Fluorescence Lifetime Imaging Images Research Articles

Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models.

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.

Bayesian Statistical Analysis for Bacterial Detection in Pulmonary Endomicroscopic Fluorescence Lifetime Imaging.

Pneumonia, a respiratory disease often caused by bacterial infection in the distal lung, requires rapid and accurate identification, especially in settings such as critical care. Initiating or de-escalating antimicrobials should ideally be guided by the quantification of pathogenic bacteria for effective treatment. Optical endomicroscopy is an emerging technology with the potential to expedite bacterial detection in the distal lung by enabling in vivo and in situ optical tissue characterisation. With advancements in detector technology, optical endomicroscopy can utilize fluorescence lifetime imaging (FLIM) to help detect events that were previously challenging or impossible to identify using fluorescence intensity imaging. In this paper, we propose an iterative Bayesian approach for bacterial detection in FLIM. We model the FLIM image as a linear combination of background intensity, Gaussian noise, and additive outliers (labelled bacteria). While previous bacteria detection methods model anomalous pixels as bacteria, here the FLIM outliers are modelled as circularly symmetric Gaussian-shaped objects, based on their discrete shape observed through visual analysis and the physical nature of the imaging modality. A Hierarchical Bayesian model is used to solve the bacterial detection problem where prior distributions are assigned to unknown parameters. A Metropolis-Hastings within Gibbs sampler draws samples from the posterior distribution. The proposed method's detection performance is initially measured using synthetic images, and shows significant improvement over existing approaches. Further analysis is conducted on real optical endomicroscopy FLIM images annotated by trained personnel. The experiments show the proposed approach outperforms existing methods by a margin of +16.85% ( F1 ) for detection accuracy.

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM)

The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings.Graphical

Multimodal fluorescence lifetime imaging and optical coherence tomography for longitudinal monitoring of tissue-engineered cartilage maturation in a preclinical implantation model.

Cartilage tissue engineering is a promising strategy for effective curative therapies for treatment of osteoarthritis. However, tissue engineers depend predominantly on time-consuming, expensive, and destructive techniques as quality control to monitor the maturation of engineered cartilage. This practice can be impractical for large-scale biomanufacturing and prevents spatial and temporal monitoring of tissue growth, which is critical for the fabrication of clinically relevant-sized cartilage constructs. Nondestructive multimodal imaging techniques combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) hold great potential to address this challenge. The feasibility of using multimodal FLIm-OCT for nondestructive, spatial, and temporal monitoring of self-assembled cartilage tissue maturation in a preclinical mouse model is investigated. Self-assembled cartilage constructs were developed for 4 weeks in vitro followed by 4 weeks of in vivo maturation in nude mice. Sterile and nondestructive in situ multispectral FLIm and OCT imaging were carried out at multiple time points ( , 4, and 8 weeks) during tissue development. FLIm and 3D volumetric OCT images were reconstructed and used for the analysis of tissue biochemical homogeneity, morphology, and structural integrity. A biochemical homogeneity index was computed to characterize nonhomogeneous tissue growth at different time points. OCT images were validated against histology. FLIm detects heterogenous extracellular matrix (ECM) growth of tissue-engineered cartilage. The outer edge of the tissue construct exhibited longer fluorescence lifetime in 375 to 410 and 450 to 485nm spectral channels, indicating increase in collagen content. Significant ( ) decrease of construct homogeneity index was observed between weeks and weeks. Both FLIm and OCT images revealed defects (voids) at the center of the tissue construct during in vitro culture ( and 4 weeks). Cyst formation during in vivo culture was detected by OCT and confirmed with histology. The ability of multimodal FLIm-OCT to nondestructively monitor the heterogenous growth of engineered tissue constructs in situ is demonstrated. Spatial and temporal variation of construct ECM component was detected by FLIm. OCT reveals structural defects (voids and cysts). This multimodal approach has great potential to replace costly destructive tests in the manufacturing of tissue-engineered medical products, facilitating their clinical translation.

Fluorescence lifetime imaging is able to recognize different hematopoietic precursors in unstained routine bone marrow films.

Fluorescence lifetime imaging (FLIM) has been used in living cells to measure metabolic activity and demonstrate cell differentiation. The aim of this study was to investigate whether the FLIM technique could be able to demonstrate cell maturation during myelopoiesis and erythropoiesis in unlabeled routine bone marrow (BM) preparations. Air-dried, unstained smears of BM aspiration samples of 32 patients without BM disease and a normal morphology on May-Grünwald-Giemsa (MGG) stained smears entered the study. FLIM images were captured with a Zeiss LSM 780 NLO multiphoton microscope equipped with a Becker & Hickl SPC-830 TCSPC FLIM module and HPM-100-40 hybrid detector. The samples were irradiated by two-photon excitation at 800 nm with a titanium-sapphire laser of the LSM 780 NLO. FLIM images were compared with those obtained by autofluorescence high resolution imaging. FLIM images of unstained smears were highly contrasted. Different cell types could be easily recognized as they were similar to those seen in MGG stained preparations. Cytoplasm of cells from the erythroid lineage revealed relatively short fluorescence lifetimes due to the presence of hemoglobin, and therefore could easily be distinguished from granulocytic precursors. Nuclear fluorescence lifetimes of all cell types were higher than those of the corresponding cytoplasm. So, FLIM of unstained BM smears obtained under routine real-life conditions permits an easy identification of BM cells, by highlighting differences of their physicochemical properties.

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.

Investigating Origins of FLIm Contrast in Atherosclerotic Lesions Using Combined FLIm-Raman Spectroscopy.

Background: Fluorescence lifetime imaging (FLIm) is a spectroscopic imaging technique able to characterize the composition of luminal surface of arterial vessels. Studies of human coronary samples demonstrated that distinct atherosclerotic lesion types are characterized by FLIm features associate with distinct tissue molecular makeup. While conventional histology has provided indications about potential sources of molecular contrast, specific information about the origin of FLIm signals is lacking. Here we investigate whether Raman spectroscopy, a technique able to evaluate chemical content of biological samples, can provide additional insight into the origin of FLIm contrast.Methods: Six human coronary artery samples were imaged using FLIm (355 nm excitation)-Raman spectroscopy (785 nm excitation) via a multimodal fiber optic probe. The spatial distribution of molecular contrast in FLIm images was analyzed in relationship with histological findings. Raman data was investigated using an endmember technique and compared with histological findings. A descriptive modeling approach based on multivariate regression was used to identify Raman bands related with changes in lifetime in four spectral channels (violet: 387/35 nm, blue: 443/29 nm, green: 546/38 nm, and red: 628/53 nm).Results: Fluorescence lifetime variations in the violet, blue and green spectral bands were observed for distinct areas of each tissue sample associated with distinct pathologies. Analysis of Raman signals from areas associated with normal, pathological intimal thickening, and fibrocalcific regions demonstrated the presence of hydroxyapatite, collagenous proteins, carotene, cholesterol, and triglycerides. The FLIm and Raman descriptive modeling analysis indicated that lifetime increase in the violet spectral band was associated with increased presence of cholesterol and carotenes, a new finding consistent with LDL accumulation in atherosclerotic lesions, and not with collagen proteins, as expected from earlier studies.Conclusions: The systematic, quantitative analysis of the multimodal FLIm-Raman dataset using a descriptive modeling approach led to the identification of LDL accumulation as the primary source of lifetime contrast in atherosclerotic lesions in the violet spectral range. Earlier FLIm validation studies relying on histopathological findings had associated this contrast to increased collagen content, also present in advanced lesions, thus demonstrating the benefits of alternative validation methods.

Review: Clinical in vivo multiphoton FLIM tomography

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.

MP54-18 QUANTITATIVE AND QUALITATIVE MULTIMODAL OPTICAL ANALYSIS TO DISCRIMINATE UROTHELIAL CARCINOMA GRADES

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2018MP54-18 QUANTITATIVE AND QUALITATIVE MULTIMODAL OPTICAL ANALYSIS TO DISCRIMINATE UROTHELIAL CARCINOMA GRADES Benjamin PRADERE, Fanny Poulon, Steeve Doizi, Olivier Cussenot, Eva Compérat, Darine Abi Haidar, and Olivier Traxer Benjamin PRADEREBenjamin PRADERE More articles by this author , Fanny PoulonFanny Poulon More articles by this author , Steeve DoiziSteeve Doizi More articles by this author , Olivier CussenotOlivier Cussenot More articles by this author , Eva CompératEva Compérat More articles by this author , Darine Abi HaidarDarine Abi Haidar More articles by this author , and Olivier TraxerOlivier Traxer More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1709AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the framework of urologic oncology, mini-invasive procedures have increased the last decades particularly for urothelial carcinoma. One of the essential step in the management of this disease stay the diagnostic, which strongly impacts the treatment choice. The histopathologic evaluation of the grade of the tumor is a keystone of the diagnosis, and its recognition is not possible with a macroscopic evaluation. Nowadays, this specific intraoperative characteristic evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore. In our study, we assessed the use of an optical multimodal technique based on endogenous fluorescence combining qualitative and quantitative analysis for the diagnostic of urothelial carcinoma grades. METHODS Urothelial samples from bladder and upper urinary tract were prospectively included (IRB-00003835) and analyzed on a specific optical multimodal setup based on endogenous fluorescence.The analysis included qualitative analyses with two-photons fluorescence imaging (TPF) and quantitative analyses with spectral analyses and fluorescence lifetime imaging (FLIM). Qualitative analyses were compared with pathological examination. Quantitative analyses was performed with a 870nm excitation wavelength, in the spectral analysis we evaluated the spectra, the redox ratio (NADH and FAD) and the fluorescence lifetime for each sample. RESULTS We identified 3 major urothelial aspects by TPF. All this samples were correlated with pathological examinations, and permitted to differentiate healthy tissue from low and high grade urothelial carcinoma. In the quantitative analysis, the spectral results shown that the intensity level of the emitted spectra was correlated with the histopathological characteristics: the higher the grade was, the lower was the fluorescence signal. The redox ratio analysis was significantly higher in the healthy urothelium compared with tumors samples (p<0.001). Moreover, we were able to discriminate low grade from high grade tumors, the low grade had a significantly higher redox ratio (p=0.002). We were able to identify the cells structure in the FLIM images and to compare the different grade. This analysis also permitted to discriminate the different origins. The average lifetime in low grade tumor appeared shorter on our images and in the histogram shown significant differences between healthy, low grade and high grade urothelial carcinoma: healthy vs. low grade (p = 0.002), healthy vs. high grade (p < 0.001) and high grade vs. low grade (p < 0.001). CONCLUSIONS Those results show that multimodal optical analysis was able to discriminate low grade from high grade urothelial carcinoma without using exogenous fluorophore. This is a promising technology for the development of an optical fiber setup designed for an intraoperative diagnosis of urothelial carcinoma in the area of endourology. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e719 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Benjamin PRADERE More articles by this author Fanny Poulon More articles by this author Steeve Doizi More articles by this author Olivier Cussenot More articles by this author Eva Compérat More articles by this author Darine Abi Haidar More articles by this author Olivier Traxer More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Simultaneous, label-free, multispectral fluorescence lifetime imaging and optical coherence tomography using a double-clad fiber.

We present a novel fiber-based imaging platform that allows simultaneous fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) using a double-clad fiber. This platform acquires co-registered images showing structural and compositional contrast in unlabeled biological samples by scanning the fiber tip across the sample surface. In this Letter, we report a characterization of each modality and show examples of co-registered FLIm and OCT images acquired from a lemon segment and a section of human coronary artery. The close comparison between the combined FLIm and OCT images and a co-registered histology section provides a qualitative validation of the technique and highlights its potential for minimally invasive, multimodal imaging of tissue structure and composition.

445 - Estimation of Mitochondrial NADH/NAD+ Ratio from FLIM

Mitochondrial matrix NADH/NAD+ ratio determines superoxide formation at the proximity of Complex I flavin site. To estimate this ratio within intact cells, we employed confocal fluorescence lifetime imaging (FLIM) with 140 fs pulse width, Leica TSC-SP2 confocal microscope plus a Becker & Hickl attachment for two-channel FLIM. Imaging of HepG2 and INS-1E cells, within mito-chondrial (mt) region of interests yielded parameters of NAD(P)H autofluorescence decay at 467–499 nm and FAD decay at 500–550 nm, while two-photon excitation was at 700 nm. At each 4x4 pixels, the iterative reconvolution yielded two lifetimes for free and bound molecules τF, τB, of 0.4–0.5 ns, ~2.6 ns and their normalized amplitudes αF and αB. Reconstructed FLIM images yielded e.g. different responses to rotenone in remaining unfragmented tubules of mt network and co-existing small fragments. Average parameters for each location were analyzed and ratio of bound NADPH/NADH was estimated using empirical term according to Duchen and colleagues [1]. Free NADH was approximated from Scatchard equation. Free NAD+ was derived from its Stern-Volmer quenching of FAD (longer FAD lifetime of free molecules). Approximating association constants, we estimated absolute NADH/NAD+ ratios being higher in HepG2 cells forced to oxidative phosphorylation. Without constants, we got exact changes in NADH/NAD+ between two conditions (e.g. intact vs. rotenone). Support: GACR grant No. 16-04788S Reference [1] T.S. Blacker, Z.F. Mann, J.E. Gale, M. Ziegler, A.J. Bain, G. Szabadkai, M.R. Duchen, Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat. Commun. 5 (2014) 3936.

Characterizing Fibrosis in Mouse Kidney Using Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model

Renal tubulointerstitial fibrosis is considered to be the final common pathway for most forms of chronic kidney disease (CKD). Fibrosis has been traditionally characterized by histological studies such as Picro‐Sirius Red (PSR) staining, Masson Trichrome staining or collagen immunohistochemistry. Such methods are limited due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to unilateral ureteral obstruction (UUO), a well‐characterized model of fibrosis. At 21 days, both kidneys were collected. Serial sections of both kidneys were analyzed by PSR staining or FLIM with SHG. Quantification of a whole kidney section from the PSR staining showed 34.32±0.99% area of fibrosis in the injured kidney compared to 5.55±1.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.44±4.21% compared to 2.59±1.98% control kidney). In conclusion, the combined FLIM and SHG images let us establish a criterion for quantitative determination of fibrosis directly from the microscope images.

Characterizing Fibrosis in Mouse Kidney using Auto Fluorescence Flim and SHG in UUO Model

2410-Pos Board B547 Characterizing Fibrosis in Mouse Kidney using Auto Fluorescence Flim and SHG in UUO Model Moshe Levi 1 , Evgenia Dobrinskikh 1 , Suman Ranjit 2 , John Montford 1 , Alexander Dvornikov 3 , Allison Lehman 1 , Seth Furgeson 1 , Raphael Nemenoff 1 , Enrico Gratton 2 . Medicine, U of Colorado, Aurora, CO, USA, 2 Biomedical Engineering, U of California, Irvine, CA, USA, 3 Biomedical Engineering, U of California, Aurora, CA, USA. Chronic kidney disease (CKD) arises from a diverse list of renal injuries. Renal fibrosis is considered to be the final common pathway for most forms of CKD and involves glomerular sclerosis and/or interstitial fibrosis. Because of that there is great interest in identifying renal fibrosis in the early stages of CKD to prevent progression. Unilateral ureteral obstruction (UUO) is a well- described model of CKD and renal fibrosis. Kidney injury and fibrosis usually are assessed by histology. Histological methods include Picro-Sirius Red stain- ing, Masson’s Trichrome staining or immunohistochemistry for collagen iso- forms. Quantitation of fibrosis using histologic techniques may be difficult due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to UUO of right kidney. At 21 days, both kidneys were collected, fixed, and paraffin-embedded. The uninjured left kidney was used as a control. Serial sections of both kidneys were analyzed by Picro-Sirius Red staining or FLIM with SHG. Quantification of a whole kidney section from the Picro- Sirius Red staining showed 34.3250.99% area of fibrosis in the left kidney compare to 5.5551.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.4454.21% compared to 2.5951.98% control kidney). Finally the combined FLIM and SHG images let us create a criterion to separate fibrosis directly from the microscope images.

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

Repeatability of Autofluorescence Lifetime Imaging at the Human Fundus in Healthy Volunteers

Purpose: We aim to evaluate the repeatability of a new fluorescence lifetime imaging (FLIM) technique which measures time-resolved autofluorescence to assess metabolism of the retina.Materials and methods: We performed FLIM with two spectral channels (channel 1: 490–560 nm and channel 2: 560–700 nm) on 10 healthy volunteers, with 10 replicates per volunteer. From the 30° fundus FLIM images, we selected three regions: the fovea, the optic disc and the papillo-macular bundle. For each channel in these regions, we determined an average multi-exponential approximation with three components, and the six resulting parameters, α1–α3 (amplitudes) and τ1–τ3 (fluorescence lifetimes), were analyzed in terms of the coefficient of variation (CV).Results: Repeatability was highest in the papillo-macular bundle, followed by the fovea and the optic disc. Repeatability was higher in channel 1 (mean CV of 7.9%) than in channel 2 (mean CV of 17.7%). The average CV for the diagnostically most relevant channel 1 and the most relevant parameters was as follows: τ1 (5.5%) and τ2 (4.7%) in the papillo-macular bundle, and τ1 (6.8%) and τ2 (6.9%) in the fovea.Conclusions: We demonstrated repeatability of FLIM measurement results within acceptable ranges of variation. Based on the detailed coefficients of variation, we derived recommendations for parameter ranges suitable for diagnostic applications.

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays.

Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments, we found the depth of the polymer matrix to be 1-2 μm. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2 μm. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform.

Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: morphologic features for non‐invasive diagnostics

Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus 'other lesions' was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of 'other lesion' extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.

A fluorescence lifetime imaging classification method to investigate the collagen to lipid ratio in fibrous caps of atherosclerotic plaque

This study describes a novel fluorescence lifetime imaging (FLIM) classification method to determine the ratio of collagen to lipid content in the fibrous cap of atherosclerotic plaques. Additionally, an analytical process to assess risk of plaque rupture based on this ratio is proposed. Collagen to lipid ratio has been shown to be an important parameter to evaluate structural integrity of the fibrous cap. FLIM and other time-resolved fluorescence techniques have recently been applied to the study of atherosclerosis based on the ability to assess biochemical composition. Autofluorescence of specimens retrieved during carotid endarterectomy procedures was measured through three optical filters, F377: 377/50 nm, F460: 460/66 nm, and F510: 510/84 nm (center wavelength/bandwidth). A Laguerre deconvolution technique was used for the evaluation of fluorescence decay dynamics. The resulting decay parameters (average fluorescence lifetime and 4 Laguerre coefficients at each of the recorded bandwidths) were used for sample characterization. Linear discriminant analysis (LDA) was used to classify each image into collagen or lipid-rich regions based on these parameters. Ultimately, a risk-level was assigned based on the ratio of collagen to lipid on the surface of the fibrous cap. FLIM images were acquired in 18 carotid plaque specimens at 43 locations. Classification of collagen and lipid-rich regions within the fibrous cap was performed with sensitivity and specificity of 80% and 82%, respectively. Results from this study show that an LDA method of classifying regions of FLIM images of carotid plaque into collagen and lipid-rich regions is capable of being automated and used to rate the risk of plaque rupture based on autofluorescence decay dynamics and without the need for fluorescence intensity or contrast agents.

Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatially distinct NADH binding

Two-photon fluorescence lifetime imaging (FLIM) of molecules can reveal important information on the local microenvironment. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic co-enzyme, has a lifetime that depends strongly on enzymatic binding. We present a custom image-processing algorithm for raw fluorescence lifetime and amplitude data that produces an image showing spatially distinct NADH fluorescence lifetimes in slices of rat and human brain. NADH FLIM images were collected in control and epileptic rat tissue. Differences in spatial patterns of NADH lifetimes support the hypothesis that NADH binding, and thus metabolic capacity, is significantly different between groups. This type of analysis can provide information on metabolic states in pathological material.

Imaging of cancer cells by multiphoton microscopy using gold nanoparticles and fluorescent dyes

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.