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

Mitochondrial G-quadruplexes are components that are potentially involved in regulating mitochondrial function and play crucial roles in the replication and transcription of mitochondrial genes. Consequently, it is imperative to develop probes that can detect mitochondrial G-quadruplexes to understand their functions and mechanisms. In this study, a triphenylamine fluorescent probe, TPPE, which has excellent cytocompatibility and does not affect the natural state of G-quadruplexes, was designed and demonstrated to localize primarily to the mitochondria. Owing to the unique binding mode between TPPE and G-quadruplexes, TPPE was able to distinguish G-quadruplexes from other substances due to the higher fluorescence lifetime and quantum yield. On the basis of the photon counts determined via fluorescence lifetime imaging microscopy, we analyzed the differences in the numbers of mitochondrial G-quadruplexes in various cell lines. We observed reductions in the number of mitochondrial G-quadruplexes during apoptosis, ferroptosis and glycolysis inhibition. This study shows the great potential of using TPPE to track and analyze mitochondrial G-quadruplexes and presents a novel perspective in the development of probes to detect mitochondrial G-quadruplexes in live cells.

Non-invasive screening for bladder cancer is crucial for treatment and postoperative follow-up. This study combines digital microfluidics (DMF) technology with fluorescence lifetime imaging microscopy (FLIM) for urine analysis and introduces a novel non-invasive bladder cancer screening technique. Initially, the DMF was utilized to perform preliminary screening and enrichment of urine exfoliated cells from 54 participants, followed by cell staining and FLIM analysis to assess the viscosity of the intracellular microenvironment. Subsequently, a deep learning residual convolutional neural network was employed to automatically classify FLIM images, achieving a three-class prediction of high-risk (malignant), low-risk (benign), and minimal risk (normal) categories. The results demonstrated a high consistency with pathological diagnosis, with an accuracy of 91% and a precision of 93%. Notably, the method is sensitive for both high-grade and low-grade bladder cancer cases. This highly accurate non-invasive screening method presents a promising approach for bladder cancer screening with significant clinical application potential.

The effective measurement of temperature in living systems at the nano and microscopic scales continues to be a challenge to this day. Here, we study the use of 2-(anthracen-2-yl)-1,3-diisopropylguanidine, 1, as a nanothermometer based on fluorescence lifetime measurements and its bioimaging applications. In aqueous solution, 1 is shown in aggregated form and the equilibrium between the two main aggregate types (T-shaped and π-π) is highly sensitive to the temperature. The heating of the medium shifts the equilibrium toward the formation of highly emissive T-shaped aggregates. This species shows a high fluorescence emission and a long lifetime in comparison with the π-π aggregates and the freé monomer. A linear relationship between the fluorescence lifetime and the temperature both in aqueous solution and in a synthetic intracellular buffer was found. Fluorescence lifetime imaging microscopy (FLIM) also showed a linear relationship between lifetime and temperature with an excellent sensitivity in MCF7 breast cancer cells, which opens the door for its potential use as FLIM nanothermometer in the biomedical field.

Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for measuring fast changes in MP in excitable cells, there is a dearth of such methods for absolute and precise measurements of steady-state membrane potentials (MPs) in bacterial cells. Conventional electrode-based methods for the measurement of MP are not suitable for calibrating optical methods in small bacterial cells. While optical measurement based on Nernstian indicators have been successfully used, they do not provide absolute or precise quantification of MP or its changes. We present a novel, calibrated MP recording approach to address this gap. In this study, we used a fluorescence lifetime-based approach to obtain a single-cell resolved distribution of the membrane potential and its changes upon extracellular chemical perturbation in a population of bacterial cells for the first time. Our method is based on (i) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer (PeT) and (ii) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily visualized, recorded and quantified. By artificially modulating potassium concentration gradients across the membrane using an ionophore, we have obtained a Bacillus subtilis-specific MP versus VF lifetime calibration and estimated the MP for unperturbed B. subtilis cells to be -65 mV and that for chemically depolarized cells as -14 mV. We observed a population level MP heterogeneity of ~6-10 mV indicating a considerable degree of diversity of physiological and metabolic states among individual cells. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.

Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.

Cancer-associated fibroblasts (CAFs) play a key role in metabolic reprogramming and are well-established contributors to drug resistance in colorectal cancer (CRC). To exploit this metabolic crosstalk, we integrated a systems biology approach that identified key metabolic targets in a data-driven method and validated them experimentally. This process involved high-throughput computational screening to investigate the effects of enzyme perturbations predicted by a computational model of CRC metabolism to understand system-wide effects efficiently. Our results highlighted hexokinase (HK) as one of the crucial targets, which subsequently became our focus for experimental validation using patient-derived tumor organoids (PDTOs). Through metabolic imaging and viability assays, we found that PDTOs cultured in CAF conditioned media exhibited increased sensitivity to HK inhibition. Our approach emphasizes the critical role of integrating computational and experimental techniques in exploring and exploiting CRC-CAF crosstalk.

We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 μm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.

OBJECTIVE: Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos?
CONCLUSIONS: FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not.
BACKGROUND: CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART.
METHODS: A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied.
METHODS: This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1-50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P-values of <0.05 were considered statistically significant.
RESULTS: A total of 851 CC clusters from 851 cumulus-oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not.
CONCLUSIONS: Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection.
CONCLUSIONS: Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART.
BACKGROUND: National Institutes of Health grant NIH R01HD092550-03 (to C.R., and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1.
BACKGROUND: N/A.

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.