Special Issue on Fluorescence Lifetime Imaging (FLIM): From Fundamentals to Applications

The present manuscript gives a short overview on Förster Resonance Energy Transfer (FRET) of molecular interactions in the nanometre range. First, its principle is described and a short historical overview is given. Subsequently some principal methods and applications of FRET sensing and imaging are described (with some emphasis on fluorescence lifetime imaging, FLIM), and finally two innovative FRET techniques are presented in more detail. Applications are focused on measurements of living cells.

Intracellular refractive index (RI) is an essential biophysical parameter, which best represents the mass and the distribution of proteins in the cell interior, including high-density accumulations in membraneless organelles. For RI measurements, a number of sophisticated techniques have been developed; however most of the new approaches are either insufficiently sensitive to intracellular variations of proteins distribution or are not compatible with live cell studies. Here, we outline the fluorescence lifetime imaging (FLIM) strategy for high resolution mapping of subcellular RI. We provide an example of our recent studies in which we utilize FLIM for measurements and monitoring of local RI in the major membraneless organelles within live cultured cells.

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

The growing demand for tissue engineered vascular grafts (TEVG) motivates the development of optimized fabrication and monitoring procedures. Bioreactors which provide physiologically-relevant conditions are important for improving holistic TEVG properties and performance. Herein we describe a fiber-based intraluminal imaging system that allows for in situ assessment of vascular materials and re-cellularization processes inside a bioreactor by simultaneous and co-registered measurements of endogenous fluorescence lifetime and exogenous marker fluorescence intensity. The lumen of 6 vascular grafts (∼4 mm diameter) were scanned by reciprocally rotating a 41° angle polished multimode optical fiber inside a protective glass tube with outer diameter of 3 mm. Tubular bovine pericardium constructs were recellularized using enhanced Green Fluorescent Protein (eGFP) transfected cells in a custom bioreactor. The imaging system has resolved consistently the cellular autofluorescence from that of tissue matrix in situ based on the lifetime fluorescence properties of endogenous molecular species. The location of the re-cellularized area was validated by the eGFP emission. Current results demonstrate the potential of this system as a valuable tool in tissue engineering for in situ studies of cell-tissue interactions in cylindrical or other 3-dimensional structures.

Autofluorescence based fluorescence lifetime imaging microscopy (AF-FLIM) techniques have come a long way from early studies on cancer characterization and have now been widely employed in several cellular and animal studies covering a wide range of diseases. The majority of research in autofluorescence imaging (AFI) study metabolic fluxes in live biological samples. However, tissues from clinical or scientific studies are often chemically fixed for preservation and stabilization of tissue morphology. Fixation is particularly crucial for enzymatic, functional, or histopathology studies. Interpretations of metabolic imaging such as optical redox intensity imaging and AF-FLIM, have often been viewed as potentially unreliable in a fixed sample due to lack of studies in this field. In this study, we carefully evaluate the possibility of extracting microenvironment information in fixed tissues using reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) endogenous fluorescence. The ability to distinguish changes such as metabolism and pH using intrinsic fluorescence in fixed tissues has great pathological value. In this work, we show that the lifetime based metabolic contrast in a sample is preserved after chemical fixation. The fluorescence lifetime of a sample increases with an additive fixative like formaldehyde; however, the fixed tissues retain metabolic signatures even after fixation. This study presents an opportunity to successfully image archived unstained histopathology tissues, and generate useful AF-FLIM signatures. We demonstrate the capability to draw metabolic interpretations in fixed tissues even after long periods of storage.

Exploring metabolism in human tumors at the cellular level remains a challenge. The reduced form of metabolic cofactor NAD(P)H is one of the major intrinsic fluorescent components in tissues and a valuable indicator of cellular metabolic activity. Fluorescence lifetime imaging (FLIM) enables resolution of both the free and protein-bound fractions of this cofactor, and thus, high sensitivity detection of relative changes in the NAD(P)H-dependent metabolic pathways in real time. However, the clinical use of this technique is still very limited. The applications of metabolic FLIM could be usefully expanded by probing cellular metabolism in tissues ex vivo. For this, however, the development of appropriate tissue preservation protocols is required in order to maintain the optical metabolic characteristics in the ex vivo sample in a state similar to those of the tumor in vivo. Using mouse tumor models of different histological types—colorectal cancer, lung carcinoma and melanoma—we tested eight different methods of tissue handling by comparing NAD(P)H fluorescence decay parameters ex vivo and in vivo as measured with two-photon excited FLIM microscopy. It was found that the samples placed in 10% BSA on ice immediately after excision maintained the same fluorescence lifetimes and free/bound ratios as measured in vivo for at least 3 hours. This protocol was subsequently used for metabolic assessments in fresh postoperative samples from colorectal cancer patients. A high degree of inter- and intra-tumor heterogeneity with a trend to a more oxidative metabolism was detected in T3 colorectal tumors in comparison with normal tumor-distant colon samples. These results suggest that the methodology developed on the basis of FLIM of NAD(P)H in tissues ex vivo show promise for interrogating the metabolic state of patients' tumors.

Biological proteins are understood in terms of five structural levels–primary, secondary, tertiary, quaternary and quinary. The quinary structure is defined as the set of macromolecular interactions that are transient in vivo. This includes non-covalent protein-protein interactions occurring within the crowded intracellular environment. For much of twentieth century science, the canonical approach to studying biological proteins involved test tube environments. These uncrowded in vitro studies inadvertently failed to replicate and observe the quinary structures present within the original cells. Consequently, contemporary literature surrounding the fifth level of protein organisation is lacking. In particular, there is a lack of literature on the size of transient clusters within living cells. In an attempt to reconcile this gap in knowledge, we propose a quantitative method for estimating the average quinary stoichiometry in living cells. The method is based on lifetime self-quenching of fluorescently-labelled proteins in living cells. Close approach of two or more proteins in a quinary complex will result in self-quenching of the fluorescence lifetime from the fluorescent labels. Our method utilises the random mixing of proteins during cell division to mix fluorescently labelled with unlabelled proteins. Such mixing reduces the probability of adjacency between labelled proteins and, hence, decreases the probability of fluorescence lifetime quenching from labels. By monitoring fluorescence lifetime dequenching during multiple cell divisions, we can determine the average quinary structure in living proliferating cells. We demonstrate this method with a case study on cultured HeLa cells. The average quinary stoichiometry was found to be between five and six. That is, at any given point in time, there are five or six weakly interacting partners in the immediate neighbourhood of any given protein.

We report a multidimensional luminescence microscope providing hyperspectral imaging and time-resolved (luminescence lifetime) imaging for the study of luminescent diamond defects. The instrument includes crossed-polariser white light transmission microscopy to reveal any birefringence that would indicate strain in the diamond lattice. We demonstrate the application of this new instrument to detect defects in natural and synthetic diamonds including N3, nitrogen and silicon vacancies. Hyperspectral imaging provides contrast that is not apparent in conventional intensity images and the luminescence lifetime provides further contrast.

Photon pressure has been used to induce the aggregation from solution of a series of photoluminescent conjugated polyelectrolytes containing tetraphenylethene units. These polymers show steady-state and time-resolved emission properties that are dependent on the local chromophore environment that can be influenced by the degree of intra- and inter-molecular interactions, which enables the photoaggregation process to be monitored by time-resolved fluorescence imaging techniques. Structural differences in the polymer lead to variations in the photo-induced aggregation behaviour.

Many molecular processes within a cell are carried out by molecular machines built from a large number of proteins organized by their protein-protein interactions (PPIs). Exploring PPIs in their cellular context is critical to better understand the proteins functions. Förster resonance energy transfer measured by fluorescence lifetime imaging (FLIM-FRET) enables to monitor PPIs and to map their spatial organization in a living cell with high spatial and temporal specificity. But both the accurate measurement and the interpretation of multi-exponential FLIM-FRET data associated to mixtures of interacting and non-interacting proteins are difficult. Here we show that a simple diagram plot can find interesting visualization properties by clustering pixels with similar decay signatures. FLIM diagram plot can be used to provide valuable information about stoichiometry and binding mode in PPIs, even in the presence of large differences in protein expression levels of the different interacting partners. The proposed FLIM diagram plot is a useful visual approach for a more straightforward interpretation of complex lifetime data. This approach was applied for revealing critical features of PPIs in live Pseudomonas aeruginosa.

The oocytes from Xenopus laevis are well known for their polarity, presenting a distinct animal and vegetal pole. Other heterogeneities are less known. To study the heterogeneity of the Xenopus oocyte, we expressed eGFP and analyzed the protein distribution with fluorescence lifetime microscopy. The vegetal pole exhibited higher levels of fluorescence, than the animal pole. However, the fluorescence lifetimes between the two areas were indistinguishable, suggesting similar environments. In contrast, we observed a substantial and gradual decrease in the fluorescence lifetime from 2.9 ns to 2.6 ns as slices approached the periphery. This has an important implication for future oocyte studies as it demonstrates the environment inside the oocyte is not uniform and might affect the fluorescence intensity. As a result, it cannot be assumed that the observed fluorescence intensity reflects the expression of the proteins but might reflect the environment within the oocyte.

Increasingly, the auto-fluorescent coenzymes NAD(P)H and FAD are being tracked by multi-photon fluorescence lifetime microscopy (FLIM) and used as versatile markers for changes in mammalian metabolism. The cellular redox state of different cell model systems, organoids and tissue sections is investigated in a range of pathologies where the metabolism is disrupted or reprogrammed; the latter is particularly relevant in cancer biology. Yet, the actual optimized process of acquiring images by FLIM, execute a correct lifetime fitting procedure and subsequent processing and analysis can be challenging for new users. Questions remain of how to optimize FLIM experiments, whether any potential photo-bleaching affects FLIM results and whether fixed specimens can be used in experiments. We have broken down the multi-step sequence into best-practice application of FLIM for NAD(P)H and FAD imaging, with images generated by a time-correlated-single-photon-counting (TCSPC) system, fitted with Becker & Hickl software and further processed with open-source ImageJ/Fiji and Python software.

We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512 × 512 pixels), even in the case of small number of gates and limited photon budget.

In this review, we discuss methods and advancements in fluorescence lifetime imaging microscopy that permit measurements to be performed at faster speed and higher resolution than previously possible. We review fast single-photon timing technologies and the use of parallelized detection schemes to enable high-throughput and high content imaging applications. We appraise different technological implementations of fluorescence lifetime imaging, primarily in the time-domain. We also review combinations of fluorescence lifetime with other imaging modalities to capture multi-dimensional and correlative information from a single sample. Throughout the review, we focus on applications in biomedical research. We conclude with a critical outlook on current challenges and future opportunities in this rapidly developing field.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.

To better understand pH-dependence of endogenous fluorescence of algae, we employed spectroscopy and microscopy methods, including advanced time-resolved fluorescence imaging microscopy (FLIM), using green algae Chlorella sp. as a model system. Absorption spectra confirmed two peaks, at 400–420 nm and 670 nm. Emission was maximal at 680 nm, with smaller peaks between 520 and 540 nm. Acidification led to a gradual decrease in the red fluorescence intensity with the maximum at 680 nm when excited by 450 nm laser. FLIM measurements, performed using 475 nm picoseconds excitation, uncovered that this effect is accompanied by a shortening of the tau1 fluorescence lifetime. Under severe acidification, we also noted an increase in the green fluorescence with a maximum between 520–540 nm and a shift toward 690–700 nm of the red fluorescence, accompanied by prolongation of the tau2 fluorescence lifetime. Gathered data increase our knowledge on the responsiveness of algae to acidification and indicate that endogenous fluorescence derived from chlorophylls can potentially serve as a biosensing tool for monitoring pH change in its natural environment.

We have implemented polarization-resolved fluorescence lifetime measurement through stimulated emission based pump-probe technique, which promises much higher temporal resolution (∼4 ps) than conventional time-correlated single-photon counting (TCSPC). The depolarization of ATTO 647N fluorescent dye is resolved through anisotropy fluorescence lifetime measurements, with variable time delay introduced between the pump and the probe beams. Importantly, the polarization anisotropy measurement and the corresponding rotational correlation time characterization of the fluorescent dye are carried out at various temperatures. We have also demonstrated the need of high temporal resolution via hetero Förster energy transfer (Hetero-FRET) through the interaction between the gold nanorods (GNRs) and the fluorescent dye ATTO 647N. Notably, our results compare highly favorably with conventional TCSPC method, which is rather limited in temporal resolution, for the above characterization. Additionally, this technique is applicable even under ambient light while being very cost-effective and robust.

Facultative intracellular pathogens are able to live inside and outside host cells. It is highly desirable to differentiate their cellular locations for the purposes of fundamental research and clinical applications. In this work, we developed a novel analysis platform that allows users to choose two analysis models: amplitude weighted lifetime (τ_A) and intensity weighted lifetime (τ_I) for fluorescence lifetime imaging microscopy (FLIM). We applied these two models to analyse FLIM images of mouse Raw macrophage cells that were infected with bacteria Shigella Sonnei, adherent and invasive E. coli (AIEC) and Lactobacillus. The results show that the fluorescence lifetimes of bacteria depend on their cellular locations. The τ_A model is superior in visually differentiating bacteria that are in extra- and intra-cellular and membrane-bounded locations, whereas the τ_I model show excellent precision. Both models show speedy performances that analysis can be performed within 0.3 s. We also compared the proposed models with a widely used commercial software tool (τ_C, SPC Image, Becker & Hickl GmbH), showing similar τ_I and τ_C results. The platform also allows users to perform phasor analysis with great flexibility to pinpoint the regions of interest from lifetime images as well as phasor plots. This platform holds the disruptive potential of replacing z-stack imaging for identifying intracellular bacteria.

Fluorescence Lifetime Imaging (FLIM) in life sciences based on ultrashort laser scanning microscopy and time-correlated single photon counting (TCSPC) started 30 years ago in Jena/East-Germany. One decade later, first two-photon FLIM images of a human finger were taken with a lab microscope based on a tunable femtosecond Ti:sapphire laser. In 2002/2003, first clinical non-invasive two-photon FLIM studies on patients with dermatological disorders were performed using a novel multiphoton tomograph. Current in vivo two-photon FLIM studies on human subjects are based on TCSPC and focus on (i) patients with skin inflammation and skin cancer as well as brain tumors, (ii) cosmetic research on volunteers to evaluate anti-ageing cremes, (iii) pharmaceutical research on volunteers to gain information on in situ pharmacokinetics, and (iv) space medicine to study non-invasively skin modifications on astronauts during long-term space flights. Two-photon FLIM studies on volunteers and patients are performed with multiphoton FLIM tomographs using near infrared femtosecond laser technology that provide rapid non-invasive and label-free intratissue autofluorescence biopsies with picosecond temporal resolution.

Multiphoton fluorescence lifetime microscopy has revolutionized studies of pathophysiological and xenobiotic dynamics, enabling the spatial and temporal quantification of these processes in intact organs in vivo. We have previously used multiphoton fluorescence lifetime microscopy to characterise the morphology and amplitude weighted mean fluorescence lifetime of the endogenous fluorescent metabolic cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) of mouse livers in vivo following induction of various disease states. Here, we extend the characterisation of liver disease models by using nonlinear regression to estimate the unbound, bound fluorescence lifetimes for NAD(P)H, flavin adenine dinucleotide (FAD), along with metabolic ratios and examine the impact of using multiple segmentation methods. We found that NAD(P)H amplitude ratio, and fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis and biliary fibrosis. Hepatocellular carcinoma was associated with an increase in spatial heterogeneity and redox ratio coupled with a decrease in mean fluorescence lifetime. We conclude that multiphoton fluorescence lifetime microscopy parameters and metabolic ratios provided insights into the in vivo redox state of diseased compared to normal liver that were not apparent from a global, mean fluorescence lifetime measurement alone.