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BACKGROUND AND OBJECTIVE: Cilia-driven mucociliary clearance is an important self-defense mechanism of great clinical importance in pulmonary research. Conventional light microscopy possesses the capability to visualize individual cilia and its beating pattern but lacks the throughput to assess the global ciliary activities and flow dynamics. Optical coherence tomography (OCT), which provides depth-resolved cross-sectional images, was recently introduced to this area. MATERIALS AND METHODS: Fourteen de-identified human tracheobronchial tissues are directly imaged by two OCT systems: one system centered at 1,300 nm with 6.5 µm axial resolution and 15 µm lateral resolution, and the other centered at 800 nm with 2.72 µm axial resolution and 5.52 µm lateral resolution. Speckle variance images are obtained in both cross-sectional and volumetric modes. After imaging, sample blocks are sliced along the registered OCT imaging plane and processed with hematoxylin and eosin (H&E) stain for comparison. Quantitative flow analysis is performed by tracking the path-lines of microspheres in a fixed cross-section. Both the flow rate and flow direction are characterized. RESULTS: The speckle variance images successfully segment the ciliated epithelial tissue from its cilia-denuded counterpart, and the results are validated by corresponding H&E stained sections. A further temporal frequency analysis is performed to extract the ciliary beat frequency (CBF) at cilia cites. By adding polyester microspheres as contrast agents, we demonstrate ex vivo imaging of the flow induced by cilia activities of human tracheobronchial samples. CONCLUSION: This manuscript presents an ex vivo study on human tracheobronchial ciliated epithelium and its induced mucous flow by using OCT. Within OCT images, intact ciliated epithelium is effectively distinguished from cilia-denuded counterpart, which serves as a negative control, by examining the speckle variance images. The cilia beat frequency is extracted by temporal frequency analysis. The flow rate, flow direction, and particle throughput are obtained through particle tracking. The availability of these quantitative parameters provides us with a powerful tool that will be useful for studying the physiology, pathophysiology and the effectiveness of therapies on epithelial cilia function, as well as serve as a diagnostic tool for diseases associated with ciliary dysmotility. Lasers Surg. Med. 49:270-279, 2017. © 2017 Wiley Periodicals, Inc.
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Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Sistema Respiratório/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Biópsia por Agulha , Cílios/patologia , Epitélio/diagnóstico por imagem , Epitélio/patologia , Humanos , Imageamento Tridimensional/métodos , Imuno-Histoquímica , Técnicas In Vitro , Depuração Mucociliar/fisiologia , Sistema Respiratório/patologia , Estudos de Amostragem , Sensibilidade e Especificidade , Técnicas de Cultura de TecidosRESUMO
BACKGROUND AND OBJECTIVE: The effect of photodynamic therapy (PDT) is dependent on the localization of photosensitizer in the treatment volume at the time of illumination. Investigation of photosensitizer pharmacokinetics in and around the treatment volume aids in determining the optimal drug light interval for PDT. MATERIALS AND METHODS: In this paper we have investigated the distribution of the photosensitizers chlorin e6 and Bremachlorin in the oral squamous cell carcinoma cell-line OSC19-Luc-Gfp in a tongue tumor, tumor boundary, invasive tumor boundary, and normal tongue tissue by the use of confocal microscopy of frozen sections. Tongues were harvested at t = [3, 4.5, 6, 24, 48] hours after injection. RESULTS: Both photosensitizers showed a decreasing fluorescence with increasing incubation time, and at all time points higher fluorescence was measured in tumor boundary than in tumor itself. For short incubation times, a higher fluorescence intensity was observed in the invasive tumor border and normal tissue compared to tumor tissue. Bremachlorin showed a small increase in tumor to normal ratio at 24 and 48 hours incubation time. Ce6 was undetectable at 48 hours. We did not find a correlation between photosensitizer localization and the presence of vasculature. CONCLUSION: The modest tumor/tumor boundary to normal selectivity of between 1.2 and 2.5 exhibited by Bremachlorin 24 and 48 hours after administration may allow selective targeting of tongue tumors. Further studies investigating the relationship between Bremachlorin concentration and therapeutic efficacy PDT with long incubation times are warranted.
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Carcinoma de Células Escamosas/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacocinética , Porfirinas/farmacocinética , Neoplasias da Língua/tratamento farmacológico , Animais , Clorofilídeos , Combinação de Medicamentos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/uso terapêutico , Distribuição AleatóriaRESUMO
This study utilizes experimentally validated Monte Carlo simulations to identify a mathematical formulation of the reflectance intensity collected by a single fiber probe expressed in terms of the reduced scattering coefficient (µs'), fiber diameter d(fiber), and a property of the first two moments of the scattering phase function (γ). This model is then utilized to accurately obtain wavelength-dependent estimates of µs'(λ) and γ(λ) from multiple single fiber spectral measurements of a turbid medium obtained with different diameters. This method returns accurate descriptions (mean residual <3%) of both µs' and γ across the biologically relevant range.
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Método de Monte Carlo , Fenômenos Ópticos , Análise Espectral , Absorção , FótonsRESUMO
HYPOTHESIS: Intracochlear pressure measurements in one cochlear scala are sufficient as reference to determine the output of an active middle ear implant (AMEI) in terms of "equivalent sound pressure level" (eqSPL). BACKGROUND: The performance of AMEIs is commonly calculated from stapes velocities or intracochlear pressure differences (PDiff). However, there are scenarios where measuring stapes velocities or PDiff may not be feasible, for example when access to the stapes or one of the scalae is impractical. METHODS: We reanalyzed data from a previous study of our group that investigated the performance of an AMEI coupled to the incus in 10 human temporal bones. We calculated eqSPL based on stapes velocities according to the ASTM standard F2504-05 and based on intracochlear pressures in scala vestibuli, scala tympani, and PDiff. RESULTS: The AMEI produced eqSPL of â¼100 to 120âdB at 1 Vrms. No significant differences were found between using intracochlear pressures in scala vestibuli, scala tympani, or PDiff as a reference. The actuator performance calculated from stapes displacements predicted slightly higher eqSPLs at frequencies above 1000âHz, but these differences were not statistically significant. CONCLUSION: Our findings show that pressure measurements in one scala can be sufficient to evaluate the performance of an AMEI coupled to the incus. The method may be extended to other stimulation modalities of the middle ear or cochlea when access to the stapes or one of the scalae is not possible.
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Cóclea , Som , Orelha Média , Humanos , Rampa do Tímpano , Rampa do VestíbuloRESUMO
INTRODUCTION: This study aims to investigate the performance of an active middle ear implant actuator for various coupling configurations. Actuator output and conductive losses were measured, and the stability of coupling was evaluated by challenging the link between actuator and ossicles through pressure events in magnitudes that occur in daily life. METHODS: Actuator coupling efficiency and the occurrence of conductive losses were measured in 10 temporal bones through laser Doppler vibrometry on the stapes footplate for various coupling types (incus short process with and without laser hole, incus long process, stapes head). To test the stability of coupling, actuator output was measured before and after daily-life pressure events that were simulated; Valsalva maneuvers (500 cycles of -40 to +60 hPa) and jumping into a swimming pool and diving 3 m deep (a step change of 300 hPa). RESULTS: Actuator output was similarly high for all types of coupling to the incus (short process and long process) and most efficient for coupling to the stapes head. Conductive losses occurred in two temporal bones (TBs) for short process coupling but for seven TBs for coupling to the incus long process. All coupling types were stable and did not lose efficiency after pressure events in the low-frequency range (<1âkHz). Losses in output of 13 to 24âdB were observed in one TB at frequencies from 3 to 6âkHz. CONCLUSION: Actuator output was similarly high for all types of coupling to the incus but coupling to the incus long process led to a higher occurrence of conductive losses. All three coupling configurations connected the actuator securely to the ossicular chain, under variations of barometric pressure that can be expected in daily life.
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Prótese Ossicular , Orelha Média/cirurgia , Humanos , Bigorna/cirurgia , Estribo , Osso Temporal/cirurgia , VibraçãoRESUMO
INTRODUCTION: The desired outcome of the implantation of active middle ear implants is maximum coupling efficiency and a minimum of conductive loss. It has not been investigated yet, which loading forces are applied during the process of coupling, which forces lead to an optimum actuator performance and which forces occur when manufacturer guidelines for coupling are followed. METHODS: Actuator output was measured by laser Doppler vibrometry of stapes motion while the actuator was advanced in 20âµm steps against the incus body while monitoring static contact force. The occurrence of conductive losses was investigated by measuring changes in stapes motion in response to acoustic stimulation for each step of actuator displacement. Additionally, the electrical impedance of the actuator was measured over the whole frequency range at each actuator position. RESULTS: Highest coupling efficiency was achieved at forces above 10âmN. Below 1âmN no efficient coupling could be achieved. At 30âmN loading force, which is typical when coupling according to manufacturer guidelines, conductive losses of more than 5âdB were observed in one out of nine TBs. The electrical impedance of the actuator showed a prominent resonance peak which vanished after coupling. CONCLUSION: A minimum coupling force of 10âmN is required for efficient coupling of the actuator to the incus. In most cases, coupling forces up to 100âmN will not result in clinically relevant conductive losses. The electrical impedance is a simple and reliable metric to indicate contact.
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Condução Óssea/fisiologia , Orelha Média/fisiologia , Prótese Ossicular , Osso Temporal/fisiologia , Estimulação Acústica , Impedância Elétrica , Humanos , Bigorna/fisiologia , Estribo/fisiologia , VibraçãoRESUMO
Optical coherence tomography (OCT) is an emerging technology for in vivo airway and lung imaging. However, OCT lacks sensitivity to the metabolic changes caused by inflammation, which drives chronic respiratory diseases such as asthma and chronic obstructive pulmonary disorder. Redox imaging (RI) is a label-free technique that uses the autofluorescence of the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide (FAD) to probe cellular metabolism and could provide complimentary information to OCT for airway and lung imaging. We demonstrate OCT and RI of respiratory ciliated epithelial function in ex vivo mouse tracheae. We applied RI to measure cellular metabolism via the redox ratio [intensity of NAD(P)H divided by FAD] and particle tracking velocimetry OCT to quantify cilia-driven fluid flow. To model mitochondrial dysfunction, a key aspect of the inflammatory process, cyanide was used to inhibit oxidative metabolism and reduce ciliary motility. Cyanide exposure over 20 min significantly increased the redox ratio and reversed cilia-driven fluid flow. We propose that RI provides complementary information to OCT to assess inflammation in the airway and lungs.
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Cílios/patologia , Oxirredução , Mucosa Respiratória/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Traqueia/diagnóstico por imagem , Animais , Cianetos/química , Feminino , Inflamação , Pulmão/diagnóstico por imagem , Camundongos , Microscopia de Fluorescência/métodos , Estresse Oxidativo , Reologia/métodosRESUMO
Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. Disruption of ciliary flow can lead to respiratory infections. Multiple factors, from drugs to disease can cause an alteration in ciliary flow. However, less attention has been given to injury of the ciliated epithelium. In this study, we show how optical coherence tomography (OCT) can be used to investigate injury to the ciliated epithelium in a multi-contrast setting. We used particle tracking velocimetry (PTV-OCT) to investigate the cilia-driven flow field and 3D speckle variance imaging to investigate size and extent of injury caused to the skin of Xenopus embryos. Two types of injuries are investigated, focal injury caused by mechanical damage and diffuse injury by a calcium chloride shock. We additionally investigate injury and regeneration of cilia to calcium chloride on ex vivo mouse trachea. This work describes how OCT can be used as a tool to investigate injury and regeneration in ciliated epithelium.
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Cílios/fisiologia , Epitélio/fisiopatologia , Pele/fisiopatologia , Traqueia/fisiopatologia , Animais , Epitélio/embriologia , Epitélio/lesões , Camundongos Endogâmicos C57BL , Regeneração , Reologia , Pele/embriologia , Pele/lesões , Tomografia de Coerência Óptica , Traqueia/diagnóstico por imagem , Traqueia/lesões , XenopusRESUMO
We present a new OCT method for flow speed quantification and directional velocimetry: particle streak velocimetry-OCT (PSV-OCT). PSV-OCT generates two-dimensional, 2.5-vector component (vx ,|vy |,vz ) maps of microscale flow velocity fields. Knowledge of 2.5-vector components also enables the estimation of total flow speed. The enabling insight behind PSV-OCT is that tracer particles in sparsely-seeded fluid flow trace out streaks in (x,z,t)-space. The streak orientations in x-t and z-t yield vx and vz , respectively. The in-plane (x-z plane) residence time yields the out-of-plane speed |vy |. Vector component values are generated by fitting streaks to a model of image formation that incorporates equations of motion in 3D space. We demonstrate cross-sectional estimation of (vx ,|vy |,vz ) in two important animal models in ciliary biology: Xenopus embryos (tadpoles) and mouse trachea.
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[This corrects the article on p. 1590 in vol. 7.].
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Cilia-driven fluid flow is a critical yet poorly understood aspect of pulmonary physiology. Here, we demonstrate that optical coherence tomography-based particle tracking velocimetry can be used to quantify subtle variability in cilia-driven flow performance in Xenopus, an important animal model of ciliary biology. Changes in flow performance were quantified in the setting of normal development, as well as in response to three types of perturbations: mechanical (increased fluid viscosity), pharmacological (disrupted serotonin signaling), and genetic (diminished ciliary motor protein expression). Of note, we demonstrate decreased flow secondary to gene knockdown of kif3a, a protein involved in ciliogenesis, as well as a dose-response decrease in flow secondary to knockdown of dnah9, an important ciliary motor protein.
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Cílios/fisiologia , Depuração Mucociliar/fisiologia , Tomografia de Coerência Óptica/métodos , Animais , Fenótipo , Serotonina/metabolismo , Transdução de Sinais , XenopusRESUMO
Microscale quantification of cilia-driven fluid flow is an emerging area in medical physiology, including pulmonary and central nervous system physiology. Cilia-driven fluid flow is most completely described by a three-dimensional, three-component (3D3C) vector field. Here, we generate 3D3C velocimetry measurements by synthesizing higher dimensional data from lower dimensional measurements obtained using two separate optical coherence tomography (OCT)-based approaches: digital particle image velocimetry (DPIV) and dynamic light scattering (DLS)-OCT. Building on previous work, we first demonstrate directional DLS-OCT for 1D2C velocimetry measurements in the sub-1 mm/s regime (sub-2.5 inch/minute regime) of cilia-driven fluid flow in Xenopus epithelium, an important animal model of the ciliated respiratory tract. We then extend our analysis toward 3D3C measurements in Xenopus using both DLS-OCT and DPIV. We demonstrate the use of DPIV-based approaches towards flow imaging of Xenopus cerebrospinal fluid and mouse trachea, two other important ciliary systems. Both of these flows typically fall in the sub-100 µm/s regime (sub-0.25 inch/minute regime). Lastly, we develop a framework for optimizing the signal-to-noise ratio of 3D3C flow velocity measurements synthesized from 2D2C measures in non-orthogonal planes. In all, 3D3C OCT-based velocimetry has the potential to comprehensively characterize the flow performance of biological ciliated surfaces.
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Oxygen supplementation [hyperoxia, increased fraction of inspired oxygen (FiO 2 )] is an indispensable treatment in the intensive care unit for patients in respiratory failure. Like other treatments or drugs, hyperoxia has a risk-benefit profile that guides its clinical use. While hyperoxia is known to damage respiratory epithelium, it is unknown if damage can result in impaired capacity to generate cilia-driven fluid flow. Here, we demonstrate that quantifying cilia-driven fluid flow velocities in the sub-100 µm/s regime (sub-0.25 in./min regime) reveals hyperoxia-mediated damage to the capacity of ciliated respiratory mucosa to generate directional flow. Flow quantification was performed using particle tracking velocimetry optical coherence tomography (PTV-OCT) in ex vivo mouse trachea. The ability of PTV-OCT to detect biomedically relevant flow perturbations in the sub-100 µm/s regime was validated by quantifying temperature- and drug-mediated modulation of flow performance in ex vivo mouse trachea. Overall, PTV-OCT imaging of cilia-driven fluid flow in ex vivo mouse trachea is a powerful and straightforward approach for studying factors that modulate and damage mammalian respiratory ciliary physiology.
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Oxigenoterapia Hiperbárica/efeitos adversos , Muco/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/fisiopatologia , Reologia/métodos , Tomografia de Coerência Óptica/métodos , Animais , Hiperóxia , Interpretação de Imagem Assistida por Computador/métodos , Camundongos , Reprodutibilidade dos Testes , Mucosa Respiratória/lesões , Sensibilidade e EspecificidadeRESUMO
Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.
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Clorofila/análogos & derivados , Fármacos Fotossensibilizantes/química , Animais , Carcinoma de Células Escamosas/patologia , Clorofila/química , Clorofilídeos , Feminino , Fluorescência , Proteínas de Fluorescência Verde , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Neoplasias Bucais/patologia , Distribuição Normal , Óptica e Fotônica , Fotoquimioterapia , Porfirinas/química , Pele/patologia , Espectrometria de Fluorescência , Espectrofotometria , Língua/patologiaRESUMO
We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 µm, and a fluorescence spectrum at an effective diameter of 1000 µm. From these spectra, we measure the absolute absorption coefficient, µ(a), reduced scattering coefficient, µ'(s'), phase function parameter, γ, and intrinsic fluorescence, Qµ(a,x)(f), across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qµ(a,x)(f).