Dual modality intravascular catheter system combining pulse-sampling fluorescence lifetime imaging and polarization-sensitive optical coherence tomography.

The clinical management of coronary artery disease and the prevention of acute coronary syndromes require knowledge of the underlying atherosclerotic plaque pathobiology. Hybrid imaging modalities capable of comprehensive assessment of biochemical and morphological plaques features can address this need. Here we report the first implementation of an intravascular catheter system combining fluorescence lifetime imaging (FLIm) with polarization-sensitive optical coherence tomography (PSOCT). This system provides multi-scale assessment of plaque structure and composition via high spatial resolution morphology from OCT, polarimetry-derived tissue microstructure, and biochemical composition from FLIm, without requiring any molecular contrast agent. This result was achieved with a low profile (2.7 Fr) double-clad fiber (DCF) catheter and high speed (100 fps B-scan rate, 40 mm/s pullback speed) console. Use of a DCF and broadband rotary junction required extensive optimization to mitigate the reduction in OCT performance originating from additional reflections and multipath artifacts. This challenge was addressed by the development of a broad-band (UV-visible-IR), high return loss (47 dB) rotary junction. We demonstrate in phantoms, ex vivo swine coronary specimens and in vivo swine heart (percutaneous coronary access) that the FLIm-PSOCT catheter system can simultaneously acquire co-registered FLIm data over four distinct spectral bands (380/20 nm, 400/20 nm, 452/45 nm, 540/45 nm) and PSOCT backscattered intensity, birefringence, and depolarization. The unique ability to collect complementary information from tissue (e.g., morphology, extracellular matrix composition, inflammation) with a device suitable for percutaneous coronary intervention offers new opportunities for cardiovascular research and clinical diagnosis.

Pulse-sampling fluorescence lifetime imaging: evaluation of photon economy.

This Letter presents an experimental study comparing the photon rate and photon economy of pulse sampling fluorescence lifetime imaging (PS-FLIm) with the conventional time-correlated single photon counting (TCSPC) technique. We found that PS-FLIm has a significantly higher photon detection rate (200 MHz) compared with TCSPC (2-8 MHz) but lower photon economy (4-5 versus 1-1.3). The main factor contributing to the lower photon economy in PS-FLIm is laser pulse variability. These results demonstrate that PS-FLIm offers 25× faster imaging speed than TCSPC while maintaining room light rejection in clinical settings. This makes PS-FLIm a robust technique for clinical applications.

In vivo characterization of the human glioblastoma infiltrative edge with label-free intraoperative fluorescence lifetime imaging.

Challenges in identifying a glioblastoma's infiltrative edge during neurosurgical procedures result in rapid recurrence. A label-free fluorescence lifetime imaging (FLIm) device was used to evaluate glioblastoma's infiltrative edge in vivo in 15 patients (89 samples). FLIm data were analyzed according to tumor cell density, infiltrating tissue type (gray and white matter), and diagnosis history (new or recurrent). Infiltrations in white matter from new glioblastomas showed decreasing lifetimes and a spectral red shift with increasing tumor cell density. Areas of high versus low tumor cell density were separated through a linear discriminant analysis with a ROC-AUC=0.74. Current results support the feasibility of intraoperative FLIm for real-time in vivo brain measurements and encourage refinement to predict glioblastoma infiltrative edge, underscoring the ability of FLIm to optimize neurosurgical outcomes.

Anatomy-Specific Classification Model Using Label-Free FLIm to Aid Intraoperative Surgical Guidance of Head and Neck Cancer.

Intraoperative identification of head and neck cancer tissue is essential to achieve complete tumor resection and mitigate tumor recurrence. Mesoscopic fluorescence lifetime imaging (FLIm) of intrinsic tissue fluorophores emission has demonstrated the potential to demarcate the extent of the tumor in patients undergoing surgical procedures of the oral cavity and the oropharynx. Here, we report FLIm-based classification methods using standard machine learning models that account for the diverse anatomical and biochemical composition across the head and neck anatomy to improve tumor region identification. Three anatomy-specific binary classification models were developed (i.e., "base of tongue," "palatine tonsil," and "oral tongue"). FLIm data from patients (N = 85) undergoing upper aerodigestive oncologic surgery were used to train and validate the classification models using a leave-one-patient-out cross-validation method. These models were evaluated for two classification tasks: (1) to discriminate between healthy and cancer tissue, and (2) to apply the binary classification model trained on healthy and cancer to discriminate dysplasia through transfer learning. This approach achieved superior classification performance compared to models that are anatomy-agnostic; specifically, a ROC-AUC of 0.94 was for the first task and 0.92 for the second. Furthermore, the model demonstrated detection of dysplasia, highlighting the generalization of the FLIm-based classifier. Current findings demonstrate that a classifier that accounts for tumor location can improve the ability to accurately identify surgical margins and underscore FLIm's potential as a tool for surgical guidance in head and neck cancer patients, including those subjects of robotic surgery.

Establishment of a sequential dual tracer 18 F-NaF/18 F-FDG PET protocol for imaging the equine foot.

The combination of 18 F-Sodium Fluoride (18 F-NaF) and 18 F-FluoroDeoxyGlucose (18 F-FDG) for positron emission tomography (PET) imaging of the equine foot is appealing for detection of both osseous and soft tissue lesions in a single scan. As the combination of tracers could lead to a loss of information, a sequential approach, consisting in imaging with one tracer prior to injecting the second tracer, might be valuable. The goals of this prospective, methods comparison, exploratory study were to establish the order of tracer injection and timing for imaging. Six research horses were imaged under general anesthesia with 18 F-NaF PET, 18 F-FDG PET, dual 18 F-NaF/18 F-FDG PET, and CT. Proper uptake could be identified in tendon lesions as early as 10 min after 18F-FDG injection. Bone uptake was limited when 18F-NaF was injected under general anesthesia, even at 1 h after injection, when compared with 18 F-NaF injection prior to anesthesia. The sensitivity and specificity of the dual tracer scans were 0.77 (0.63 to 0.86) and 0.98 (0.96 to 0.99) respectively, to assess 18 F-NaF uptake and 0.5 (0.28 to 0.72) and 0.98 (0.95 to 0.99), respectively, for 18F-FDG uptake. These results suggest that the sequential dual tracer approach is a pertinent technique to optimize the PET data gained from a single anesthetic episode. Based on dynamics of tracer uptake, the optimal protocol consists in injecting 18F-NaF prior to anesthesia, acquire 18F-NaF data then inject 18F-FDG and start acquisition of dual tracer PET data 10 min later. This protocol should be further validated in a larger clinical study.

Intraoperative detection of IDH-mutant glioma using fluorescence lifetime imaging.

Identifying isocitrate dehydrogenase (IDH)-mutation and glioma subtype during surgery instead of days later can aid in modifying tumor resection strategies for better survival outcomes. We report intraoperative identification of IDH-mutant glioma (N = 12 patients) with a clinically compatible fluorescence lifetime imaging (FLIm) device (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm). The fluorescence-derived parameters were analyzed to study the optical contrast between IDH-mutant tumors and surrounding brain tissue. IDH-mutant oligodendrogliomas exhibited shorter lifetimes (3.3 ± 0.1 ns) than IDH-mutant astrocytomas (4.1 ± 0.1 ns). Both IDH-mutant glioma subtypes had shorter lifetimes than white matter (4.6 ± 0.4 ns) but had comparable lifetimes to cortex. Lifetimes also increased with malignancy grade within IDH-mutant oligodendrogliomas (grade 2: 2.96 ± 0.08 ns, grade 3: 3.4 ± 0.3 ns) but not within IDH-mutant astrocytomas. The current results support the feasibility of FLIm as a surgical adjuvant for identifying IDH-mutant glioma tissue.

Engineering the gain and bandwidth in avalanche photodetectors.

Avalanche and Single-Photon Avalanche photodetectors (APDs and SPADs) rely on the probability of photogenerated carriers to trigger a multiplication process. Photon penetration depth plays a vital role in this process. In silicon APDs, a significant fraction of the short visible wavelengths is absorbed close to the device surface that is typically highly doped to serve as a contact. Most of the photogenerated carriers in this region can be lost by recombination, get slowly transported by diffusion, or multiplied with high excess noise. On the other hand, the extended penetration depth of near-infrared wavelengths requires thick semiconductors for efficient absorption. This diminishes the speed of the devices due to the long transit time in the thick absorption layer that is required for detecting most of these photons. Here, we demonstrate that it is possible to drive photons to a critical depth in a semiconductor film to maximize their gain-bandwidth performance and increase the absorption efficiency. This approach to engineering the penetration depth for different wavelengths in silicon is enabled by integrating photon-trapping nanoholes on the device surface. The penetration depth of short wavelengths such as 450 nm is increased from 0.25 µm to more than 0.62 µm. On the other hand, for a long-wavelength like 850 nm, the penetration depth is reduced from 18.3 µm to only 2.3 µm, decreasing the device transit time considerably. Such capabilities allow increasing the gain in APDs by almost 400× at 450 nm and by almost 9× at 850 nm. This engineering of the penetration depth in APDs would enable device designs requiring higher gain-bandwidth in emerging technologies such as Fluorescence Lifetime Microscopy (FLIM), Time-of-Flight Positron Emission Tomography (TOF-PET), quantum communications systems, and 3D imaging systems.

Dual-modality fluorescence lifetime imaging-optical coherence tomography intravascular catheter system with freeform catheter optics.

SIGNIFICANCE: Intravascular imaging is key to investigations into atherosclerotic plaque pathobiology and cardiovascular diagnostics overall. The development of multimodal imaging devices compatible with intracoronary applications has the potential to address limitations of currently available single-modality systems. AIM: We designed and characterized a robust, high performance multimodal imaging system that combines optical coherence tomography (OCT) and multispectral fluorescence lifetime imaging (FLIm) for intraluminal simultaneous assessment of structural and biochemical properties of coronary arteries. APPROACH: Several shortcomings of existing FLIm-OCT catheter systems are addressed by adopting key features, namely (1) a custom fiber optic rotary joint based on an air bearing, (2) a broadband catheter using a freeform reflective optics, and (3) integrated solid-state FLIm detectors. Improvements are quantified using a combination of experimental characterization and simulations. RESULTS: Excellent UV and IR coupling efficiencies and stability (IR: 75.7 % ± 0.4 % , UV: 45.7 % ± 0.35 % ) are achieved; high FLIm optical performance is obtained (UV beam FWHM: 50 µm) contemporaneously with excellent OCT beam quality (IR beam FWHM: 17 µm). High-quality FLIm OCT image of a human coronary artery specimen was acquired. CONCLUSION: The ability of this intravascular imaging system to provide comprehensive structural and biochemical properties will be valuable to further our understanding of plaque pathophysiology and improve cardiovascular diagnostics.

Intraoperative delineation of p16+ oropharyngeal carcinoma of unknown primary origin with fluorescence lifetime imaging: Preliminary report.

BACKGROUND: This study evaluated whether fluorescence lifetime imaging (FLIm), coupled with standard diagnostic workups, could enhance primary lesion detection in patients with p16+ head and neck squamous cell carcinoma of the unknown primary (HNSCCUP). METHODS: FLIm was integrated into transoral robotic surgery to acquire optical data on six HNSCCUP patients' oropharyngeal tissues. An additional 55-patient FLIm dataset, comprising conventional primary tumors, trained a machine learning classifier; the output predicted the presence and location of HNSCCUP for the six patients. Validation was performed using histopathology. RESULTS: Among the six HNSCCUP patients, p16+ occult primary was surgically identified in three patients, whereas three patients ultimately had no identifiable primary site in the oropharynx. FLIm correctly detected HNSCCUP in all three patients (ROC-AUC: 0.90 ± 0.06), and correctly predicted benign oropharyngeal tissue for the remaining three patients. The mean sensitivity was 95% ± 3.5%, and specificity 89% ± 12.7%. CONCLUSIONS: FLIm may be a useful diagnostic adjunct for detecting HNSCCUP.

First in patient assessment of brain tumor infiltrative margins using simultaneous time-resolved measurements of 5-ALA-induced PpIX fluorescence and tissue autofluorescence.

SIGNIFICANCE: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide [NAD(P)H] fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection. AIM: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients. APPROACH: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid (<33 ms) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation. RESULTS: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values (τPpIX   ∼ 3 to 14 ns, τNAD(P)H = 3 to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration. CONCLUSIONS: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo. Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.

Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging.

Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50-80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between ß-oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases.

Multispectral fluorescence lifetime imaging device with a silicon avalanche photodetector.

We report the design, development, and characterization of a novel multi-spectral fluorescence lifetime measurement device incorporating solid-state detectors and automated gain control. For every excitation pulse (∼1 µJ, 600 ps), this device records complete fluorescence decay from multiple spectral channels simultaneously within microseconds, using a dedicated UV enhanced avalanche photodetector and analog to digital convert (2.5 GS/s) in each channel. Fast (<2 ms) channel-wise dynamic range adjustment maximizes the signal-to-noise ratio. Fluorophores with known lifetime ranging from 0.5-6.0 ns were used to demonstrate the device accuracy. Current results show the clear benefits of this device compared to existing devices employing microchannel-plate photomultiplier tubes. This is demonstrated by 5-fold reduction of lifetime measurement variability in identical conditions, independent gain adjustment in each spectral band, and 4-times faster imaging speed. The use of solid-state detectors will also facilitate future improved performance and miniaturization of the instrument.

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

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

Label-Free Visualization and Quantification of Biochemical Markers of Atherosclerotic Plaque Progression Using Intravascular Fluorescence Lifetime.

OBJECTIVES: This study aimed to systematically investigate whether plaque autofluorescence properties assessed with intravascular fluorescence lifetime imaging (FLIm) can provide qualitative and quantitative information about intimal composition and improve the characterization of atherosclerosis lesions. BACKGROUND: Despite advances in cardiovascular diagnostics, the analytic tools and imaging technologies currently available have limited capabilities for evaluating in situ biochemical changes associated with luminal surface features. Earlier studies of small number of samples have shown differences among the autofluorescence lifetime signature of well-defined lesions, but a systematic pixel-level evaluation of fluorescence signatures associated with various histological features is lacking and needed to better understand the origins of fluorescence contrast. METHODS: Human coronary artery segments (n = 32) were analyzed with a bimodal catheter system combining multispectral FLIm with intravascular ultrasonography compatible with in vivo coronary imaging. Various histological components present along the luminal surface (200-µm depth) were systematically tabulated (12 sectors) from each serial histological section (n = 204). Morphological information provided by ultrasonography allowed for the accurate registration of imaging data with histology data. The relationships between histological findings and FLIm parameters obtained from 3 spectral channels at each measurement location (n = 33,980) were characterized. RESULTS: Our findings indicate that fluorescence lifetime from different spectral bands can be used to quantitatively predict the superficial presence of macrophage foam cells (mFCs) (area under the receiver-operator characteristic curve: 0.94) and extracellular lipid content in advanced lesions (lifetime increase in 540-nm band), detect superficial calcium (lifetime decrease in 450-nm band area under the receiver-operator characteristic curve: 0.90), and possibly detect lesions consistent with active plaque formation such as pathological intimal thickening and healed thrombus regions (lifetime increase in 390-nm band). CONCLUSIONS: Our findings indicate that autofluorescence lifetime provides valuable information for characterizing atherosclerotic lesions in coronary arteries. Specifically, FLIm can be used to identify key phenomena linked with plaque progression (e.g., peroxidized-lipid-rich mFC accumulation and recent plaque formation).

Intraoperative Margin Assessment in Oral and Oropharyngeal Cancer Using Label-Free Fluorescence Lifetime Imaging and Machine Learning.

OBJECTIVE: To demonstrate the diagnostic ability of label-free, point-scanning, fiber-based Fluorescence Lifetime Imaging (FLIm) as a means of intraoperative guidance during oral and oropharyngeal cancer removal surgery. METHODS: FLIm point-measurements acquired from 53 patients (n = 67893 pre-resection in vivo, n = 89695 post-resection ex vivo) undergoing oral or oropharyngeal cancer removal surgery were used for analysis. Discrimination of healthy tissue and cancer was investigated using various FLIm-derived parameter sets and classifiers (Support Vector Machine, Random Forests, CNN). Classifier output for the acquired set of point-measurements was visualized through an interpolation-based approach to generate a probabilistic heatmap of cancer within the surgical field. Classifier output for dysplasia at the resection margins was also investigated. RESULTS: Statistically significant change (P 0.01) between healthy and cancer was observed in vivo for the acquired FLIm signal parameters (e.g., average lifetime) linked with metabolic activity. Superior classification was achieved at the tissue region level using the Random Forests method (ROC-AUC: 0.88). Classifier output for dysplasia (% probability of cancer) was observed to lie between that of cancer and healthy tissue, highlighting FLIm's ability to distinguish various conditions. CONCLUSION: The developed approach demonstrates the potential of FLIm for fast, reliable intraoperative margin assessment without the need for contrast agents. SIGNIFICANCE: Fiber-based FLIm has the potential to be used as a diagnostic tool during cancer resection surgery, including Transoral Robotic Surgery (TORS), helping ensure complete resections and improve the survival rate of oral and oropharyngeal cancer patients.

FLImBrush: dynamic visualization of intraoperative free-hand fiber-based fluorescence lifetime imaging.

A free-hand scanning approach to medical imaging allows for flexible, lightweight probes to image intricate anatomies for modalities such as fluorescence lifetime imaging (FLIm), optical coherence tomography (OCT) and ultrasound. While very promising, this approach faces several key challenges including tissue motion during imaging, varying lighting conditions in the surgical field, and sparse sampling of the tissue surface. These challenges limit the coregistration accuracy and interpretability of the acquired imaging data. Here we report FLImBrush as a robust method for the localization and visualization of intraoperative free-hand fiber optic fluorescence lifetime imaging (FLIm). FLImBrush builds upon an existing method while employing deep learning-based image segmentation, block-matching based motion correction, and interpolation-based visualization to address the aforementioned challenges. Current results demonstrate that FLImBrush can provide accurate localization of FLIm point-measurements while producing interpretable and complete visualizations of FLIm data acquired from a tissue surface. Each of the main processing steps was shown to be capable of real-time processing (> 30 frames per second), highlighting the feasibility of FLImBrush for intraoperative imaging and surgical guidance. Current findings show the feasibility of integrating FLImBrush into a range of surgical applications including cancer margins assessment during head and neck surgery.

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.

Real-time augmented reality for delineation of surgical margins during neurosurgery using autofluorescence lifetime contrast.

Current clinical brain imaging techniques used for surgical planning of tumor resection lack intraoperative and real-time feedback; hence surgeons ultimately rely on subjective evaluation to identify tumor areas and margins. We report a fluorescence lifetime imaging (FLIm) instrument (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm and 629/53 nm) that integrates with surgical microscopes to provide real-time intraoperative augmentation of the surgical field of view with fluorescent derived parameters encoding diagnostic information. We show the functionality and safety features of this instrument during neurosurgical procedures in patients undergoing craniotomy for the resection of brain tumors and/or tissue with radiation damage. We demonstrate in three case studies the ability of this instrument to resolve distinct tissue types and pathology including cortex, white matter, tumor and radiation-induced necrosis. In particular, two patients with effects of radiation-induced necrosis exhibited longer fluorescence lifetimes and increased optical redox ratio on the necrotic tissue with respect to non-affected cortex, and an oligodendroglioma resected from a third patient reported shorter fluorescence lifetime and a decrease in optical redox ratio than the surrounding white matter. These results encourage the use of FLIm as a label-free and non-invasive intraoperative tool for neurosurgical guidance.

Broadband, freeform focusing micro-optics for a side-viewing imaging catheter.

Successful implementation of a catheter-based imaging system relies on the integration of high-performance miniaturized distal end optics. Typically, compensation of chromatic dispersion, as well as astigmatism introduced by the device's sheath, can be addressed only by combining multiple optical elements, adversely impacting size and manufacturability. Here, we present a 300×300×800 µm3 monolithic optic that provides high optical performances over an extended wavelength range (near UV-visible-IR) with minimal chromatic aberrations. The design of the optic, fully optimized using standard optical simulation tools, provides the ability to freely determine aperture and working distance. Manufacturing is cost effective and suited for prototyping and production alike. The experimental characterization of the optic demonstrates a good match with simulation results and performances well suited to both optical coherence tomography and fluorescence imaging, thus paving the way for high-performance multimodal endoscopy systems.

Simultaneous intraluminal imaging of tissue autofluorescence and eGFP-labeled cells in engineered vascular grafts inside a bioreactor.

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