A novel OCT otoscope device is needed for measuring thickness changes of tympanic membrane, a structure that separates outer and middle ear. While the traditional otoscope imaging can only obtain a monocular view, the new otoscope is aimed at obtaining depth perception. This information will be crucial to early detect abnormal ear condition and prevent hearing loss (one third of older adults encounter it). Planar waveguide array structure, presented here, is designed for snapshot imaging spectrometer and full field optical OCT system. 2-photon polymerization (2PP) three dimensional (3D) printing is used to fabricate the structure. The benefits of using 2PP 3D printing include high degree of freedom in design, short manufacture time, and lower cost.
Point-of-care depth resolved 3D imaging of the tympanic membrane and middle ear with OCT, combined with quantitative image analysis, could improve the diagnosis and management of patients in the clinical setting. We imaged the TMs and MEs of 55 patients in a neurotology clinic, using a custom-built hand-held OCT (HHOCT) device. Patients with a diagnosis of TM retraction pockets, perforations, cholesteatomas, and postoperative states were included in this study. Healthy volunteers were also imaged to provide a baseline for quantitative metrics. Images were post processed to perform segmentation of the TM and create thickness maps of the TM, derive mean TM thickness values, and conduct ear symmetry analysis. The normal mean TM thickness was found to be significantly different from every other condition explored. Ear symmetry of healthy subjects was found to be 80% between left and right ears. Quantitative metrics derived from OCT images can be used to characterize TM pathologies and potentially aid in diagnosis and management.
SignificancePathologies within the tympanic membrane (TM) and middle ear (ME) can lead to hearing loss. Imaging tools available in the hearing clinic for diagnosis and management are limited to visual inspection using the classic otoscope. The otoscopic view is limited to the surface of the TM, especially in diseased ears where the TM is opaque. An integrated optical coherence tomography (OCT) otoscope can provide images of the interior of the TM and ME space as well as an otoscope image. This enables the clinicians to correlate the standard otoscopic view with OCT and then use the new information to improve the diagnostic accuracy and management.AimWe aim to develop an OCT otoscope that can easily be used in the hearing clinic and demonstrate the system in the hearing clinic, identifying relevant image features of various pathologies not apparent in the standard otoscopic view.ApproachWe developed a portable OCT otoscope device featuring an improved field of view and form-factor that can be operated solely by the clinician using an integrated foot pedal to control image acquisition. The device was used to image patients at a hearing clinic.ResultsThe field of view of the imaging system was improved to a 7.4 mm diameter, with lateral and axial resolutions of 38 μm and 33.4 μm, respectively. We developed algorithms to resample the images in Cartesian coordinates after collection in spherical polar coordinates and correct the image aberration. We imaged over 100 patients in the hearing clinic at USC Keck Hospital. Here, we identify some of the pathological features evident in the OCT images and highlight cases in which the OCT image provided clinically relevant information that was not available from traditional otoscopic imaging.ConclusionsThe developed OCT otoscope can readily fit into the hearing clinic workflow and provide new relevant information for diagnosing and managing TM and ME disease.
We have been investigating Optical Coherence Tomography (OCT) as a tool to measure the tympanic membrane and middle ear morphology and vibrational response. The hand-held OCT ostoscope system, based on a 1.3 µm swept laser, is integrated into an endoscopy cart. It has an ~ 8 mm diameter field of view, 38 µm lateral resolution, 35 µm axial resolution, A-line rate of 200 kHz, and subnanometer sensitivity to vibration within the tympanic membrane and middle ear. The system has been used in the clinic at USC Keck Medical Center to image over 100 patients and healthy volunteers. Total imaging time is ~2 minutes, which allows it to easily fit into the clinic workflow, while providing high-resolution images and vibrometric assessment of the tympanic membrane and middle ear. The functional and morphological features visible within these image sets that allow us to readily differentiate among pathologies, will be discussed.
Early diagnosis of ear disorders is difficult in part because patients do not seek out an otologist until they have significant hearing loss. Early detection could happen in the primary care provider’s office, however the sensitivity of an otoscopic examination by a primary care provider during an annual physical is very low. On the other hand, Optical Coherence Tomography (OCT) imaging of the tympanic membrane and middle ear can provide detailed volumetric images of the structure and function. These detailed images can form the basis for an approach for finding early signs of ear disease. Our hypothesis is that asymmetry between the ears could be used for early diagnosis. In order to test this, we need to understand the naturally occurring asymmetry in healthy volunteers. We have collected volumetric OCT images from 8 healthy subjects using a hand-held otoscopic OCT system. As part of a registration algorithm, we crop and down sample the data before finding the transformation matrix that registers the volumes. This matrix is then used to register the original volumes. Then the quantitative analysis of the symmetry between the left and right ears was applied through the similarity coefficient and overall, the left and right ears similarity of 8 healthy subjects has a mean of 0.7892, and a standard deviation of 0.0186. From a scientific perspective, this is the first quantitative measure of how symmetric the right and left ears are in humans. From a diagnostic perspective, this approach could provide a simple method to find early signs of ear disease.
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.
Optical coherence tomography (OCT) of the human cochlea has potential to reveal pathophysiological details of hearing disorders and cochlear function via vibrometry and angiography. However, the ability of 1.3μm OCT to image the detailed microanatomy inside the cochlea is limited by light scattering in the tympanic membrane and otic capsule. Since light scattering in biological tissues is reduced at longer wavelengths, we investigated the use of a 1.7μm swept-source laser for OCT imaging of an ex-vivo human cochlea to compare with 1.3μm OCT imaging. We found that 1.7μm OCT could provide sharper details and greater contrast inside the cochlea compared to 1.3μm OCT due to reduced multiple scattering from the bony otic capsule. These results support the continued development of 1.7μm OCT for cochlear imaging.
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.
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.
Optical coherence tomography (OCT) has been shown to provide detailed images of the morphology and vibratory response in the living cochlea. As a part of the cochlea, the organ of Corti (OC) has a complex tissue structure including three rows of outer hair cells which act to amplify sound, supporting cells and one row of inner hair cells which transduce sound-induced vibrations into electrical signals. Unfortunately, OCT images of the OC have relatively low contrast, in spite of the fact that the microstructures have very different function and morphology. That fact has led us to explore alternative approaches to extracting contrast from these OCT images. In this paper, we propose a contrast-enhanced method based on spatial frequency to identify structures within the cochlea, including the OC. In total, 15 mice have been imaged with our customed OCT system and analyzed. A two-dimension spatial frequency analysis was performed over subregions of the images, using a sliding window. Then the power spectral density was fit to a 2-D Gaussian. Finally, we extracted several Gaussian fitting coefficients and constructed a coefficients map to enhance the visualization of the cochlea and identify structures within the OC. This method improves our ability to identify specific microstructures within the cochlea and ultimately map the functional vibratory response to these microstructures. Application of this approach can elucidate the micromechanical function of the cochlea.
We present a flexible catheter endoscope using a 3D printed miniature side-viewing reflective lens for OCT imaging. A reflective lens with a depth of field (DOF) of 3mm and a maximum spot size of 80 microns was first designed and optimized in OpticStudio (ZEMAX). The structure of the lens and the fiber mating part of the optic was then constructed using 2-photon polymerization technique, and a gold reflective coating was added as the final step for the reflective lens. The reflective lens was then glued to cleaved optical fiber, and the assembly was covered by a torque coil to allow rotation of the catheter endoscope. This endoscope was used with a fiber-optic rotary joint and custom-built Mach-Zehnder interferometer to acquire OCT images.
SignificanceThe vocal folds are critically important structures within the larynx which serve the essential functions of supporting the airway, preventing aspiration, and phonation. The vocal fold mucosa has a unique multilayered architecture whose layers have discrete viscoelastic properties facilitating sound production. Perturbations in these properties lead to voice loss. Currently, vocal fold pliability is inferred clinically using laryngeal videostroboscopy and no tools are available for in vivo objective assessment.AimThe main objective of the present study is to evaluate viability of Brillouin microspectroscopy for differentiating vocal folds’ mechanical properties against surrounding tissues.ApproachWe used Brillouin microspectroscopy as an emerging optical imaging modality capable of providing information about local viscoelastic properties of tissues in noninvasive and remote manner.ResultsBrillouin measurements of the porcine larynx vocal folds were performed. Elasticity-driven Brillouin spectral shifts were recorded and analyzed. Elastic properties, as assessed by Brillouin spectroscopy, strongly correlate with those acquired using classical elasticity measurements.ConclusionsThese results demonstrate the feasibility of Brillouin spectroscopy for vocal fold imaging. With more extensive research, this technique may provide noninvasive objective assessment of vocal fold mucosal pliability toward objective diagnoses and more targeted treatments.
Cross-sectional 4-D imaging of vocal fold morphology and function is desirable for accurate diagnosis of many vocal fold pathologies, which occur throughout the epithelial layer and alter the mucosal wave. Clinical videoendoscopy provides qualitative diagnostic information but remains limited to surface visualization of layered vocal fold structure and two-dimensional mucosal vibration. While OCT has been investigated to address the shortcomings of standard 2-D endoscopy, challenges remain in reconstruction of the 4-D mucosal wave over the entire vocal fold structure. To address these challenges, we have developed a fast-scanning OCT laryngoscope to enable asynchronous Nyquist sampling of the human voice fundamental frequency range (and its harmonics, up to 1 kHz). We present a new algorithm for reconstruction of the 4-D vocal fold dynamics during phonation using OCT volume data of the entire anterior-posterior vocal fold structure. Reconstruction of the vibration of a vocal fold phantom confirmed feasibility of the algorithm and preliminary reconstruction of the in vivo vocal fold glottal cycle is presented. This work represents the first cross-sectional Nyquist sampling of the in vivo human mucosal wave using an OCT system with hardware capable of encompassing the human fundamental frequency range (i.e., 90-260 Hz). The developed OCT laryngoscope and algorithm will enable volumetric representations of vocal fold dynamics in the clinic and development of quantitative metrics for diagnostic and interventional guidance.
Custom fiber arrays can be used to encode 3-dimensional data for snapshot imaging techniques like imaging spectrometry or volumetric spectral domain OCT. This is achieved if array’s input is dense, while its output creates void spaces for spectral information. Here we present fiber arrays with entirely automatic development process based on 2-Photon Polymerization (2PP) additive manufacturing using Nanoscribe GmbH Quantum X system. Specifically, We developed two types of array prototypes: 10x10 to 1x100 and 20x20 - dense fiber spacing (1-2 microns fiber gap) to 20x20 – sparse fiber spacing (30-40 microns fiber gap). Fiber arrays were incorporated into prism-based imaging spectrometer system to demonstrate proof of concept spectral imaging experiments.
2-photon lithography enables custom fabrication of optical waveguides at a sub-micron resolution and millimeter scale. Custom optical fiber architecture is a powerful component for development of fiber coupler systems and advancement of fiber based imaging technology. Here we present an exploration of methods for 2-photon fabrication of optical fibers and fiber coupler systems.
OCT has been exploited extensively in studies of cochlear mechanics due to its ability to non-invasively measure vibrations of various cochlear structures. A key limitation has been the ability to measure only in one dimension, along the optical axis. However, recent evidence suggests the organ of Corti has complex, three-dimensional vibratory micromechanics. Therefore, a 3D-OCT vibrometry system has been developed to measure the vector of motion within the cochlea and hopefully shed light on the underlying mechanics that lead to cochlear amplification and the exquisite sensitivity and frequency selectivity of mammalian hearing. The system uses three independent sample arms (channels) with a single reference arm to acquire vibrations, exploiting the long coherence length of the laser to depth encode the three channels. The system was first validated using a piezoelectric actuator. This yielded an RMS error of ≤0.3° in both polar angles with expected sensitivity to vibrational amplitude. Preliminary measurements in the cochlea of a live mouse demonstrate direction-dependent differences in vibratory responses.
We describe the optimization and application of a multi-window approach for improved resolution, side-lobe suppression, and phase sensitivity. Using the Hann window as a reference, we show that 10 windows are sufficient to achieve 42% resolution improvement, -31 dB side-lobe intensity, and a 20% improvement in phase sensitivity. We explored the benefits of this windowing technique for OCT imaging using a prototype narrow-band laser, OCT vibrometry, and Doppler OCT for angiography. Experimental data are in good agreement with simulation. We believe it will be possible using this optimized approach to achieve real-time processing and display, despite the added computational load.
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.
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.
The exquisite phase sensitivity of Optical Coherence Tomography (OCT) has enabled the development of sensitive spatially resolved vibrometers. Using this technology, it is possible to measure vibratory response in live animals and humans down to a few picometers in amplitude. We are employing this technology in animal models to probe the mechanics of the cochlea, the part of the inner ear responsible for hearing. OCT based Vibrometry (OCTV) can image through the bone to capture morphological and functional images of the soft tissues within the cochlea. These measurements have led to new understandings of the mechanical processing of sound. Through recent advances it is now possible to make completely noninvasive OCTV measurements in awake mice. This has enabled studies including feedback from the efferent nerves that are shutdown in an anesthetized animal. These and related optical technologies are making a significant impact on our understanding of the mechanics of hearing.
A principal tool for the visual inspection of the middle ear in the hearing clinic is the surgical stereo-microscope. We have developed a compact accessory for the surgical microscope that enables volumetric optical coherence tomography (OCT) imaging of the middle ear as well as functional vibratory imaging with subnanometer sensitivity. The sensitivity to vibration is achieved by careful engineering of the microscope attachment and frequency-domain processing. The microscope attachment integrates the entire OCT interferometer onto a custom aluminum base that mounts directly to the accessory area at the foot of most surgical microscopes. This approach is effective at removing high-frequency phase-noise, thus enabling near shot-noise limited sensitivity above ~2 kHz even though the OCT system is suspended above the patient by a boom arm. We analyze the vibratory response in the frequency domain, hence our ability to measure vibrations in the tympanic membrane and ossicles is near the shot-noise limit. As a demonstration of this system we have recorded in vivo volumetric images of a healthy human patient as well as the vibratory response at the tympanic membrane down to the hearing threshold. We also show that it is possible to use time averaging to drive the noise floor down below 2 pm which allowed us to make the first measure of distortion product otoacoustic emissions (DPOAE) using OCT. Finally, the system can easily be taken on and off of the surgical microscope and when in use does not impinge on the normal view through the surgical microscope.
Atherosclerosis, a condition in which plaque accumulates on the inner wall of arteries, is often recognized as a precursor to cardiovascular diseases (CVDs), the most common causes of death in the US. Optical Coherence Tomography (OCT) is an intravascular optical diagnosis tool, which can be used to obtain high resolution morphological images of atherosclerotic plaque. However, atherosclerotic plaque components, such as macrophages, can be misclassified due to their signal similarities to fibrin accumulations, cholesterol crystals and microcalcifications. To overcome these challenges, we develop a biocompatible contrast agent to enhance molecular imaging of a Pump-Probe OCT (PPOCT) system. Methylene blue (MB) was encapsulated into poly lactic-co-glycolic acid (PLGA) particles by an emulsion/solvent evaporation technique. Fabrication parameters were controlled to synthesize particles with desired properties such as: size, encapsulation efficiency, degradation rate, and particle surface functionalization. The encapsulation of MB protects it from the enzymatic reduction to leuco-methylene blue (92.8 % protection), and reduces the singlet oxygen generation by the excited MB molecules by 78.3%. Likewise, the PLGA shells improve the OCT signal by enhancing the scattering of light. The surface of particles was modified with ligands that can target molecular biomarkers involved in atherosclerotic plaque formation such as vascular cell adhesion molecules (VCAM-1) and apoptotic macrophages. This modification is expected to enhance tissue selectivity, provides detailed information on the local biochemistry and yields visualization of pathological processes. PLGA-based contrast agents were tested in human postmortem artery sections to study particles permeability as a function of particle size and its molecular selectivity.
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.
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.
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.
Diseases that cause hearing loss and/or vertigo in humans such as Meniere’s disease are often studied using animal models. The volume of endolymph within the inner ear varies with these diseases. Here, we used a mouse model of increased endolymph volume, endolymphatic hydrops, to develop a computer-aided objective approach to measure endolymph volume from images collected in vivo using optical coherence tomography. The displacement of Reissner’s membrane from its normal position was measured in cochlear cross sections. We validated our computer-aided measurements with manual measurements and with trained observer labels. This approach allows for computer-aided detection of endolymphatic hydrops in mice, with test performance showing sensitivity of 91% and specificity of 87% using a running average of five measurements. These findings indicate that this approach is accurate and reliable for classifying endolymphatic hydrops and quantifying endolymph volume.
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.
Zebrafish, an auditory specialist among fish, offer analogous auditory structures to vertebrates and is a model for hearing and deafness in vertebrates, including humans. Nevertheless, many questions remain on the basic mechanics of the auditory pathway. Phase-sensitive Optical Coherence Tomography has been proven as valuable technique for functional vibrometric measurements in the murine ear. Such measurements are key to building a complete understanding of auditory mechanics. The application of such techniques in the zebrafish is impeded by the high level of pigmentation, which develops superior to the transverse plane and envelops the auditory system superficially. A zebrafish double mutant for nacre and roy (mitfa-/- ;roya-/- [casper]), which exhibits defects for neural-crest derived melanocytes and iridophores, at all stages of development, is pursued to improve image quality and sensitivity for functional imaging. So far our investigations with the casper mutants have enabled the identification of the specialized hearing organs, fluid-filled canal connecting the ears, and sub-structures of the semicircular canals. In our previous work with wild-type zebrafish, we were only able to identify and observe stimulated vibration of the largest structures, specifically the anterior swim bladder and tripus ossicle, even among small, larval specimen, with fully developed inner ears. In conclusion, this genetic mutant will enable the study of the dynamics of the zebrafish ear from the early larval stages all the way into adulthood.
Molecular contrast imaging can target specific molecules or receptors to provide detailed information on the local biochemistry and yield enhanced visualization of pathological and physiological processes. When paired with Optical Coherence Tomography (OCT) it can simultaneously supply the morphological context for the molecular information. We recently demonstrated in vivo molecular contrast imaging of methylene blue (MB) using a 663 nm diode laser as a pump in a Pump-Probe OCT (PPOCT) system. The simple addition of a dichroic mirror in the sample arm enabled PPOCT imaging with a typical 830-nm band spectral-domain OCT system. Here we report on the development of a microencapsulated MB contrast agent. The poly lactic-co-glycolic acid (PLGA) microspheres loaded with MB offer several advantages over bare MB. The microsphere encapsulation improves the PPOCT signal both by enhancing the scattering and preventing the reduction of MB to leucomethylene blue. The surface of the microsphere can readily be functionalized to enable active targeting of the contrast agent without modifying the excited state dynamics of MB that enable PPOCT imaging. Both MB and PLGA are used clinically. PLGA is FDA approved and used in drug delivery and tissue engineering applications. 2.5 μm diameter microspheres were synthesized with an inner core containing 0.01% (w/v) aqueous MB. As an initial demonstration the MB microspheres were imaged in a 100 μm diameter capillary tube submerged in a 1% intralipid emulsion.
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.
KEYWORDS: Field programmable gate arrays, Demodulation, Clocks, Signal processing, Image processing, Transducers, Photoacoustic microscopy, Heart, Real time imaging, Imaging systems
The ability to collect data in real time is important in all biological imaging modalities that aim to image dynamic
processes. Photoacoustic Microscopy (PAM) is a rapidly growing biomedical imaging technique that is often used to
image microvasculature and melanoma, and is capable of fully rendering three-dimensional images. However, due to the
bi-polar nature of the PAM signal, post processing through demodulation is required to accurately display morphological
data. Typically, demodulation requires post processing of the data, limiting its use in real-time applications. This results
in many PAM systems displaying data through maximum amplitude projection (MAP) images, completely ignoring the
axial dimension of their scans and throwing away useful data. We overcome this processing limit by utilizing a
configurable integrated circuit known as a Field Programmable Gate Array (FPGA). The FPGA allows us to perform
quadrature demodulation of the photoacoustic signal as it is being collected. The result is a PAM system capable of
producing continuous, morphologically accurate B-scans and volumes at a rate limited only by the repetition rate of the
laser. This allows us to generate accurately rendered volumes at the same speed as MAP images. With a 100 KHz
actively q-switched laser we are able to generate 200 by 200 pixel b-scans at a rate of 500 Hz. The imaging potential of
the system has been demonstrated in volumes of human hair phantoms and chick embryo vasculature. This system is
capable of 50 x 50 x 50 volume stacks processed and displayed at better than video rate.
Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue
with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation
followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and
objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we
present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance
and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12
mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that
our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can
potentially serve as a guide in early oral cancer detection..
Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.
We have recently developed Transient Absorption Ultrasonic Microscopy (TAUM) as an ultrahigh-resolution
photoacoustic microscopy technique. The amplitude of the multiphoton pump-probe interaction is dependent on the
interpulse delay between the pump and probe pulses. Measuring the interpulse delay dependent TAUM amplitude maps
out the ground state recovery time of the chromophore. The ground state recovery time is a molecular signature that may
be used to differentiate multiple chromophores, analogous to fluorescence lifetime. We have used TAUM to measure the
ground state recovery time of Rhodamine 6G to be 3.65 ns, which matches well with known literature values. Whole
blood is also investigated, with measured ground state recovery times of 3.74 ns for oxygenated blood and 7.9 ns for
deoxygenated blood. The distinct difference in lifetimes for the oxidized and reduced forms suggests the feasibility of
subcellular SO2 images maps in future iterations of TAUM.
Several factors are spurring the development of hardware and software to accomplish high-speed
processing for Optical Coherence Tomography (OCT). The two most prevalent architectures incorporate
either an FPGA or a GPU. While GPUs have faster clock-speed the fact an FPGA can be pipelined
makes a direct comparison based simply on system specifications difficult. We have undertaken an effort
to make a direct comparison on the same host and consider the total time from digitization to rendering of
the image. In addition to making quantitative comparisons between the two architectures we hope to
derive useful benchmarks that will inform the design of an optimal high-speed processing system.
Clinical diagnosis of cochlear dysfunction typically remains incomplete due to a lack of proper diagnostic methods.
Medical imaging modalities can only detect gross changes in the cochlea, and non-invasive in vivo cochlear
measurements are scarce. As a result, extensive efforts have been made to adapt optical coherence tomography (OCT)
techniques to analyze and study the cochlea. Herein, we detail the methods for measuring vibration using OCT. We
used spectral domain OCT with ~950 nm as the center wavelength and a bandwidth of ~80 nm. The custom
spectrometer used was based on a high speed line scan camera which is capable of line rates up to 28 kHz. The signal-to-
noise ratio of the system was ~90 dB. The data collection and processing software was written in LabVIEW and
MATLAB. We tested whether streaming directly from the camera, writing the data to multiple hard drives in the RAID-
0 configuration, and processing using the GPU shortened experiment times. We then analyzed the A-line phase noise
over several hundred milliseconds and growth curves from a piezoelectric element. We believe this is the first step
towards a diagnostic device which generates vibration information of cochlear structures.
Optical coherence tomography (OCT) is becoming a popular tool for imaging morphology in the
middle and inner ear. Vibratory measurements of the structures of the ear facilitate better
understanding of the function and limitations of the ear. We have developed an algorithm that
enables a standard spectrometer based OCT system to measure the full spectrum (90 kHz) frequency
response of the mouse ear by incorporating coherently interleaved sampling, increasing the effective
Nyquist rate of the system by a factor of 5+. The algorithm is evaluated by measuring the frequency
response of a mouse tympanic membrane to a pure tone stimulus.
We present chromatic confocal microscopy as a technique to axially scan the sample by spectrally encoding depth
information to avoid mechanical scanning of the lens or sample. We have achieved an 800 μm focal shift over a range of
680-1080 nm using a hyperchromat lens as the imaging lens. A more complex system that incorporates a water
immersion objective to improve axial resolution was built and tested. We determined that increasing objective
magnification decreases chromatic shift while improving axial resolution. Furthermore, collimating after the
hyperchromat at longer wavelengths yields an increase in focal shift.
We have developed a novel, hybrid imaging modality, Transient Absorption Ultrasonic Microscopy
(TAUM), which fuses photoacoustic microscopy with non-linear microscopy. Photoacoustic
microscopy is well known for its ability to image chromophores deep (> 1 mm) in scattering media
with spatial resolutions in the 10s of microns. Non-linear microscopy is well known for its exquisite
spatial resolution in three dimensions. This superior spatial resolution is attributed to the fact that
the collected signal has a non-linear dependence on the light intensity. This dependence confines the
signal to a very small focal volume, producing optically resolved voxels. Transient absorption is a
non-linear process often used to map the excited state lifetimes of molecules exhibiting low
fluorescence quantum efficiency. This sensitivity to non-radiative transitions makes transient
absorption an attractive process to combine with photoacoustic imaging. We have built a prototype
transient absorption ultrasonic microscope, implementing off-axis photoacoustic detection to allow
the use of a high-quality, high numerical aperture objective. This high-quality, commercial lens is
required to provide the tight focusing needed to optimize non-linear effects. We have demonstrated
the increased spatial resolution of TAUM by imaging Rhodamine 6G in a capillary tube. The
capillary cross-section is fully resolved, suggesting an axial resolution of < 10 microns. A 6 MHz
transducer was used in this experiment, which results in an axial resolution of ~ 400 microns when
used in a traditional photoacoustic microscope. Boasting the superior penetration depth and
absorption contrast offered by photoacoustic emission and complemented by spatial resolutions
comparable to confocal microscopy, we believe that Transient Absorption Ultrasonic Microscopy
has excellent potential for producing volumetric images with cellular/subcellular resolution in three
dimensions deep inside living tissue.
Current medical imaging modalities, such as MRI and CT, do not provide high enough resolution to detect many
changes within the cochlea that cause hearing loss. We sought to develop the technique of optical coherence
tomography (OCT) to image the cochlea noninvasively and within its native environment. We used spectral domain
OCT with 950 nm as the center wavelength and a bandwidth of ~100 nm to image freshly excised normal mouse cochlea
at different developmental ages. The OCT system has an axial resolution of ~4 μm (in air) and a lateral resolution of
~10 μm. When we imaged normal adult mouse cochleae through the round window membrane, Reissner's membrane,
the basilar membrane, the tectorial membrane, the spiral ligament, the spiral limbus, and the modiolus could be clearly
identified. When we imaged intact adult cochleae, we were able to image through ~130 μm of bone and tissue to see up
to a depth of ~600 μm, and all of the previously identified structures were still visible. Imaging of early postnatal mice
during the timeline of cochlear development permitted visualization of the expected structural differences from adult
cochleae. Therefore, we conclude that spectral domain OCT is an effective technique for noninvasive imaging of the
murine cochlea.
Photoacoustic microscopy (PAM) is a high-contrast, high-resolution imaging modality used primarily for imaging
hemoglobin and melanin. Important applications include mapping of the microvasculature and melanoma tumor
margins. We have developed a novel photoacoustic microscope design, which substantially simplifies construction by
enabling the use of unmodified commercial optics and ultrasonic transducers. Moreover, the simple design may be
readily incorporated into a standard light microscope, thus providing a familiar imaging platform for clinical researchers.
A proof-of-concept Off-Axis PAM system with a lateral resolution of 26 μm and a modest axial resolution of 410 μm
has been assembled and characterized using tissue samples. We have derived the appropriate equations to describe the
relevant design parameters and verified the equations via measurements made on our prototype Off-Axis PAM system.
A consequence of the simple design is a reduction in axial resolution compared to coaxial designs. The reduction is
inversely proportional to the cosine of the angle between excitation and detection and equal to 15% and 41% for angles
of 30º and 45º, respectively. While resolution is negatively affected by off-axis detection, the ability to measure weak
signals at depth is enhanced. Off-axis detection has an inherent dark-field quality; chromophores excited outside the
numerical aperture of the ultrasonic detector will not be detected. The physical geometry of Off-Axis PAM enables the
placement of the ultrasonic transducer at the minimum distance from the sample with no obstructions between the
sample and transducer. This may prove to be an additional advantage of Off-Axis PAM over designs that incorporate
long working distance ultrasonic transducers and/or require the propagation of the acoustic wave through the laser
excitation optics to achieve co-axial detection.
Diseased states of tissue are accompanied by changes in both the microanatomy and biochemistry. Optical Coherence
Tomography (OCT) is a high resolution imaging technique that allows micron scale high contrast volumetric imaging of
tissue microanatomy to a depth of up to 2 mm. Fluorescence Lifetime Imaging Microscopy (FLIM) provides 2-D maps
of the concentration of fluorescent biomolecules identified with their corresponding lifetime map. The combination of
OCT and FLIM into one imaging system holds promise for identifying diseased states of tissue with improved sensitivity
and specificity. We have developed a combined OCT/FLIM system that capable of simultaneous high-speed coregistered
micro-anatomical and biochemical real-time imaging of tissues. The OCT/FLIM system is capable of a
maximum A-line rate of 59 kHz for OCT and a maximum pixel rate of 30 kHz for FLIM. A 40 nm bandwidth 830 nm
SLED provides 7.6 μm axial resolution for OCT. The OCT sample light and FLIM excitation co-propagate through an
ultra-wide bandwidth achromatic objective lens to provide co-registered images with 15 μm and 100 μm resolution for
OCT and FLIM, respectively. The FLIM subsystem was validated using dyes with well characterized spectral properties.
The combined system has been tested and optimized on human postmortem coronary arteries and in vivo hamster cheek
pouch. Once optimized, real-time data acquisition and processing would be possible with the system. Future applications
of this technology include early detection and diagnosis of oral cancer and the characterization of arterial plaques.
High-resolution optical molecular imaging has become a vital tool for understanding and measuring physiologically
important biometrics on the cellular and subcellular level. In spite of significant recent advances in microscopy,
molecular imaging of most endogenous biomolecular species remains elusive. Directly imaging endogenous
biomolecules without the aid of exogenous tags is highly desirable. We developed pump-probe optical coherence
microscopy (PPOCM) based on our previous success in integrating pump-probe absorption spectroscopy with
optical coherence tomography. A fixed human skin tissue with melanoma was imaged by the PPOCM system. The
preliminary results show that PPOCM can provide better can clear contrast between normal tissue and melanoma
than OCM. This system also can be used to image other chromophores.
Here we report on our current efforts to simultaneously quantify both morphological and biochemical tissue information
by combining optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM). The Fourier domain
OCT module is built around a custom designed high-speed spectrometer (bandwidth of 102 nm, 3 dB rolloff of 1.2 mm,
lines rates of up to 59 kHz). A 40 nm bandwidth SLED centered at 830 nm provided an axial resolution of 7.6 mm for
OCT. The objective lens provided 10 um lateral resolution for OCT and 100 um for FLIM. Lateral OCT and FLIM beam
scanning was accomplished using a set of galvo mirrors. The FLIM module excites and collects the fluorescence decay
signal pixel by pixel coincident with OCT A-line collection. Each 2-D FLIM image has a corresponding coregistered
OCT volume. Fluorescence excitation for FLIM was provided by a solid-state pulse laser (355 nm, 1 ns FWHM, 50 kHz
rep rate). The fluorescence signal was detected with a MCP-PMT coupled to a 1.5 GHz digitizer (250 ps temporal
resolutions). In addition, simultaneous multispectral time-resolved fluorescence detection was achieved by separating the
fluorescence emission in three bands using a series of dichroic mirrors and bandpass filters, and launching each band into
three fibers of different lengths (providing a time delay of 50 ns among bands) focused onto the MCP-PMT. The
resulting OCT/FLIM system is capable of a maximum A-line rate of 59 kHz for OCT and a maximum pixel rate of at
least 30 kHz for FLIM. The multimodality OCT/FLIM imaging system was validated on biological tissue. Future efforts
include evaluating its potential for oral cancer diagnosis and intravascular imaging.
The detailed 3-D mapping of tissue microcirculation, including blood oxygen saturation
and flow, would provide important biometrics needed to understand the cause and
progression of numerous diseases. To that end we have started developing a two-color
Fourier domain Pump-Probe Optical Coherence Tomography (PPOCT) system designed
specifically to image hemoglobin with the eventual goal of measuring blood oxygen
saturation. This system utilizes a two-color pump-probe scheme chosen to maximize the
potential imaging depth by probing in the near IR where the tissue scattering properties are
most favorable and pumping in the visible where the hemoglobin light absorption is most
efficient. A sample consisting of pure hemoglobin placed between two coverslips has been
used for the initial demonstration and to begin the process of optimizing the system.
We present spectral domain phase microscopy (SDPM) as a new tool for measurements at the cellular scale. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity in real time. Our goal was to use SDPM to investigate the mechanical properties of the cytoskeleton of MCF-7 cells. Magnetic tweezers were designed to apply a vertical force to ligand-coated magnetic beads attached to integrin receptors on the cell surfaces. SDPM was used to resolve cell surface motions induced by the applied stresses. The cytoskeletal response to an applied force is shown for both normal cells and those with compromised actin networks due to treatment with Cytochalasin D. The cell response data were fit to several models for cytoskeletal rheology, including one- and two-exponential mechanical models, as well as a power law. Finally, we correlated displacement measurements to physical characteristics of individual cells to better compare properties across many cells, reducing the coefficient of variation of extracted model parameters by up to 50%.
We have undertaken an effort to further develop ground state recovery Pump-Probe Optical Coherence Tomograpy (gsrPPOCT) to specifically target and measure 3-D images of hemoglobin concentration with the goals of mapping tissue vasculature, total hemoglobin, and hemoglobin oxygen saturation. As a first step toward those goals we have measured the gsrPPOCT signal from the hemoglobin in the filament arteries of a zebra danio fish. We have further processed the resulting signal to extract a qualitative map of the hemoglobin concentration. We have also demonstrated the potential to use ground state recovery times to differentiate between two chromophores which may prove to be an effective tool for differentiating between oxy and deoxy hemoglobin.
We present a full-field phase microscopy technique, motivated by swept-source Fourier-domain optical coherence tomography, for quantitative nanoscale two-dimensional profiling of sample surfaces and internal structures. The optical configuration consisted of a common path interferometer, illuminating the sample with a collimated beam and detecting the back-scattered light on a 2D CCD camera. A tunable fiber Fabry Perot filter was used to sweep a narrow band (0.07nm) through the 47nm FWHM bandwidth of a superluminescent diode source. The full field of view was recorded for each discrete wavelength step, generating a spectrally indexed interferometric data cube mapping each pixel to a point on the sample. A three dimensional volume was generated by performing the discrete Fourier transform along the spectral axis. Sub-coherence length variation across a depth slice was obtained by examining the phase of the Fourier transformed data set at the selected depth. The phase stability of the system was measured to be 1.3nm for high SNR surface features. The nanoscale imaging potential of this system was demonstrated by measuring the height of patterned chrome on a USAF resolution target, the location of receptor sites on a DNA assay biochip, and the surface topography of erythrocytes in a blood smear.
We have developed a novel imaging technique, ground state recovery pump-probe optical coherence tomography (gsrPPOCT), which measures the transient absorption and recovery time of the ground state of a target chromophore or chromophores. By imaging the molecular absorption as opposed to the fluorescence of the target contrast agent, gsrPPOCT helps to fill a valuable niche for imaging biologically important molecules which do not fluoresce. The majority of biologically active molecules are non fluorescent. Here we present gsrPPOCT imaging of natural and transfected chromophores in living animal and plant tissues, as well as the first gsrPPOCT measurements of the ground state recovery time of a molecular chromophore, which may be used to deduce the relative concentrations of a mixture of chromophores.
Molecular contrast OCT (MCOCT) is an extension of OCT in which specific molecular species are imaged based on their spectroscopic characteristics. In order to improve the sensitivity and specificity of MCOCT, several techniques have recently been introduced which depend upon coherent detection of inelastically scattered light from molecules of interest in a sample. These techniques include harmonic generation, coherent anti-Stokes Raman scattering, and several different forms of pump-probe spectroscopy. We have developed a theoretical framework to facilitate the comparison of different inelastic scattering-based contrast mechanisms for molecular contrast OCT. This framework is based upon the observation that since the noise floor is defined by the reference arm power in a shot-noise limited heterodyne detection system, the relevant comparison among the techniques is isolated to the available molecular-specific signal power. We have derived the value of the molecular contrast signal power for second harmonic generation OCT (SHOCT) and three different pump-probe OCT (PPOCT) techniques. Motivated by this analysis, we have constructed a preliminary ground state recovery pump-probe OCT system, and demonstrated its performance using rhodamine 6G as the MCOCT contrast agent.
Optical coherence tomography (OCT) provides micrometer scale structural imaging by coherent detection of light backscattered from a sample. The significance of OCT would be greatly enhanced by the capability to measure molecular specific signals. Observation of fluorescent markers is unfortunately not possible using OCT because fluorescence is not a coherent process. Instead, the methods being researched to extend OCT to include molecular contrast are, amongst others: transient absorption (pump-probe imaging), Coherent Anti-Stokes Raman Scattering (CARS), and second harmonic generation. With any of these techniques, the quality of the images obtained is limited by the conversion efficiency of the nonlinear process, which is inevitably much less than 1 and thus has resulted in low SNR and long acquisition times in previously reported work. Recent publications have demonstrated a sensitivity advantage of 20-30dB for spectral domain (SD) techniques in OCT over conventional time domain acquisition. The increased sensitivity of SD OCT systems stands to benefit the small signal powers observed in molecular contrast OCT. We have constructed a prototype spectral domain second harmonic OCT system utilizing a 130 femtosecond Nd:Glass laser and a pair of custom spectrometers for simultaneous acquisition of the fundamental and second harmonic signals. We report a 10dB increase in sensitivity while imaging 100 times faster than in initial reports of second harmonic OCT using time domain systems.
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