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Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Reduced nicotinamide adenine dinucleotide (NADH) is the principal electron donor in glycolysis and oxidative metabolism and is thus recognized as a key biomarker for probing metabolic state. While the fluorescence characteristics of NADH have been investigated extensively, there are discrepancies in the published data due to diverse experimental conditions, instrumentation and microenvironmental parameters that can affect NADH fluorescence. Using a cuvette-based time-resolved spectrofluorimeter employing one-photon excitation at 375 nm, we characterized the fluorescence intensity, lifetime, spectral response, anisotropy and time-resolved anisotropy of NADH in aqueous solution under varying microenvironmental conditions, namely temperature, pH, and binding to lactate dehydrogenase (LDH). Our results demonstrate how temperature, pH, and binding partners each impact the fluorescence signature of NADH and highlight the complexity of the fluorescence data when different parameters produce competing effects. We hope that the data presented in this study will provide a reference for potential sources of variation in experiments measuring NADH fluorescence.

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 mu g/mL bacterial collagenase at 37 degrees C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm(-1). Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

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.

The inability to efficiently separate autofluorescence signals from bright background illumination is a key issue that hinders widespread deployment of optical techniques such as time-correlated single photon counting (TCSPC) in clinical settings. We introduce a novel approach to TCSPC measurements that synchronizes the fluorescence acquisition with an externally modulated light source at 50 Hz, thus guaranteeing background-free fluorescence detection and “continuous” eye-perceived specimen illumination. A color camera synchronized with fluorescence detection provides spatial resolution to the single-point measurements, to generate autofluorescence lifetime maps in real-time. We believe this method provides a new research pathway for clinical translation of optical techniques.

Tumor-free surgical margins are critical in breast-conserving surgery. In up to 38% of the cases, however, patients undergo a second surgery since malignant cells are found at the margins of the excised resection specimen. Thus, advanced imaging tools are needed to ensure clear margins at the time of surgery. The objective of this study was to evaluate a random forest classifier that makes use of parameters derived from point-scanning label-free fluorescence lifetime imaging (FLIm) measurements of breast specimens as a means to diagnose tumor at the resection margins and to enable an intuitive visualization of a probabilistic classifier on tissue specimen. FLIm data from fresh lumpectomy and mastectomy specimens from 18 patients were used in this study. The supervised training was based on a previously developed registration technique between autofluorescence imaging data and cross-sectional histology slides. A pathologist’s histology annotations provide the ground truth to distinguish between adipose, fibrous, and tumor tissue. Current results demonstrate the ability of this approach to classify the tumor with 89% sensitivity and 93% specificity and to rapidly (∼ 20 frames per second) overlay the probabilistic classifier overlaid on excised breast specimens using an intuitive color scheme. Furthermore, we show an iterative imaging refinement that allows surgeons to switch between rapid scans with a customized, low spatial resolution to quickly cover the specimen and slower scans with enhanced resolution (400 μm per point measurement) in suspicious regions where more details are required. In summary, this technique provides high diagnostic prediction accuracy, rapid acquisition, adaptive resolution, nondestructive probing, and facile interpretation of images, thus holding potential for clinical breast imaging based on label-free FLIm.

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.

Tumor removal typically involves electrocautery, but no studies to date have quantified the effect of electrocautery on fluorescence emission. Electrocautery was applied to N=4 locations of the oral cavity and striated leg muscle of a live Yorkshire pig. Autofluorescence of cauterized tissues and surrounding regions was measured at distinct time points up to 120 minutes following cauterization. The fluorescence lifetime was spectrally resolved in four spectral detection channels that maximized the signal emanating from endogenous fluorophores of interest. The autofluorescence emission (355 nm excitation) was temporally resolved using a high-speed digitizer; resulting fluorescence decay characteristics were retrieved using the Laguerre deconvolution technique. Histology was performed and co-registered with the autofluorescence data. Results show that cauterized tissue presents a distinct autofluorescence signature from surrounding regions immediately after cauterization. Differences become less evident with time. The autofluorescence-derived parameters suggest altered metabolism in peripheral regions compared to the region of maximal damage. Within the time-frame of this study, tissues investigated show variable degrees of recovery from the effects of electrocautery that can be monitored by changes in fluorescence lifetime characteristics. Our findings suggest delineation of pathologic conditions could be affected by tissue cauterization and that future studies in this area will be necessary.

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development. Electronic supplementary material The online version of this article (doi:10.1007/s10895-017-2101-7) contains supplementary material, which is available to authorized users.

Osteoarthritis (OA) is the most common arthritis and its hallmark is degradation of articular cartilage by proteolytic enzymes leading to loss of joint function. It is challenging to monitor the status of cartilage in vivo and this study explores the use of autofluorescence lifetime (AFL) measurements to provide a label-free optical readout of cartilage degradation that could enable earlier detection and evaluation of potential therapies. We previously reported that treatment of ex vivo porcine cartilage with proteolytic enzymes resulted in decreased AFL. Here we report changes in AFL of ex vivo mouse knee joints, porcine metacarpophalangeal joints, normal human metatarsophalangeal articular tissue and human OA tibial plateau tissues measured with or without treatment using a compact single-point time resolved spectrofluorometer. Our data show that proteolytically damaged areas in porcine metacarpophalangeal joints present a reduced AFL and that inducing aggrecanases in mouse and human joints also significantly reduces AFL. Further, human cartilage from OA patients presents a significantly lower AFL compared to normal human cartilage. Our data suggest that AFL can detect areas of cartilage erosion and may potentially be utilised as a minimally-invasive diagnostic readout for early stage OA in combination with arthroscopy devices.

Autofluorescence spectroscopy is a promising label-free approach to characterize biological samples with demonstrated potential to report structural and biochemical alterations in tissues in a number of clinical applications. We report a characterization of the ex vivo autofluorescence fingerprint of cardiac tissue, exploiting a Langendorff-perfused isolated rat heart model to induce physiological insults to the heart, with a view to understanding how metabolic alterations affect the autofluorescence signals. Changes in the autofluorescence intensity and lifetime signatures associated with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized during oxygen- or glucose-depletion protocols. Results suggest that both NAD(P)H and FAD autofluorescence intensity and lifetime parameters are sensitive to changes in the metabolic state of the heart owing to oxygen deprivation. We also observed changes in NAD(P)H fluorescence intensity and FAD lifetime parameter on reperfusion of oxygen, which might provide information on reperfusion injury, and permanent tissue damage or changes to the tissue during recovery from oxygen deprivation. We found that changes in the autofluorescence signature following glucose-depletion are, in general, less pronounced, and most clearly visible in NAD(P)H related parameters. Overall, the results reported in this investigation can serve as baseline for future investigations of cardiac tissue involving autofluorescence measurements.

A correction is proposed to the Delta function convolution method (DFCM) for fitting a multiexponential decay model to time-resolved fluorescence decay data using a monoexponential reference fluorophore. A theoretical analysis of the discretised DFCM multiexponential decay function shows the presence an extra exponential decay term with the same lifetime as the reference fluorophore that we denote as the residual reference component. This extra decay component arises as a result of the discretised convolution of one of the two terms in the modified model function required by the DFCM. The effect of the residual reference component becomes more pronounced when the fluorescence lifetime of the reference is longer than all of the individual components of the specimen under inspection and when the temporal sampling interval is not negligible compared to the quantity (τR (-1) - τ(-1))(-1), where τR and τ are the fluorescence lifetimes of the reference and the specimen respectively. It is shown that the unwanted residual reference component results in systematic errors when fitting simulated data and that these errors are not present when the proposed correction is applied. The correction is also verified using real data obtained from experiment.

Multi-Spectral Time-Resolved Fluorescence Spectroscopy (ms-TRFS) can provide label-free real-time feedback on tissue composition and pathology during surgical procedures by resolving the fluorescence decay dynamics of the tissue. Recently, an ms-TRFS system has been developed in our group, allowing for either point-spectroscopy fluorescence lifetime measurements or dynamic raster tissue scanning by merging a 450 nm aiming beam with the pulsed fluorescence excitation light in a single fiber collection. In order to facilitate an augmented real-time display of fluorescence decay parameters, the lifetime values are back projected to the white light video. The goal of this study is to develop a 3D real-time surface reconstruction aiming for a comprehensive visualization of the decay parameters and providing an enhanced navigation for the surgeon. Using a stereo camera setup, we use a combination of image feature matching and aiming beam stereo segmentation to establish a 3D surface model of the decay parameters. After camera calibration, texture-related features are extracted for both camera images and matched providing a rough estimation of the surface. During the raster scanning, the rough estimation is successively refined in real-time by tracking the aiming beam positions using an advanced segmentation algorithm. The method is evaluated for excised breast tissue specimens showing a high precision and running in real-time with approximately 20 frames per second. The proposed method shows promising potential for intraoperative navigation, i.e. tumor margin assessment. Furthermore, it provides the basis for registering the fluorescence lifetime maps to the tissue surface adapting it to possible tissue deformations.

Fluorescence Lifetime Imaging (FLIm) was used to study the zonal and depth-dependent changes in the structure, and biochemistry of bovine articular cartilage to evaluate its potential for nondestructively measuring cartilage health.

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine.

Degradation of articular cartilage extracellular matrix (ECM) by proteolytic enzyme is the hallmark of arthritis that leads to joint destruction. Detection of early biochemical changes in cartilage before irreversible structural damages become apparent is highly desirable. Here we report that the autofluorescence decay profile of cartilage is significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A multidimensional fluorometer utilizing ultraviolet excitation at 355 nm or 375 nm coupled to a fibreoptic probe was developed for single point time-resolved AFL measurements of porcine articular cartilage explants treated with different proteinases. Degradation of cartilage matrix components by treating with bacterial collagenase, matrix metalloproteinase 1, or trypsin resulted in significant reduction of AFL of the cartilage in both a dose and time dependent manner. Differences in cartilage AFL were also confirmed by fluorescence lifetime imaging microscopy (FLIM). Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may be utilized for diagnosis of arthritis as well as monitoring the efficacy of anti-arthritic therapeutic agents.

We report the development and application of instrumentation to measure and image tissue autofluorescence lifetime for the study and diagnosis of disease including cancer and osteoarthritis.

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.

We are developing an autofluorescence (AFL) lifetime-based technique to characterise the signatures associated with histological, metabolic and functional changes in myocardial disease. AFL spectroscopy exploits the properties of a number of endogenous molecules and offers the potential to avoid exogenous label use that could potentially alter the physiological environment. AFL measurements of molecules such as NADH and flavins may report energetic state changes, whilst signatures from matrix such as collagen inform of structural alterations. We investigated the application of a fibre-optic based single-point instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to a doxorubicin cardiomyopathy heart failure model (DOX-HF) in vivo . Sprague-Dawley male rats received 1.25 mg/kg doxorubicin or vehicle (0.9% NaCl) via tail vein injection weekly for 8 weeks. AFL signals were measured in vivo in week 11, once heart failure phenotype was well established. DOX-HF(n = 8) LVEF 49.6% vs. 80.4% in controls (n = 4)(**p = 0.006). At 11 weeks, significant differences in AFL signals between DOX-HF(n = 6) and control (n = 3) in LV, RV and LV posterior wall were observed (*p Our instrument has sensitivity to characterise in vivo changes in DOX-HF model without use of exogenous compounds. Incorporation into coronary catheters or pacing leads could offer additional diagnostic information and monitoring. Identification of earlier changes could permit development of a screening strategy to identify cancer patients developing cardiomyopathy to permit judicious adjustment of chemotherapy.

We investigate the potential of an instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to measure structural and metabolic changes in cardiac tissue in vivo in a 16 week post-myocardial infarction heart failure model in rats. In the scar region, we observed changes in the fluorescence signal that can be explained by increased collagen content, which is in good agreement with histology. In areas remote from the scar tissue, we measured changes in the fluorescence signal (p < 0.001) that cannot be explained by differences in collagen content and we attribute this to altered metabolism within the myocardium. A linear discriminant analysis algorithm was applied to the measurements to predict the tissue disease state. When we combine all measurements, our results reveal high diagnostic accuracy in the infarcted area (100%) and border zone (94.44%) as well as in remote regions from the scar (> 77%). Overall, our results demonstrate the potential of our instrument to characterize structural and metabolic changes in a failing heart in vivo without using exogenous labels.

Time-resolved autofluorescence spectroscopy permits characterization of biological tissues and has already shown the potential in a number of disease applications. It harnesses the photo-physical properties of a number of endogenous molecules, e.g. NADH and flavoproteins, to offer an opportunity to discern the energetic state of the myocardium without the issues of toxicity or consequences associated with the introduction of exogenous compounds. Additionally, autofluorescence from extra-cellular matrix molecules, e.g. collagen, can provide information on structural changes to the heart. We report the development and application of a custom fibre-optic probe-based time-resolved spectrofluorometer combining time-correlated single photon counting detection and white-light reflectometry to characterize the autofluorescence ‘fingerprint’ associated with the changes in histology, morphology, metabolism and function in heart health and pathological states. Measurement of fluorescence lifetime is inherently ratiometric and unaffected by molecule concentration and excitation intensity amongst other experimental variables. Furthermore fluorescence lifetime is affected by changes in the microenvironment e.g. protein binding states. Explanted hearts from male SD rats perfused using the ex vivo Langendorff system demonstrated stable fluorescence lifetimes. Fluorescence lifetime signals were successfully derived from fluorescence decays with acquisition times as low as 1 second. The changes in the autofluorescence signal of ex vivo Langendorff preparations using perfusate column switching were characterised for an ischaemia-reperfusion model and for a change of substrate between glucose and mannitol. This technology has the potential to be a label-free method to observe biochemical changes associated with disease mechanisms in the heart, with clear potential application to cardiac research and clinical diagnostics.

Fluorescence spectroscopy is a promising tool for the characterisation of biological tissues and shows the potential for in vivo clinical diagnosis in many diseases. It exploits the inherent photo-physical properties of a number of endogenous molecules. Fluorescence lifetime measurements of these fluorophores such as NADH and flavoproteins present an opportunity to discern functional information concerning myocardial energetics without issues of toxicity or systemic effects associated with the introduction of exogenous compounds. Additionally, autofluorescence from extra-cellular matrix molecules e.g. collagen may provide information on structural changes to the heart. Measurements of fluorescence lifetime are independent of fluorophore concentration, excitation intensity, sample attenuation and other experimental artefacts and can also report on changes to the fluorophore microenvironment e.g. pH and protein binding state. Thus we are interested to develop autofluorescence lifetime (AFL)-based techniques as a myocardial "optical biopsy". We report the application of a custom fibre-optic probe-based time-resolved spectrofluorometer utilising time-correlated single photon counting that we developed to characterise the autofluorescence signatures associated with the histological, morphological, metabolic and functional changes in myocardial tissue in health and disease states. We studied an in vivo rat left anterior descending coronary artery ligation heart failure (MI-HF) model 16 weeks post-infarction, which is a model well characterised in our institution. We observed stable AFL signals across age-matched control (AMC) hearts (n = 6) in different anatomical locations. We observed significant differences in AFL signals between MI-HF (n = 6) and AMC (n = 6) in infarcted regions: left ventricle (LV) anterior wall (p in vivo application of time-resolved fluorescence spectroscopy to the study of the heart. This technique shows promise as a diagnostic tool and could be readily and rapidly translatable into clinical cardiology practice.