Application Of Fluorescence Lifetime Imaging Research Articles

Simple and Robust Deep Learning Approach for Fast Fluorescence Lifetime Imaging.

Fluorescence lifetime imaging (FLIM) is a powerful tool that provides unique quantitative information for biomedical research. In this study, we propose a multi-layer-perceptron-based mixer (MLP-Mixer) deep learning (DL) algorithm named FLIM-MLP-Mixer for fast and robust FLIM analysis. The FLIM-MLP-Mixer has a simple network architecture yet a powerful learning ability from data. Compared with the traditional fitting and previously reported DL methods, the FLIM-MLP-Mixer shows superior performance in terms of accuracy and calculation speed, which has been validated using both synthetic and experimental data. All results indicate that our proposed method is well suited for accurately estimating lifetime parameters from measured fluorescence histograms, and it has great potential in various real-time FLIM applications.

Fluorescence lifetime imaging of NAD(P)H upon oxidative stress in Kluyveromyces marxianus.

K. marxianus is a promising cell factory for producing heterologous proteins. Oxidative stresses were raised during overexpression of heterologous proteins, leading to the shift of the redox state. How to measure the redox state of live K. marxianus cells without perturbing their growth remains a big challenge. Here, a fluorescence lifetime imaging (FLIM)-based method was developed in live K. marxianus cells. During the early exponential growth, K. marxianus cells exhibited an increased mean fluorescence lifetime (τ-mean) of NAD(P)H compared with Saccharomyces cerevisiae cells, which was consistent with the preference for respiration in K. marxianus cells and that for fermentation in S. cerevisiae cells. Upon oxidative stresses induced by high temperature or H2O2, K. marxianus cells exhibited an increased τ-mean in company with decreased intracellular NAD(P)H/NAD(P)+, suggesting a correlation between an increased τ-mean and a more oxidized redox state. The relationship between τ-mean and the expression level of a heterologous protein was investigated. There was no difference between the τ-means of K. marxianus strains which were not producing a heterologous protein. The τ-mean of a strain yielding a high level of a heterologous protein was higher than that of a low-yielding strain. The results suggested the potential application of FLIM in the non-invasive screen of high-yielding cells.

Smart Wide-field Fluorescence Lifetime Imaging System with CMOS Single-photon Avalanche Diode Arrays.

Wide-field fluorescence lifetime imaging (FLIM) is a promising technique for biomedical and clinic applications. Integrating with CMOS single-photon avalanche diode (SPAD) sensor arrays can lead to cheaper and portable real-time FLIM systems. However, the FLIM data obtained by such sensor systems often have sophisticated noise features. There is still a lack of fast tools to recover lifetime parameters from highly noise-corrupted fluorescence signals efficiently. This paper proposes a smart wide-field FLIM system containing a 192×128 COMS SPAD sensor and a field-programmable gate array (FPGA) embedded deep learning (DL) FLIM processor. The processor adopts a hardware-friendly and light-weighted neural network for fluorescence lifetime analysis, showing the advantages of high accuracy against noise, fast speed, and low power consumption. Experimental results demonstrate the proposed system's superior and robust performances, promising for many FLIM applications such as FLIM-guided clinical surgeries, cancer diagnosis, and biomedical imaging.

Mesoscopic fluorescence lifetime imaging: Fundamental principles, clinical applications and future directions.

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

One-Dimensional Deep Learning Architecture for Fast Fluorescence Lifetime Imaging

We present a hardware-friendly deep learning architecture with one-dimensional convolutional neural networks (1D CNN) for fast analyzing fluorescence lifetime imaging (FLIM) data. A 1D CNN shows unparalleled advantages; they are more straightforward, quicker to train, and faster than high dimensional CNNs. 1D CNNs can be easily applied to multi-exponential fluorescence decay models. Compared with traditional least-square methods, superior performances of 1D CNNs on fluorescence lifetime image reconstruction have been validated using simulated data. We also employ the proposed 1D CNN to analyze two-photon FLIM images of functionalized gold nanoprobes in Hek293 and human prostate cancer cells. The results further demonstrate that 1D CNNs are fast and can accurately extract lifetime parameters from fluorescence signals. Our study shows that 1D CNNs have great potential in various real-time FLIM applications.

Fast Timing Techniques in FLIM Applications

Fluorescence lifetime imaging (FLIM) is increasingly used in many scientific disciplines, including biological and medical research, materials science and chemistry. The fluorescence label is not only used to indicate its location, but also to probe its immediate environment, via its fluorescence lifetime. This allows FLIM to monitor and image the cellular microenvironment including the interaction between proteins in their natural environment. It does so with high specificity and sensitivity in a nondestructive and minimally invasive manner, providing both structural and functional information. Time-Correlated Single Photon Counting (TCSPC) is a popular, widely used, robust and mature method to perform FLIM measurements. It is a sensitive, accurate and precise method of measuring photon arrival times after an excitation pulse, with the arrival times not affected by photobleaching or excitation or fluorescence intensity fluctuations. It has a very large dynamic range, and only needs a low illumination intensity. Different methods have been developed to advance fast and accurate timing of photon arrival. In this review a brief history of the development of these methods is given, and their merits are discussed in the context of their applications in FLIM.

Interrogation of tumor metabolism in tissue samples ex vivo using fluorescence lifetime imaging of NAD(P)H

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.

Thermally activated delayed fluorescence organic dots for two-photon fluorescence lifetime imaging

Autofluorescence is a major challenge in complex tissue imaging when molecules present in the biological tissue compete with the fluorophore. This issue may be resolved by designing organic molecules with long fluorescence lifetimes. The present work reports the two-photon absorption (TPA) properties of a thermally activated delayed fluorescence (TADF) molecule with carbazole as the electron donor and dicyanobenzene as the electron acceptor (i.e., 4CzIPN). The results indicate that 4CzIPN exhibits a moderate TPA cross-section (∼9 × 10−50 cm4 s photon−1), high fluorescence quantum yield, and a long fluorescence lifetime (∼1.47 μs). 4CzIPN was compactly encapsulated into an amphiphilic copolymer via nanoprecipitation to achieve water-soluble organic dots. Interestingly, 4CzIPN organic dots have been utilized in applications involving two-photon fluorescence lifetime imaging (FLIM). Our work aptly demonstrates that TADF molecules are promising candidates of nonlinear optical probes for developing next-generation multiphoton FLIM applications.

Applications of fluorescence lifetime imaging in clinical medicine

Fluorescence lifetime is not only associated with the molecular structure of fluorophores, but also strongly depends on the environment around them, which allows fluorescence lifetime imaging microscopy (FLIM) to be used as a tool for precise measurement of the cell or tissue microenvironment. This review introduces the basic principle of fluorescence lifetime imaging technology and its application in clinical medicine, including research and diagnosis of diseases in skin, brain, eyes, mouth, bone, blood vessels and cavity organs, and drug evaluation. As a noninvasive, nontoxic and nonionizing radiation technique, FLIM demonstrates excellent performance with high sensitivity and specificity, which allows to determine precise position of the lesion and, thus, has good potential for application in biomedical research and clinical diagnosis.

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Fluorescence lifetime imaging (FLIM) is an effective noninvasive bioanalytical tool based on measuring fluorescent lifetime of fluorophores. A growing number of FLIM studies utilizes genetically engineered fluorescent proteins targeted to specific subcellular structures to probe local molecular environment, which opens new directions in cell science. This paper highlights the unconventional applications of FLIM for studies of molecular processes in diverse organelles of live cultured cells.

Fluorescence lifetime imaging ophthalmoscopy.

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases.Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review.

Application of Fluorescence Lifetime Imaging (FLIM) to Measure Intracellular Environments in a Single Cell.

Fluorescence lifetime imaging (FLIM) has now been used in many bioscience fields, which comes from the quantification of fluorescence lifetime. The procedure for obtaining lifetime images is very similar to that used in fluorescence microscopy. However, obtaining reliable lifetime images requires an understanding of the theory of fluorescence lifetime, principle of FLIM systems, and evaluation procedure of intracellular environments. In this chapter, the materials, methods, and notes on FLIM measurements have been described, in conjunction with a brief explanation of the background of FLIM.

Blind and reference-free fluorescence lifetime estimation via consumer time-of-flight sensors

Fluorescence lifetime imaging (FLI) is a popular method for extracting useful information that is otherwise unavailable from a conventional intensity image. Usually, however, it requires expensive equipment, is often limited to either distinctly frequency- or time-domain modalities, and demands calibration measurements and precise knowledge of the illumination signal. Here, we present a generalized time-based, cost-effective method for estimating lifetimes by repurposing a consumer-grade time-of-flight sensor. By developing mathematical theory that unifies time- and frequency-domain approaches, we can interpret a time-based signal as a combination of multiple frequency measurements. We show that we can estimate lifetimes without knowledge of the illumination signal and without any calibration. We experimentally demonstrate this blind, reference-free method using a quantum dot solution and discuss the method’s implementation in FLI applications.

Time-Resolved Photoluminescence Spectroscopy and Imaging: New Approaches to the Analysis of Cultural Heritage and Its Degradation

Applications of time-resolved photoluminescence spectroscopy (TRPL) and fluorescence lifetime imaging (FLIM) to the analysis of cultural heritage are presented. Examples range from historic wall paintings and stone sculptures to 20th century iconic design objects. A detailed description of the instrumentation developed and employed for analysis in the laboratory or in situ is given. Both instruments rely on a pulsed laser source coupled to a gated detection system, but differ in the type of information they provide. Applications of FLIM to the analysis of model samples and for the in-situ monitoring of works of art range from the analysis of organic materials and pigments in wall paintings, the detection of trace organic substances on stone sculptures, to the mapping of luminescence in late 19th century paintings. TRPL and FLIM are employed as sensors for the detection of the degradation of design objects made in plastic. Applications and avenues for future research are suggested.

Handheld multispectral fluorescence lifetime imaging system for in vivo applications

There is an increasing interest in the application of fluorescence lifetime imaging (FLIM) for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed clinically compatible systems. We present a handheld probe design consisting of a small maneuverable box fitted with a rigid endoscope, capable of continuous lifetime imaging at multiple emission bands simultaneously. The system was characterized using standard fluorescent dyes. The performance was then further demonstrated by imaging a hamster cheek pouch in vivo, and oral mucosa tissue both ex vivo and in vivo, all using safe and permissible exposure levels. Such a design can greatly facilitate the evaluation of FLIM for oral cancer imaging in vivo.

Fluorescence lifetime imaging microscopy in the medical sciences

The steady improvement in the imaging of cellular processes in living tissue over the last 10-15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).

Flexible endoscope for continuous in vivo multispectral fluorescence lifetime imaging

Fluorescence lifetime imaging (FLIM) offers a noninvasive approach for characterizing the biochemical composition of biological tissue. There has been an increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed endoscopy systems. Unfortunately, the predominant multispectral FLIM approaches suffer from limitations that impede the development of endoscopy systems that are suitable for in vivo tissue imaging. We present a compact wide-field time-gated FLIM flexible endoscope capable of continuous lifetime imaging of up to three fluorescence emission bands simultaneously. This endoscope design will facilitate the evaluation of FLIM for in vivo applications.

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Time-resolved nanosecond fluorescence lifetime imaging and picosecond infrared spectroscopy of combretastatin A-4 in solution and in cellular systems**DedicationThis paper is dedicated to the memory of Professor Christopher G Morgan (1949–2011). He was a valued colleague and friend at the University of Salford and made significant contributions to the development and applications of fluorescence lifetime imaging.

Fluorescence lifetime images of intrinsic fluorescence obtained with two-photon excitation at 630 nm are shown following uptake of a series of E-combretastatins into live cells, including human umbilical vein endothelial cells (HUVECs) that are the target for the anticancer activity of combretastatins. Images show distribution of the compounds within the cell cytoplasm and in structures identified as lipid droplets by comparison with images obtained following Nile red staining of the same cells. The intracellular fluorescent lifetimes are generally longer than in fluid solution as a consequence of the high viscosity of the cellular environment. Following incubation, the intracellular concentrations of a fluorinated derivative of E-combretastatin A-4 in HUVECs are between two and three orders of magnitude higher than the concentration in the surrounding medium. Evidence is presented to indicate that at moderate laser powers (up to 6 mW), it is possible to isomerize up to 25% of the combretastatin within the femtolitre focal volume of the femtosecond laser beam. This suggests that it may be possible to activate the E-combretastatin (with low cellular toxicity) to the Z-isomer with high anticancer drug activity using two-photon irradiation. The isomerization of Z- and E-combretastatins by 266 nm irradiation has been probed by ultrafast time-resolved infrared spectroscopy. Results for the E-isomer show a rapid loss of excess vibrational energy in the excited state with a lifetime of 7 ps, followed by a slower process with a lifetime of 500 ps corresponding to the return to the ground state as also determined from the fluorescence lifetime. In contrast, the Z-isomer, whilst also appearing to undergo a rapid cooling of the initial excited state, has a much shorter overall excited state lifetime of 14 ps.

Fluorescence lifetime imaging – techniques and applications

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time-domain FLIM by multidimensional time-correlated single photon counting, time-domain FLIM by gated image intensifiers, frequency-domain FLIM by gain-modulated image intensifiers, and frequency-domain FLIM by gain-modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein-interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity-based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM-FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM-based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM-FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady-state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady-state fluorescence techniques.