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.
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.
Gastrointestinal disorders such as colorectal cancer or inflammatory bowel disease are linked to gut dysbiosis, an unbalanced gut microbiota. This early manifestation of the disease alters colon epithelial metabolism influencing the gut autofluorescence emission, which is susceptible to carry diagnostic value. We analyzed the fluorescence properties of healthy and dysbiotic ex vivo murine colons with an intraluminal fiber-based fluorescence lifetime imaging (FLIm) instrument. The results indicate that fluorescence lifetime reacts to inflammation in a spectrally dependant manner, and the full-length colon images allow to localize specific areas of activity. Imaging results were correlated to biochemical metabolic readouts (i.e. intracellular NADH, lactate) to establish the diagnostic potential of intraluminal FLIm.
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.
Fluorescence Lifetime Imaging (FLIm) is a label-free technique that reveals extracellular lipid and foam cells accumulation within atherosclerotic lesions, complementing the structural information provided by Optical Coherence Tomography (OCT). Successful intravascular implementation of FLIm-OCT requires high optical performances for both single and multimode beams, spanning the near UV to near IR range, out of reach of the current miniature optics (ball or gradient index lenses). Here, we present a 0.3 x 0.3 x 0.8 mm3 monolithic freeform optic that provides high optical performances over the UV-IR range (355-1360 nm), enabling high-performance intravascular FLIm-OCT imaging catheter designs.
Endogenous and exogenous fluorescence imaging have shown great values for monitoring and studying all kind of biological processes including vascular tissue regeneration and disease progression. In this study, we present a fiber-based optical imaging instrument that is able to simultaneously acquire endogenous and exogenous fluorescence images from tissue samples using a reflective optical chopper wheel to temporally interleave the two modalities. The functionality of the system was demonstrated by imaging native tissue constructs seeded with cells labeled with different dyes. The cellular regions were clearly resolved in both modalities, providing an exogenous and in-situ validation for endogenous fluorescence lifetime images.
Tissue engineered vascular graft (TEVG) are used when native vessels are not available to repair vascular damage. At the time of implantation in human body, these constructs present poor cellularity. To understand the cellularization kinetics under physiological conditions in a setting suitable for experimentation, bioreactors are often used in laboratory setting because of its controllable culture parameters including seeding conditions, flow type, pressure and temperature. Therefore, a non-destructive, label-free imaging modality that is capable of evaluating cell migration on luminal surfaces of TEVGs inside bioreactors is valuable for studying cellularization kinetics and providing a potential quality control method for manufacturing mature TEVGs. A multispectral Fluorescence Lifetime Imaging (ms-FLIm) using 355 nm excitation was configured to accommodate a rotating side-firing scanning probe for intraluminal imaging of tubular-shaped bovine pericardium (BP) scaffolds. The scanning was realized by reciprocal rotation and pullback of the fiber probe. Mesenchymal stem cells were seeded on BP-based TEVGs and cultured in the prototype bioreactor for up to one week. Distinct experimental conditions including the seeding side (i.e. BP serious and fibrous side) and media flow (i.e. static and dynamic pulsatile flow) were evaluated. Using ms-FLIm, the migration of cells on antigen removed BP TEVGs was periodically examined over a week; and the migration rates under different conditions were analyzed. Current results suggest helical ms-FLIm has potential to monitor in situ tissue recellularization process in bioreactors.
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.
KEYWORDS: Collagen, Fluorescence lifetime imaging, In vivo imaging, Luminescence, Proteins, Molecules, Tissue engineering, Nondestructive evaluation, In vitro testing, Imaging systems
Bovine pericardium (BP) exhibits distinct biochemical and biomechanical properties that are dominant by the structural protein collagen. The enzymatic degradation of collagen molecules is critical for in vivo incorporation and remodeling of BP in tissue engineering applications. A non-destructive method for monitoring BP during degradation would provide a valuable tool for quantifying functional changes initially in vitro and ultimately in vivo. In this study, we demonstrated the sensitivity of multi-spectral fluorescence lifetime imaging system (ms-FLIm) developed by our group to collagen content and compressive modulus of BP during collagenase degradation. A pairwise study was performed using bacterial collagenase to partially digest BP. We measured the biomaterials properties with ms-FLIm and destructive conventional measurements including collagen assay, compressive test and histology. A single factor study design was utilized. Test group samples were digested by bacterial collagenase for 0, 8, 16 and 24 hours, while control group samples were prepared in the Hank’s balanced salt solution to control for time in solution. Statistical analysis was performed using the Kendall τB correlation test. The results demonstrate that fluorescence parameters measured by ms-FLIm are significantly correlated with collagen content and compressive modulus (|τB| > 0.45, p < 0.05). Based on these findings, we aim to predict BP’s collagen content and mechanical properties using fluorescence metrics, and ultimately apply ms-FLIm for non-destructively monitoring of in vivo remodeling of BP.
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