Fluorescence Lifetime Maps Research Articles

Building Fluorescence Lifetime Maps Photon-by-Photon by Leveraging Spatial Correlations.

Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of fluorophore species contributing to the signal is unknown, while excited state lifetimes with <1 ns difference are difficult to discriminate. Finally, existing analyses require high photon budgets and often cannot rigorously propagate experimental uncertainty into values over lifetime maps and number of species involved. To overcome all of these challenges simultaneously and self-consistently at once, we propose the first doubly nonparametric framework. That is, we learn the number of species (using Beta-Bernoulli process priors) and absolute maps of these fluorophore species (using Gaussian process priors) by leveraging information from pulses not leading to observed photon. We benchmark our framework using a broad range of synthetic and experimental data and demonstrate its robustness across a number of scenarios including cases where we recover lifetime differences between species as small as 0.3 ns with merely 1000 photons.

High Resolution Fluorescence Lifetime Maps from Minimal Photon Counts.

Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report direct blind unmixing of lifetimes with subnanosecond resolution and subpixel spatial resolution using standard raster scan FLIM images. We demonstrate our method using a wide range of simulated and experimental data.

Fluorescence Lifetime Imaging and Spectroscopic Co-Validation for Protoporphyrin IX-Guided Tumor Visualization in Neurosurgery

Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

Dual phase two-color emission observed in van der Waals GaTe planes

After the eagerly study on two-dimensional (2D) electronic devices made by layered semiconductors, light-emitting devices based on 2D semiconductors have also flourished in recent years due to their special characters of flexibility, flatness surface, and thickness-tunable band gap modulation, etc. Among the 2D materials, III-VI layer compounds with hexagonal structure like GaSe and InSe are essentially direct semiconductors that own high band-edge luminescence efficiency and wide thickness tunable range from bulk to thin few-layer scale. In this work, dual-phase two-color emissions of multilayered gallium monotelluride positioned at 1.588 and 1.652 eV are simultaneously detected by micro-photoluminescence (μPL) measurement at 300 K. The lower-energy peak may originate from the hexagonal GaTe (H-GaTe) phase while the higher-energy luminescence might come from monoclinic GaTe (M-GaTe) phase and which are verified by micro-thermoreflectance (μTR) and high-resolution transmission electron microscopy (HRTEM). Micro-time-resolved photoluminescence (TRPL) and area fluorescence lifetime mapping (AFLM) of the multilayer GaTe indicated that the luminescence decay time constant of the H-GaTe is larger than that of the M-GaTe for verification of the dominant phase in multilayered GaTe is the monoclinic phase. In-plane structural and optical anisotropy of the multilayered GaTe were characterized by using polarized micro-Raman and polarized μPL measurements. The results clearly indicate that the monoclinic-phase GaTe possesses higher polarized extinction ratio with respect to that of the hexagonal GaTe for fully extinction of the Raman vibration mode and band-edge emission in the dual-phase gallium telluride. The existence of dual crystalline phases in multilayer GaTe may render it possible for fabrication of two-color emission device available for near-infrared (NIR) optical communication use.

Real-time multispectral fluorescence lifetime imaging using Single Photon Avalanche Diode arrays

Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is passed through a transmission grating and both spectral and temporal information are encoded in the SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial information is provided by an external camera together with a guiding beam that provides a visual reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence lifetime maps are augmented on white light images to provide feedback of the measurements in real-time. We validate and demonstrate the practicality of this technique in the reference fluorophores and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.

Simultaneous fluorescence lifetime and Raman fiber-based mapping of tissues.

We report the development of a novel, to the best of our knowledge, fiber-based system to realize coregistered simultaneous acquisition of fluorescence lifetime (FL) data and Raman spectra from the same area. FL measurements by means of time-correlated single photon counting are realized with periodic out-of-phase external illumination of the field of view, enabling acquisition of data under bright illumination of the specimen. Raman measurements in the near-infrared are realized asynchronously. We present a detailed characterization of this technique and validate its potential to report intrinsic contrast. Fiber-based FL and Raman maps report complementary structural, compositional, and molecular contrast in biological tissues with diverse compositional features.

Real-time fiber-based fluorescence lifetime imaging with synchronous external illumination: A new path for clinical translation.

Time-correlated single photon counting is the "gold-standard" method for fluorescence lifetime measurements and has demonstrated potential for clinical deployment. However, the translation of the technology into clinic is hindered by the use of ultrasensitive detectors, which make the fluorescence acquisition impractical with bright lighting conditions such as in clinical settings. We address this limitation by interleaving periodic fluorescence detection with synchronous out-of-phase externally modulated light source, thus guaranteeing specimen illumination and a fluorescence signal free from bright background light upon temporal separation. Fluorescence lifetime maps are generated in real-time from single-point measurements by tracking a reference beam and using the phasor approach. We demonstrate the feasibility and practicality of this technique in a number of biological specimens, including real-time mapping of degraded articular cartilage. This method is compatible and can be integrated with existing clinical microscopic, endoscopic and robotic modalities, thus offering a new pathway towards label-free diagnostics and surgical guidance in a number of clinical applications.

Fluorescence Lifetime Mapping of NADH Reveals DNA Repair Activity in Live Cells

Reduced nicotinamide adenine dinucleotide (NADH) is an important coenzyme that catalyzes many cellular functions including energy production and DNA damage repair. The ratio of free and bound NADH has recently emerged as a regulator of transcriptional activity and age-associated diseases. The DNA damage response, which requires NADH to gain full functionality, senses and repairs any breaks and errors in the genome. Uncovering the role of this ubiquitous molecule in response to DNA damage will further our understanding of how DNA protects itself and provide insight on how cancer cells evade these processes. In laser-induced damage experiments, HeLa cells were damaged with a femto-second pulsed two-photon laser at a wavelength of 780nm. Fluorescence lifetime imaging microscopy (FLIM) data was acquired and processed using the FLIM-phasor method. Induction of DNA damage and initiation of DNA repair were confirmed via immunostaining for γH2AX and PAR. The lifetime of NADH species in each pixel of FLIM images were transformed into polar coordinates on the phasor plot. The relative fraction of bound NADH was then calculated from the phasor plot and plotted over time. Low laser power damage resulted in a transient change toward a high fraction of bound NADH that was restored in approximately 8 hours. High laser power damage resulted in an immediate (∼5 minutes) and persistent (after 12 hours) shift toward a high fraction of bound NADH. The data show that the extent of DNA damage correlates to increases in the fraction of bound NADH in the nucleus owing to increased binding to DNA repair-associated substrates. Thus, monitoring NADH activity can be an intrinsic biosensor for DNA repair activity. Future studies will explore the role of NADH in chromatin remodeling to facilitate transcriptional regulation of DNA repair.

Early-photon guided reconstruction method for time-domain fluorescence lifetime tomography

A reconstruction method guided by early-photon fluorescence yield tomography is proposed for time-domain fluorescence lifetime tomography (FLT) in this study. The method employs the early-arriving photons to reconstruct a fluorescence yield map, which is utilized as a priori information to reconstruct the FLT via all the photons along the temporal-point spread functions. Phantom experiments demonstrate that, compared with the method using all the photons for reconstruction of fluorescence yield and lifetime maps, the proposed method can achieve higher spatial resolution and reduced crosstalk between different targets without sacrificing the quantification accuracy of lifetime and contrast between heterogeneous targets.

Quantitative Analysis of Fluorescence Lifetime Measurements of the Macula Using the Fluorescence Lifetime Imaging Ophthalmoscope in Healthy Subjects

Fundus autofluorescence (FAF) cannot only be characterized by the intensity or the emission spectrum, but also by its lifetime. As the lifetime of a fluorescent molecule is sensitive to its local microenvironment, this technique may provide more information than fundus autofluorescence imaging. We report here the characteristics and repeatability of FAF lifetime measurements of the human macula using a new fluorescence lifetime imaging ophthalmoscope (FLIO). A total of 31 healthy phakic subjects were included in this study with an age range from 22 to 61 years. For image acquisition, a fluorescence lifetime ophthalmoscope based on a Heidelberg Engineering Spectralis system was used. Fluorescence lifetime maps of the retina were recorded in a short- (498-560 nm) and a long- (560-720 nm) spectral channel. For quantification of fluorescence lifetimes a standard ETDRS grid was used. Mean fluorescence lifetimes were shortest in the fovea, with 208 picoseconds for the short-spectral channel and 239 picoseconds for the long-spectral channel, respectively. Fluorescence lifetimes increased from the central area to the outer ring of the ETDRS grid. The test-retest reliability of FLIO was very high for all ETDRS areas (Spearman's ρ = 0.80 for the short- and 0.97 for the long-spectral channel, P < 0.0001). Fluorescence lifetimes increased with age. The FLIO allows reproducible measurements of fluorescence lifetimes of the macula in healthy subjects. By using a custom-built software, we were able to quantify fluorescence lifetimes within the ETDRS grid. Establishing a clinically accessible standard against which to measure FAF lifetimes within the retina is a prerequisite for future studies in retinal disease.

Electrogenerated Chemiluminescence and Fluorescence Lifetime Spatial Heterogeneity of Poly (2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) in Presence of [6,6]-phenyl-C61-butyric Acid Methyl Ester

ABSTRACTWe present electrogenerated chemiluminescence (ECL) and fluorescence lifetime mapping of MEH-PPV/PCBM thin films. The ECL results show that the oxidation peak of MEH-PPV near 0.7 V (vs. SCE) and ECL response of films shifted positively towards 1.2 V in the presence of PCBM. At the same time, the oxidation current density of MEH-PPV increases along with the decrease of ECL intensity in the presence of PCBM. The fluorescence lifetime images clearly show that the lifetime spatial heterogeneities are affected by different substrates and MEH-PPV/PCBM ratios. Meanwhile, the lifetime of MEH-PPV decreases with the increasing of film thickness. The lifetimes of MEH-PPV films on TiO2 substrate are lower than films on glass.

The Influence of Silver Nanostructures Formed in situ in Silica Sol–Gel Derived Films on the Rate of Förster Resonance Energy Transfer

The efficiency of Förster resonance energy transfer (FRET) can be enhanced in the presence of a metal. Herein, we demonstrate the increased efficiency for a novel model sensor system where FRET is shown to occur between Rhodamine 6G in the bulk sol-gel matrix and Texas Red, which is held a fixed distance away by covalent attachment onto a silane spacer. Silver colloids are formed using light to initiate the reduction of a silver salt, which can be achieved at controlled locations within the film. Both the fluorescence intensity and lifetime maps and analysis indicate that an enhanced FRET efficiency has been achieved in the presence of silver nanoparticles. An increase in efficiency of 1.2-1.5 times is demonstrated depending on the spacer used. The novelty of our approach lies in the method of silver-nanoparticle formation, which allows for the accurate positioning of the silver nanoparticles and hence selective fluorescence enhancement within a biocompatible host material. Our work gives a practical demonstration of metal-enhanced FRET and demonstrates the ability of such systems to be developed for molecular-recognition applications that could find use in lab-on-a-chip technologies.

A photo-multiplier tube-based hybrid MRI and frequency domain fluorescence tomography system for small animal imaging

Fluorescence tomography (FT) is a promising molecular imaging technique that can spatially resolve both fluorophore concentration and lifetime parameters. However, recovered fluorophore parameters highly depend on the size and depth of the object due to the ill-posedness of the FT inverse problem. Structural a priori information from another high spatial resolution imaging modality has been demonstrated to significantly improve FT reconstruction accuracy. In this study, we have constructed a combined magnetic resonance imaging (MRI) and FT system for small animal imaging. A photo-multiplier tube is used as the detector to acquire frequency domain FT measurements. This is the first MR-compatible time-resolved FT system that can reconstruct both fluorescence concentration and lifetime maps simultaneously. The performance of the hybrid system is evaluated with phantom studies. Two different fluorophores, indocyanine green and 3-3′ diethylthiatricarbocyanine iodide, which have similar excitation and emission spectra but different lifetimes, are utilized. The fluorescence concentration and lifetime maps are both reconstructed with and without the structural a priori information obtained from MRI for comparison. We show that the hybrid system can accurately recover both fluorescence intensity and lifetime within 10% error for two 4.2 mm-diameter cylindrical objects embedded in a 38 mm-diameter cylindrical phantom when MRI structural a priori information is utilized.

Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells

Traditional cuvette-based enzyme studies lack spatial information and do not allow real-time monitoring of the effects of modulating enzyme functions in vivo. In order to probe the realistic timescales of steric modifications in enzyme-substrate complexes and functional binding-unbinding kinetics in living cells without losing spatial information, it is imperative to develop sensitive imaging strategies that can report enzyme kinetics in real time over a wide dynamic range of timescales. Here we present a multi-photon excitation-based, ultra-fast photon detection using a streak camera and Laguerre expansion-based fast deconvolution approach for achieving high spatio-temporal resolution in monitoring real-time enzyme kinetics in single cells. In particular, we report spatially resolved, nanosecond-scale fluorescence dynamics associated with binding-unbinding kinetics of endogenous metabolic co-factor nicotinamide adenine dinucleotide with enzymes in intact living cells. By monitoring real-time kinetics of NAD(P)H-enzyme kinetics in primary hepatocytes isolated from young and aged mouse models, we observed that the mechanism of inhibition of mitochondrial respiration at complex I site is mediated by redistribution of free and protein-bound nicotinamide adenine dinucleotide pools and that this equilibrium redistribution is affected by age-related modifications in mitochondrial function. We describe unique advantages of Laguerre deconvolution algorithm in comparison with conventional lifetime analysis approaches. Non-invasive monitoring of metabolic dysfunctions in intact animal models is an attractive strategy for gaining insight into the dynamics of tissue metabolism in health and in various metabolic syndromes such as cancer, diabetes and aging-induced metabolic dysfunctions. Besides the example demonstrated above, we envisage that the proposed method can find applications in a variety of other situations where intensity-based approaches fall short owing to spectroscopic artefacts.

In Vivo Resolution of Multiexponential Decays of Multiple Near-Infrared Molecular Probes by Fluorescence Lifetime-Gated Whole-Body Time-Resolved Diffuse Optical Imaging

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.

Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo1

Multiphoton excitation fluorescent microscopy is a laser-based technology that allows subcellular resolution of native tissues in situ. We have recently applied this technology to the structural and photochemical imaging of cultured glioma cells and experimental gliomas ex vivo. We demonstrated that high microanatomical definition of the tumor, invasion zone, and normal adjacent brain can be obtained down to single-cell resolution in unprocessed tissue blocks. In this study, we used multiphoton excitation and four-dimensional microscopy to generate fluorescence lifetime maps of the murine brain anatomy, experimental glioma tissue, and biopsy specimens of human glial tumors. In murine brain, cellular and noncellular elements of the normal anatomy were identified. Distinct excitation profiles and lifetimes of endogenous fluorophores were identified for specific brain regions. Intracranial grafts of human glioma cell lines in mouse brain were used to study the excitation profiles and fluorescence lifetimes of tumor cells and adjacent host brain. These studies demonstrated that normal brain and tumor could be distinguished on the basis of fluorescence intensity and fluorescence lifetime profiles. Human brain specimens and brain tumor biopsies were also analyzed by multiphoton microscopy, which demonstrated distinct excitation and lifetime profiles in glioma specimens and tumor-adjacent brain. This study demonstrates that multiphoton excitation of autofluorescence can distinguish tumor tissue and normal brain based on the intensity and lifetime of fluorescence. Further technical developments in this technology may provide a means for in situ tissue analysis, which might be used to detect residual tumor at the resection edge.

Fluorescence lifetime imaging and spectroscopy as tools for nondestructive analysis of works of art.

A system for advanced fluorescence investigation of works of art has been assembled and integrated in a characterization procedure that allows one to localize and identify organic compounds that are present in artworks. At the beginning of the investigation, fluorescence lifetime imaging and spectroscopy address a selective microsampling of the artwork. Then analytical measurements of microsamples identify the chemical composition of the materials under investigation. Finally, on the basis of fluorescence lifetime and amplitude maps, analytical data are extended to the whole artwork. In such a way, information on the spatial distribution of organic materials can be inferred. These concepts have been successfully applied in an extensive campaign for analysis of Renaissance fresco paintings in Castiglione Olona, Italy. Residue of various types of glue and stucco left from a restoration carried out in the early 1970s was localized and classified. Insight into the technique used by the painter to make gilded reliefs was also obtained.

A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning

This article describes a wide-field time-domain fluorescence lifetime imaging (FLIM) microscope with optical sectioning. The FLIM system utilizes a wide-field time-gated optical image intensifier, with a minimum gate width of 85 ps, to achieve high temporal resolution of fluorescence decays induced by ultrashort laser pulses. Different configurations, using excitation pulses of picojoule energy at 80 MHz repetition rate and of nanojoule energy at 10 kHz, are compared. The instrument has a temporal dynamic range spanning from 100 ps to tens of μs and is shown to have a temporal discrimination better than 10 ps. When applied to laser dye samples, it has produced FLIM maps demonstrating sensitivity to variations in both chemical species and local environment, e.g., viscosity. Wide-field optical sectioning is achieved using the technique of structured illumination, which is applied to remove out-of-focus light that can result in lifetime artifacts. The sectioning strength, which may be adjusted by choosing an appropriate spatial modulation frequency, is characterized and shown to be comparable to that of a confocal microscope. Practical considerations concerned with improving the quality of sectioned fluorescence lifetime maps, including using a large bit depth camera, are discussed.

Truncated Newton's optimization scheme for absorption and fluorescence optical tomography: Part II Reconstruction from synthetic measurements

Using two dimensional synthetic frequency-domain measurements, the inverse imaging problem is solved for absorption and fluorescence lifetime mapping with the truncated Newtons optimization scheme developed in Part I of this contribution. Herein, we present reconstructed maps of absorption owing to a fluorophore from excitation and emission measurements which detail the presence of tissue heterogeneities characterized by tenfold increase in fluorescent contrast agent. Our results confirm that fluorescence provides superior mapping of heterogeneities over excitation measurements. Using emission measurements we then map fluorescent lifetime under conditions of tenfold uptake of contrast agent in tissue heterogeneities. The ability to map fluorescent quenching and lengthening of contrast agents facilitates the solution of the inverse problem and further improves the ability to reconstruct tissue heterogeneities.