel–Crafts alkylation over an FeCl3 catalyst and imprinted with 3,5-dinitrosalicylic acid (DNS). The DNS-molecularly-imprinted HCPs (MIHCPs) were characterized as having IUPAC Type I mesoporosity, a specific surface area of 1134 m2 g−1, a monolayer adsorption capacity of 116 cm2 g−1, pore sizes ranging from 5 to 8.5 Å, amorphous frameworks, and distinctive absorption and emission bands by N2 adsorption/desorption analyses, scanning and transmission electron microscopies, and FTIR, UV–Vis, and fluorescence spectrometries. The π-conjugated imprinted framework endowed the MIHCPs with 405-nm fluorescent emission at a 330-nm excitation and dynamic quenching, which was confirmed by changes in fluorescence lifetime and followed a linear Stern-Volmer plot against 1.0–200 μM DNS template molecules under optimized conditions of a pH 7.0 buffer, an MIHCP concentration of 125 μg mL−1, and a 3.0-min equilibration time. The MIHCPs exhibited a high imprinted factor of 8.7 against nonimprinted HCP and a selectivity of 8.63 against reduced DNS, which enabled fluorometric detection of DNS molecules produced by the hydrolysis of starch with microbial, salivary, and pancreatic α-amylases and the subsequent redox incubation with the DNS oxidant. The fluorometric measurement of α-amylase activity was higher in accuracy and precision (RSD: 2.6–2.8% vs. 3.9–4.0%) than conventional UV–Vis spectrometry because the excellent fluorescent sensitivity and imprinting selectivity of the MIHCP probes enabled the use of higher dilution factors with weaker matrix effects.