A strong roll-off performance in Optical Coherence Tomography (OCT) is essential for imaging the inner ear, as it enables better depth resolution and penetration into the delicate structures within the temporal bone. This ensures higher-quality images and improved visualization of inner ear pathologies, thereby rendering OCT a valuable non-invasive tool for accurate diagnosis and assessment of inner ear conditions. In this work, we propose a spectral-domain OCT design that incorporates a frequency comb light source to enhance the penetration depth through improved roll-off performance. A broadband (~87nm at 3dB) comb source was developed centered near 1300nm, producing ~1000 lines spaced by a constant dk (~0.83cm-1), which were coupled into a Mach-Zehnder interferometer and then detected by a spectrometer. Our initial results demonstrate an >80% improvement in 3-dB roll-off compared to the same system without the comb, i.e., with a broadband light source as used in the classical SD-OCT design.
Magnetic Resonance Imaging and x-ray Computed Tomography have limitations when applied to diseases of the human inner ear due to insufficient resolution. Key morphological features of the inner ear are below the resolving power of both modalities; thus, they are unable to measure functional aspects of the microstructures in the cochlea. Furthermore, general access to the cochlea is a challenge due to its location in the inner ear and its bony encapsulation. These limitations cause clinicians to rely on clinical history when diagnosing and managing hearing loss in patients, which is not ideal. This paper explores the application of Optical Coherence Tomography (OCT) as a diagnostic tool for inner ear diseases. OCT’s high spatial and temporal resolution allows for detailed imaging of inner ear structures and their function. To address the challenge of accessing the cochlea in humans, a hand-held endoscopic OCT device has been developed that can image through the round window membrane. The technology has been tested in cadaver temporal bone, enabling functional and morphological imaging of the cochlea when navigated to the round window. Alongside the device, we are developing an algorithm to perform subsequent stitching of volumes to overcome limitations with a small field of view. Applying this algorithm on cadaver tissue serves as a preliminary step before advancing to live human cochlear imaging. By utilizing our hand-held OCT endoscope, clinicians will have the ability to record changes in morphological and functional information, thereby improving the approach to diagnosing and treating patients with inner ear diseases.
We report dynamic optical coherence tomography (D-OCT) images of the organ of Corti (ooC) in ex vivo mouse cochleas. The ooC is responsible for transducing sound-evoked mechanical vibrations and amplifying them in the process of hearing. Thorough knowledge of the micromechanical properties of the ooC is required for understanding how hearing functions. Recently, OCT has emerged as a safe and effective tool to probe the inner workings of the cochlea and ooC. However, OCT is limited in its ability to directly resolve cellular architecture due to limited optical scattering-based contrast between different cell types. D-OCT is a label-free method capable of probing sub-resolution movements by analyzing speckle and phase information from standard OCT data as a function of time. We show that key structures in the ooC can be identified with D-OCT versus standard OCT, and that D-OCT has the potential to characterize the ooC and advance our understanding of the process of hearing.
We have recently developed a novel, cost-effective, versatile, and practical frequency-domain (FD) FLIM implementation capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. This novel technology has been adopted to implement an FD-FLIM endoscopy system for label-free metabolic imaging of epithelial tissues. The FD-FLIM endoscopy system is capable of simultaneous excitation at two wavelengths (375-nm for NADH, 445-nm for FAD). Preliminary results indicate that established metabolic autofluorescence biomarkers of epithelial pre-malignancy and malignancy can be clinically imaged with this novel FD-FLIM endoscopy technology. Future work will focus on exploring clinical applications of label-free metabolic imaging using this novel FD-FLIM endoscopy technology.
We report a new approach for label-free metabolic imaging of oral lesions using a novel fluorescence lifetime imaging endoscopy (FLIMEnd) system. The FLIMEnd system is capable of simultaneous dual-wavelength excitation and fluorescence detection at four emission spectral bands. Preliminary results indicate that established metabolic autofluorescence biomarkers of oral pre-malignancy and malignancy can be clinically imaged with this novel FLIMEnd technology. Future work will focus on identifying both established and novel autofluorescence biomarkers of oral pre-cancer and cancer that can be used to develop machine-learning driven FLIMEnd systems for early detection of oral cancer.
We present a novel, cost-effective, versatile, and practical frequency-domain fluorescence lifetime imaging/microscopy (FD-FLIM) implementation capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of ~0.4-12 ns. This novel technology has been adopted to implement an FD-FLIM endoscopy system for label-free metabolic imaging of oral lesions based on NADH (375-nm excitation, 484/37-nm emission) and FAD (445-nm excitation, 553/93-nm emission) autofluorescence imaging. Future efforts will focus on developing imaging systems for simultaneous endogenous (UV-VI excitation) and exogenous (NIR excitation) fluorescence lifetime imaging.
Increased cellular metabolic activity can be quantified by imaging the oral tissue autofluorescence originated from NADH and FAD. Clinical label-free metabolic imaging of oral epithelial cancer based on 375-nm excitation maFLIM endoscopy was recently demonstrated in patients presenting oral malignant lesions. A recently upgraded maFLIM endoscopy system enables simultaneous autofluorescence excitation at 375 nm (for NADH) and 445 nm (for FAD). This maFLIM endoscope is being used to clinically image healthy, benign, precancerous and cancerous oral lesions in oral health care settings. Comprehensive statistical analysis will be performed to identify new metabolic and biochemical autofluorescence biomarkers of oral epithelial cancer.
Increased cellular metabolic activity, a hallmark of malignant epithelial cells, can be quantified by imaging the oral tissue autofluorescence originated from the metabolic cofactors NADH and FAD. We report a novel multispectral autofluorescence lifetime imaging (maFLIM) handheld probe capable of simultaneous autofluorescence excitation at 375 nm (for NADH) and 445 nm (for FAD), and simultaneous multispectral time-resolved fluorescence measurement at four emission spectral channels. The performance of the dual-wavelength excitation maFLIM handheld probe was assessed by imaging fluorescent dye standards with well characterized fluorescence lifetimes, and the oral mucosa of human subjects in oral health care settings.
We report the development of a novel point-scanning frequency-domain (FD) fluorescence lifetime imaging (FLIM) system. The system, which was customized for label-free metabolic and biochemical imaging of oral epithelial tissue, is capable of simultaneous excitation at 375nm and 445nm, emission detection across 4 spectral bands, real-time fluorescence modulation and phase lifetime calculation, and low-cost data acquisition implementation using an Abaco FMC104 digitizer, Xilinx Zedboard FPGA, and Python software on the host computer. In this work we discuss our challenges and workflow with the goal of helping fellow researchers develop versatile and cost-effective FLIM systems for biomedical and clinical imaging applications.
Significance: Detailed biochemical and morphological imaging of the plaque burdened coronary arteries holds the promise of improved understanding of atherosclerosis plaque development, ultimately leading to better diagnostics and therapies.
Aim: Development of a dual-modality intravascular catheter supporting swept-source optical coherence tomography (OCT) and frequency-domain fluorescence lifetime imaging (FD-FLIM) of endogenous fluorophores with UV excitation.
Approach: We instituted a refined approach to endoscope development that combines simulation in a commercial ray tracing program, fabrication, and a measurement method for optimizing ball-lens performance. With this approach, we designed and developed a dual-modality catheter endoscope based on a double-clad fiber supporting OCT through the core and fluorescence collection through the first cladding. We varied the relative percent of UV excitation launched into the core and first cladding to explore the potential resolution improvement for FD-FLIM. The developed catheter endoscope was optically characterized, including measurement of spatial resolution and fluorescent lifetimes of standard fluorophores. Finally, the system was demonstrated on fresh ex vivo human coronary arteries.
Results: The developed endoscope was shown to have optical performance similar to predictions derived from the simulation approach. The FLIM resolution can be improved by over a factor of 4 by primarily illuminating through the core rather than the first cladding. However, time-dependent solarization losses need to be considered when choosing the relative percentage. We ultimately chose to illuminate with 7% of the power transmitting through the core. The resulting catheter endoscope had 40-μm lateral resolution for OCT and <100 μm lateral resolution for FD-FLIM. Images of ex vivo coronary arteries are consistent with expectations based on histopathology.
Conclusions: The results demonstrate that our approach for endoscope simulation produces reliable predictions of endoscope performance. Simulation results guided our development of a multimodal OCT/FD-FLIM catheter imaging system for investigating atherosclerosis in coronary arteries.
We present our 2nd generation handheld simultaneous multispectral frequency-domain FLIM endoscopic system for label-free metabolic imaging of oral cancer, with enhanced optical performance and system usability. Our custom-designed and 3D-printed handheld endoscope consists of an enclosure (6 x 3 x 3 cm3) with a rigid probe (1 cm diameter, 9 cm length) that weighs less than 125 g with all the system components, which, compared to our previous system, is significantly smaller and lighter, and has improved ergonomics and usability. The enclosure has mounts for a dual axes bi-directional MEMS scanner and a dichroic mirror, and plug-and-play ports for excitation, emission collection and rigid probe optics. The rigid probe used for oral mucosa imaging contains a three-lens imaging system that, compared to our previous system, has: an increased field of view (FOV) (6 x 6 mm2 vs. 16 x 16 mm2), improved lateral resolution (36 μm in the center and 65 μm at the edge, diffraction-limited performance across a central ± 5.5 mm field), and an extended working distance (10 mm vs. 40 mm). A 375 nm CW laser is used as the excitation source, and fluorescence emission is spectrally divided into three emission bands (405±10 nm, 440±20 nm, and 525±25 nm) targeting collagen, NADH, and FAD, which are relevant for early detection of oral cancer. Fluorescence emission is then detected by three APDs and further processed onboard an FPGA. Our clinically compatible handheld endoscope allows for noninvasive and fast in situ clinical metabolic imaging of the oral mucosa.
A compact handheld system for simultaneous multispectral frequency-domain (FD) FLIM imaging is presented. The handheld endoscope consists of a handheld enclosure (10 X 5 X 3 cm3 in volume) with a rigid probe (0.8 cm diameter, 12 cm length). The customized enclosure holds the MEMS scanner and a dichroic mirror whose tip and tilt angle can be adjusted. The rigid probe includes four achromatic lenses (f = 30mm). Two of the four lenses form a relay system to extend the length of the probe. The most distal lens works as an objective to focus the light onto the sample. An additional lens is placed in the intermediate image plane of the relay system to increases the imaging FOV from ~3.6 mm to ~5 mm. The excitation for the handheld FD FLIM system is a 375nm CW diode laser modulated at 1.25MHz and 20MHz. The fluorescence emission is spectrally divided in three emission bands (405/40nm, 440/40nm, and 525/50nm) and detected by three independent APDs. The multispectral signals are further digitized and processed by a FPGA. Phase shift and decreased magnitude are computed at 1.25MHz, 20MHz and its harmonic frequencies (40MHz, 60MHz, 80MHz and 100MHz) via Discrete Fourier Transform (DFT) for lifetime estimation. The current pixel rate is 12.5 KHz which is limited by the SNR. The system is validated by imaging standard fluorescent dyes and human healthy oral mucosa in vivo. This handheld FLIM system offers a cost reduction of at least 50% compared to previous time-domain implementations.
Atherosclerosis is a progressive asymptomatic disease that has the highest rate of death and morbidity in the United States. High macrophage infiltration and thin cap fibroatheromas are known to be the precursor lesions of plaque rupture. Lipid-laden macrophages called foam cells are formed by the uptake of lipids within the plaque. These foam cells eventually die forming a necrotic core. Ruptured plaques are characterized by a necrotic core with an overlying thin-ruptured cap highly infiltrated by macrophages. Imaging modalities capable of identifying macrophage clusters in atherosclerotic plaques could be used for plaque vulnerability assessment. In this study, Multispectral Fluorescence Lifetime Imaging (FLIM) is used to retrieve information of biochemical markers present in atherosclerotic tissue. Here, we present a computational methodology that makes use of FLIM-based biochemical plaque features in order to identify macrophage/foam cells in atherosclerotic plaques. In the proposed methodology, the FLIM lifetime map obtained from a spectral channel of 494 ± 20.5 nm provides information about the accumulation of macrophages, which produce long lifetimes (>6 ns). This methodology was validated against histopathological assessment (CD68 staining specific for macrophages) in terms of statistical correlation, a 10-fold cross validation (sensitivity = 88.45%; specificity= 91.21%), and receiver operating characteristic (ROC AUC = 0.91) analyses.
We report the design and validation of a novel ball lens-based imaging catheter based on dual-clad fiber for frequency-domain fluorescence lifetime imaging microscopy (FLIM) of atherosclerosis. The illumination and collection performance of the catheter endoscope was modeled and optimized with the ray-tracing program Zemax. A 1.55-m-long dual-clad fiber was spliced with a short length of coreless fiber, and then heated and polished to fabricate the angled ball lens. The fiber endoscope was enclosed in a torque cable and had a diameter of 2Fr. The catheter was affixed to a custom built lensless rotary joint which had high coupling efficiency (>90%) over a broad spectral range, accommodating both the UV (375 nm) excitation and the broad fluorescence emission (385 nm - 600 nm). The computer controlled rotary joint and translation stage for pullback imaging can routinely achieve rotation rates of 6000 rpm. The endoscope has two configurations depending on different illumination methods. Lateral resolution was improved more than twice by illuminating the core instead of the inner cladding, while SNR decreased due to higher attenuation of the core. Experiments conducted using a resolution target demonstrate a lateral resolution 80 μm at 1 mm lens-to-sample distance. Experiments conducted using a fluorescein phantom and a segment of ex vivo human coronary artery demonstrate the system performance for fluorescence lifetime imaging with pullback velocities of >10mm/s. This study demonstrates the novel design of a ball lens-based FLIM catheter system to record fluorescence in a continuous helical scanning method across broad-spectral emission bands.
We have shown in an ex vivo human coronary artery study that the biochemical information derived from FLIM interpreted in the context of the morphological information from OCT enables a detailed classification of human coronary plaques associated with atherosclerosis. The identification of lipid-rich plaques prone to erosion or rupture and associated with sudden coronary events can impact current clinical practice as well as future development of targeted therapies for “vulnerable” plaques. In order to realize clinical translation of intravascular OCT/FLIM we have had to develop several key technologies. A multimodal catheter endoscope capable of delivering near UV excitation for FLIM and shortwave IR for OCT has been fabricated using a ball lens design with a double clad fiber. The OCT illumination and the FLIM excitation propogate down the inner core while the large outer multimode core captures the fluorescence emission. To enable intravascular pullback imaging with this endoscope we have developed an ultra-wideband fiber optic rotary joint using the same double clad fiber. The rotary joint is based on a lensless design where two cleaved fibers, one fixed and one rotating, are brought into close proximity but not touching. Using water as the lubricant enabled operation over the near UV-shortwave IR range. Transmission over this bandwidth has been measured to be near 100% at rotational frequencies up to 147 Hz. The entire system has been assembled and placed on a mobile cart suitable for cath lab based imaging. System development, performance, and early ex vivo imaging results will be discussed.
Most studies evaluating the potential of optical coherence tomography (OCT) for the diagnosis of oral cancer are based on visual assessment of OCT B-scans by trained experts. Human interpretation of the large pool of data acquired by modern high-speed OCT systems, however, can be cumbersome and extremely time consuming. Development of image analysis methods for automated and quantitative OCT image analysis could therefore facilitate the evaluation of such a large volume of data. We report automated algorithms for quantifying structural features that are associated with the malignant transformation of the oral epithelium based on image processing of OCT data. The features extracted from the OCT images were used to design a statistical classification model to perform the automated tissue diagnosis. The sensitivity and specificity of distinguishing malignant lesions from benign lesions were found to be 90.2% and 76.3%, respectively. The results of the study demonstrate the feasibility of using quantitative image analysis algorithms for extracting morphological features from OCT images to perform the automated diagnosis of oral malignancies in a hamster cheek pouch model.
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