We present a study investigating fluorescence lifetime signatures of normal tissues adjacent to tumors (NATs) in head and neck squamous cell carcinoma (HNSCC) using fluorescence lifetime imaging (FLIm). Label-free FLIm offers insight into the metabolic activity and extracellular matrix composition. Understanding the metabolic activity, tissue heterogeneity and tumor-associated alterations in these transition areas can enhance the accuracy of margin delineation. Initial results show that the fluorescence lifetime is gradually increasing from shorter to longer lifetimes with increasing distance from the cancer and with varying magnitudes of change being observed in the individual emission bands.
This study assesses the sensitivity of label-free fluorescence lifetime imaging (FLIm) for detecting low-grade gastrointestinal inflammation in mice. A FLIm probe was developed for in vivo colon imaging, enabling complete colon scanning. Using the probe, low-grade inflammation induced by streptomycin was imaged, showing decreased fluorescence lifetime at wavelengths related to metabolic activity, indicating a shift to glycolytic metabolism in inflamed tissue. The potential of validating FLIm maps with spatial transcriptomics was explored. These methodologies provide a basis for further experimentation to establish FLIm as a tool to quantify colon epithelial metabolism over time and its relevance to monitor colorectal inflammatory disease.
We report the development and characterization of a multispectral FLIm/ polarization-sensitive OCT intravascular imaging catheter system. Key innovations include a high return loss rotary junction. The combined imaging contrasts target the improved characterization of inflammation and disruption of the extracellular matrix, two key contributors to atherosclerosis, by enabling the evaluation of biochemical signatures, birefringence, and depolarization in addition to lesion morphology. We present key aspects of the system’s design and performance and highlight challenges associated with the development of intravascular imaging systems suitable for clinical translation.
We report the development and characterization of a multispectral FLIm/ polarization-sensitive OCT intravascular imaging catheter system. This system targets the improved characterization of two key contributors to atherosclerosis disease development: inflammation and disruption of the extracellular matrix, by enabling the evaluation of biochemical signature, birefringence, and depolarization in addition to lesion morphology. We present key aspects of the system’s design and performance and highlight challenges associated with the development of intravascular imaging systems suitable for clinical translation.
Pulse sampling fluorescence Lifetime Imaging (PS-FLIm) is a powerful noninvasive technique with applications to basic science and translational clinical research. However, little is known about the photon-economy of PS-FLIm. We report the first study about the photon-economy of PS-FLIm. We found that for in vivo measurements, the photon rate of multispectral PS-FLIm ranges from 1 to 5 GHz (three channels), order of magnitude higher than that of TCSPC and the F-value of PS-FLIm is less performant than that of TCSPC. This study is a first step toward understanding the photon-economy of PS-FLIm and will facilitate the optimization of PS-FLIm for future clinical use.
In-situ identification of glioma subtype can enable modifications of clinical and surgical strategies. Particularly, astrocytoma benefit from more aggressive resection than oligodendroglioma, which have a more favorable response to post-surgical chemotherapy. Preoperative MRI and intraoperative histology cannot accurately determine glioma subtype. There is a need for real-time identification of adult-type diffuse glioma subtypes to aid the neurosurgeon’s decision-making during resection surgery. Fluorescence lifetime imaging (FLIm) where tissue autofluorescence can be used as an indicator to distinguish among brain tumor tissue types in real-time could aid this process. Here, we report the use of label-free FLIm in distinguishing IDH-mutant glioma subtypes (astrocytoma and oligodendroglioma). The FLIm system (excitation: 355 nm; emission bands: 390/40 nm, 470/28 nm, 542/50 nm) was used to scan brain tissue from the resection margins of glioma patients during tumor resection. Fluorescence lifetimes were extracted and analyzed by constrained least-squares deconvolution with the Laguerre expansion method. FLIm data was validated with histopathology of collected biopsies. Current results show that FLIm provides optical contrast between tumor and healthy white matter, and between IDH-mutant astrocytoma (N=7 patients) and oligodendroglioma (N=5 patients). Tumors showed shorter lifetime values (470-nm: 3.6±0.6ns; 542-nm: 3.3±0.7ns) than healthy white matter (470-nm: 4.6±0.4ns; 542-nm: 4.3±0.5ns, p<0.01). Oligodendroglioma had shorter lifetimes in the 470-nm (3.3±0.1ns) and 542-nm (2.8±0.2ns) channels, which are associated with NAD(P)H and FAD fluorescence respectively, when compared with IDH-mutant astrocytoma (470-nm: 4.1±0.1ns; 542-nm: 3.9±0.2ns, p<0.01). Together, these results demonstrate the feasibility of using FLIm as an intraoperative tool in glioma diagnosis.
SignificanceCartilage 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.AimThe 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.ApproachSelf-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 (t = 2, 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.ResultsFLIm 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 485 nm spectral channels, indicating increase in collagen content. Significant (p < 0.05) decrease of construct homogeneity index was observed between t = 2 weeks and t = 4 weeks. Both FLIm and OCT images revealed defects (voids) at the center of the tissue construct during in vitro culture (t = 2 and 4 weeks). Cyst formation during in vivo culture was detected by OCT and confirmed with histology.ConclusionsThe 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.
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.
The standard treatment for infiltrative gliomas is surgery to remove as much tumor tissue as possible without compromising neurological functions. Thus, real-time identification of infiltrative tumor tissue is necessary. Here a fluorescence lifetime imaging (FLIm) was used to distinguish between healthy brain and areas with different degrees of tumor cellularity as defined by histopathology. We conducted FLIm measurements and collected microbiopsies from tumor resection margins to identify the FLIm characteristics of tumor edges in cortex and white matter of low- and high-grade gliomas. Results from a 13-patient cohort indicate that FLIm identifies infiltrations of up to moderate tumor cellularity.
We report a novel multispectral FLIm/ swept-source OCT intravascular imaging catheter system including three key innovative features: 1) UV/NIR beam focusing is achieved with a free-form reflective distal optics that outperforms both ball and GRIN-based catheter optics, 2) stable optical coupling (single mode transmission: 75.7+-0.5% at 100 rps) by way of an air bearing rotary collimator, and 3) improvements in FLIm SNR obtained by integrating solid-state FLIm detection within the motor drive. Validation in excised human coronary artery specimens demonstrates the capabilities of FLIm to detect and quantify inflammation and characterize the extracellular matrix of atherosclerotic lesions.
5-ALA induced PpIX is increasingly used for guiding brain tumor resection surgery. The current intensity-based approach fails at detecting lower concentrations of PpIX found in low-grade gliomas or the infiltrative edge of glioblastomas. Here, we report the first results in patients of real-time, PpIX fluorescence lifetime measurements using a hand-held fiber probe. Fluorescence from different spectral channels (390/40 nm (Collagen), 470/28 nm (NADH), 629/53 nm (PPIX)), is augmented onto a video stream of the surgical field-of-view to provide intraoperative tumor visualization in real-time. In-vivo data reveals strong contrast between regions of high PpIX accumulation associated with tumor (>8 ns) and healthy brain tissue (<4 ns).
Controlling light penetration depth in Avalanche Photodiodes (APDs) and Single Photon Avalanche Diodes (SPADs) play a major role in achieving high multiplication gain by delivering light near the multiplication region where the electric field is the strongest. Such control in the penetration depth for a particular wavelength of light has been previously demonstrated using integrated photon-trapping nanostructures. In this paper, we show that an optimized periodic nanostructure design can control the penetration depth for a wide range of visible and near-infrared wavelengths simultaneously. A conventional silicon APD structure suffers from high photocarrier loss due to recombination for shorter wavelengths as they are absorbed near the surface region, while silicon has low absorption efficiency for longer wavelengths. This optimized nanostructure design allows shorter wavelengths of light to penetrate deeper into the device, circumventing recombination sites while trapping the longer wavelengths in the thin silicon device by bending the vertically propagating light into horizontal modes. This manipulation of penetration depth improves the absorption in the device, increasing light sensitivity while nanostructures reduce the reflectance from the top surface. While delivery of light near the multiplication region reduces the photogenerated carrier loss and shortens transit time, leading to high multiplication gain in APDs and SPADs over a wide spectral range. These high gain APDs and SPADs will find their potential applications in Time-Of-Flight Positron Emission Tomography (TOF-PET), Fluorescence Lifetime Imaging Microscopy (FLIM), and pulse oximetry where high detection efficiency and high gain-bandwidth is required over a multitude of wavelengths.
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.
The gain in Avalanche Photodiodes (APDs) and Single Photon Avalanche Diodes (SPADs) is dependent on the probability of photo-generated carriers to trigger an avalanche process, which is correlated to the depth where a photon is absorbed by the photodiode. For silicon photodiodes, most of the photons with wavelengths in the visible spectrum are absorbed near the surface in the highly doped contact regions where the recombination rate is high. Thus, they do not contribute significantly to the avalanche multiplication process. By integrating photon-trapping nanostructures, we facilitate deeper penetration of photons into the devices, enhancing light absorption to generate more carriers that can trigger the avalanche process. This improves the gain-bandwidth of silicon APDs and SPADs significantly. Photon-trapping nanoholes can reduce the thickness of silicon without compromising its quantum efficiency, while a perforated surface reduces the device capacitance improving the bandwidth. Therefore, the manipulation of light penetration depth using photon-trapping nanoholes leads to ultrafast high-gain photodetectors capable of detecting faint light signals particularly useful for low light applications such as fluorescent lifetime imaging microscopy and time-of-flight positron emission tomography.
We report the design, development, and characterization of the first multispectral fluorescence lifetime imaging platform designed for clinical application based on avalanche photodiodes (APD). Consistent and accurate measurement of fluorescence lifetimes is achieved. Fast dynamic gain adjustment via APD bias modulation is implemented independently for each spectral channel, maximizing photon economy and greatly improving signal quality. Compared to conventional MCP-PMT based systems, the new platform presents 5-fold reduction of lifetime measurement variability under identical experiment conditions. This improved photon economy leads to 35 times faster coverage of imaging field in clinical practice with identical tissue exposure, making the new platform better suited for clinical application.
We report a novel 355 nm multi-spectral FLIm/ 1310 nm swept-source OCT intravascular imaging catheter system with improved performance. A free-form reflective distal optics enables uncompromised UV and NIR beam focusing. Adoption of an air bearing rotary collimator (100 rps) provides stable optical coupling (single mode transmission: 70+/-2% ); the integration of solid-state multi-spectral FLIm detection within motor drive benefits both FLIm acquisition speed (30 kHz point measurement rate) and signal quality (6x reduction in lifetime standard deviation). We anticipate that this system will complement OCT’s well known capabilities with improved inflammation quantification and extracellular matrix characterization of intravascular lesions.
We demonstrated the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of engineered vascular grafts with high speed and specificity. We report FLIm guided Raman imaging as an effective multimodal technique to evaluate scaffold cross-linking and localized calcification. Current results indicate that the lifetime of AR-BP shortens upon GA cross-linking,and Raman spectroscopy reveals secondary structural changes occurring in the Amide I region of cross-linked pericardia. GA fixed vascular grafts are prone to calcification, an effect linked to graft failure. The calcified regions exhibited shorter lifetimes in fluorescence spectral bands ranging from 380 to 455 nm and Raman spectra exhibited the specific hydroxyapatite signature at 960 cm-1 co-localized with these lower lifetime regions. We conclude that FLIm guided Raman imaging can detect cross-linking signatures and areas of calcification in tissue with biochemical specificity.
A fiber-based, label-free multispectral fluorescence lifetime imaging and intravascular ultrasound (FLIm/IVUS) system was evaluated as a new tool for monitoring variations in biochemical and structural composition of vascular biomaterials, including native arteries and engineered vascular grafts both in vitro and in vivo. Fiber-based FLIm was adapted to assess the hollow geometry of vasculature, allowing for imaging of the luminal surface of vessels. The capacity of FLIm to resolve tissue cellular location (i.e. scaffold reendothelialization) and collagen to elastin ratio on the vessel wall was investigated. Quantitative imaging parameters derived from spectrally- and temporally-resolved autofluorescence (i.e. intensity ratios and fluorescence lifetime) provide benchmark indicators to identify areas of recellularized tissue, and to distinguish wall matrix compositions within and across biomaterials. In addition, fiber-based FLIm was complemented with intravascular ultrasound (IVUS) for simultaneous in vivo evaluation of biochemical and structural tissue properties. Here, we performed an in vitro evaluation of pig carotid arteries and show correlations between FLIm parameters and biochemical composition in different anatomical locations. We discuss the spectral and lifetime differences between native pig carotid artery, acellular antigen removed bovine pericardium grafts, and reendotheliarized grafts. Finally, we translate the findings to an in vivo clinical FLIm/IVUS imaging study with antigen removed bovine pericardium grafted on healthy pig native carotid artery. Upon implantation, the graft is expected to repopulate with cells, and change composition as cells remodel it. These experiments demonstrate the feasibility of fiber-based FLIm/IVUS to examine vascular engineered tissue in research and clinical settings.
Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition and function of engineered tissue equivalents. A non-destructive solution to monitor tissue quality and maturation would greatly reduce costs and accelerate the development of tissue-engineered products. A label-free multimodal system combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) via a single fiber-optic interface was used for evaluation of biochemical and structural properties of tissue-engineered articular cartilage in a murine model of cartilage maturation. Nude mice (n=5) received 2 dorsal subcutaneous tissue-engineered cartilage implants each consisting of: 1) latent transforming growth factor-beta1 (LAP) treated; and 2) untreated control (CTL) constructs. At 6 weeks post-implantation, mice were sacrificed and multimodal imaging was performed in situ. FLIm showed clear delineation of the implant in all spectral bands (SB). Quantification of the cartilage construct fluorescence lifetime (LT) showed a lower LT in SB-1 (375-410 nm) and higher SB-3 LT (515-565 nm) as compared to the surrounding muscle tissue. Comparison between treatment groups showed a significant increase in FLIm SB-3 LT in LAP-treated constructs over CTL (p < 0.01). Quantification of OCT images allowed implant morphology and 3D volume comparisons between treatment groups. These results suggest that FLIm-OCT based tools are a potential non-destructive method for quantitatively monitoring the growth and quality of tissue engineered articular cartilage. The use of optical techniques to monitor maturation could represent a significant element in reducing costs in research, meeting the FDA regulatory requirements for manufacturing, and providing novel diagnostic tools in the clinic.
Glycosaminoglycan (GAG) loss is an early marker of osteoarthritis, which is a clinical late stage disease that affects millions of people worldwide. The goal of our study was to evaluate the ability of a fiber-based fluorescence lifetime imaging (FLIm) technique to detect GAG loss in articular cartilage. Native bovine cartilage explants (n = 20) were exposed to 0 (control), 0.5 (low), or 1 U / mL (high) concentrations of chondroitinase ABC (cABC) to create samples with different levels of GAG loss. FLIm assessment (excitation: 355 nm; detection: channel 1: 375 to 410 nm, channel 2: 450 to 485 nm, channel 3: 530 to 565 nm) was conducted on depth-resolved cross-sections of the cartilage sample. FLIm images, validated with histology, revealed that loss of GAG resulted in a decrease of fluorescence lifetime values in channel 2 (Δ = 0.44 ns, p < 0.05) and channel 3 (Δ = 0.75 ns, p < 0.01) compared to control samples (channel 2: 6.34 ns; channel 3: 5.22 ns). Fluorescence intensity ratio values were lower in channel 1 (37%, p < 0.0001) and channel 2 (31% decrease, p < 0.0001) and higher in channel 3 (23%, p < 0.0001) relative to control samples. These results show that FLIm can detect the loss of GAG in articular cartilage and support further investigation into the feasibility of in vivo FLIm arthroscopy.
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