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Optical diffraction tomography algorithms typically assume weak scattering, and are therefore not applicable to multiple-scattering objects. A time-gating approach extending the validity of these algorithms is proposed and numerically demonstrated.

Combining different contrast mechanisms to achieve simultaneous multimodal imaging is always desirable but is challenging due to the various optical and hardware requirements for different imaging systems. We developed a multimodal microscopic optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissues. This imaging system integrated photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and confocal fluorescence microscopy in one platform. By taking advantage of the depth resolving capability of OCT, we developed a novel OCT-guided surface contour scanning methodology for dynamic focusing adjustment. We have conducted phantom, in vivo, and ex vivo tests to demonstrate the capability of the multimodal imaging system for providing comprehensive microscopic information of biological tissues. Integrating all the aforementioned imaging modalities with OCT-guided dynamic focusing for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the future.

We present a three beam optical Doppler tomography (ODT) technique suitable for 3-D velocity and flow measurements to evaluate total retinal blood circulation from and to the optic nerve head (ONH). The system consists of three independent ODT channels. Superluminescent diodes with a central wavelength of 840 nm and a spectral bandwidth of 50 nm were used. The sources are coupled to collimators resting in a specially designed mount to ensure a well-defined beam geometry, necessary for the full reconstruction of the three dimensional velocity vector. The reconstruction works without prior knowledge on the vessel geometry, which is normally required for ODT systems with less than three beams. The beams share a common bulk optics Michelson interferometer, while the detection comprises three identical spectrometers with a line scan rate of 50 kHz. 20 eyes of healthy volunteers were imaged with the 3 beam ODT, employing a circular scan pattern around the ONH. The mean total blood flow was calculated for arteries (47.1 ± 2.4 μl/min (mean ± SD)) and veins (47.1 ± 2.7 μl/min μl/min) independently. The two results showed no significant difference (paired t-test, p < 0.96), rendering both equally reliable for total flow measurements. Furthermore the reproducibility of the method was evaluated for the total flow and flow, velocities within each individual vessel of 6 eyes. The average variation for total flow measurements is sufficiently low to detect deviations of ~ 6% indicating high precision of the proposed method.

Optical coherence tomography is a noninvasive optical imaging modality that is rapidly gaining adoption in many clinical applications. OCT is capable of providing high-speed three-dimensional cross-sectional views into biological tissues with micrometer-scale resolutions. In this chapter, we will review the physical principles behind OCT starting early time-domain-based systems and then discuss the development of more recent high-speed Fourier-domain OCT systems. We will then discuss clinical utilization of OCT via two functional developments: Doppler-based OCT for the quantification of particulate flow rates such as blood flow and optical coherence elastography for mapping of tissue mechanical properties. Finally, we will discuss the development of endoscopic-based OCT for in vivo imaging within internal lumens such as the human airway.

In this study, the feasibility of spectral domain optical Doppler tomography for measuring blood flow characteristics in a micro-tube was demonstrated through several experiments. The use of an SD-ODT system in blood flow measurement can provide high resolution images (5 microns resolution). We prepared three capillary tubes to reveal the effect of different concentrations of hematocrit ratio (HR). One tube serves as the control. The two other tubes contained different concentrations of HR (5%, 25%). Three different capillary tube inlet flow velocities were tested in the present study. The Reynolds number (Re) which is based on the capillary tube inner diameter ranges from Re=6 to 48. We calculated a Doppler shift of the power spectrum of the temporal interference fringes with Kasai autocorrelation function to achieve the velocity profile of the flow. As a result, SD-ODT systems could not detect the cell depletion layer in the present study due to the limitation of spatial resolution. Nevertheless, these systems were proven to be capable of observing the RBCs of blood.

In this paper, the features of the intensity-based Doppler variance (IBDV) method were analyzed systemically with a flow phantom. The effects of beam scanning density, flow rate and the time interval between neighboring A-lines on the performance of this method were investigated. The IBDV method can be used to quantify the flow rate and its sensitivity can be improved by increasing the time interval between the neighboring A-lines. A higher sensitivity IBDV method that applies the algorithm along the slower scan direction was proposed. In comparison to laser speckle imaging maps of blood flow, we demonstrated the ability of the method to identify vessels with altered blood flow. In clinical measurements, we demonstrated the ability of the method to image vascular networks with exquisite spatial resolution and at depths up to 1.2 mm in human skin. These results collectively demonstrated the potential of the method to monitor the microvasculature during disease progression and in response to therapeutic intervention.

Blood flow in murine epicardial and intra-myocardial coronary arteries was measured in vivo with spectral domain optical Doppler tomography (SD-ODT). Videos at frame rates up to 180 fps were collected and processed to extract phase shifts associated with moving erythrocytes in the coronary arteries. Radial averaging centered on the vessel lumen provided spatial smoothing of phase noise in a single cross-sectional frame for instantaneous peak velocity measurement without distortion of the flow profile. Temporal averaging synchronized to the cardiac cycle (i.e., gating) was also performed to reduce phase noise, although resulting in lower flow profiles. The vessel angle with respect to incident imaging beam was measured with three-dimensional raster scans collected from the same region as the high speed cross-sectional scans. The variability in peak phase measurement was 10-15% from cycle to cycle on a single animal but larger for measurements among animals. The inter-subject variability is attributed to factors related to real physiological and anatomical differences, instrumentation variables, and measurement error. The measured peak instantaneous flow velocity in a ~40-µm diameter vessel was 23.5 mm/s (28 kHz Doppler phase shift). In addition to measurement of the flow velocity, we observed several dynamic features of the vessel and surrounding myocardium in the intensity and phase sequences, including asymmetric vessel deformation and rapid flow reversal immediately following maximum flow, in confirmation of known coronary artery flow dynamics. SD-ODT is an optical imaging tool that can provide in vivo measures of structural and functional information on cardiac function in small animals.

The traditional phase-resolved Doppler method demonstrates great success for in-vivo imaging of blood flow and blood vessels. However, the phase-resolved method always requires high phase stability of the system. In phase instable situations, the performance of the phase-resolved methods will be degraded. We propose a modified Doppler variance algorithm that is based on the intensity or amplitude value. Performances of the proposed algorithm are compared with traditional phase-resolved Doppler variance and color Doppler methods for both phase stable and phase instable systems. For the phase instable situation, the proposed algorithm demonstrates images without phase instability induced artifacts. In-vivo imaging of window-chamber hamster skin is demonstrated for phase instable situation with a spectrometer-based Fourier domain OCT system. A microelectromechanical systems (MEMS) based swept source OCT (SSOCT) system is also used to demonstrate the performance of the proposed method in a phase instable situation. The phase stability of the SSOCT system is analyzed. In-vivo imaging of the blood vessel of human skin is demonstrated with the proposed method and the SSOCT system. For the phase stable situation, the proposed algorithm also demonstrates comparable performance with traditional phase-resolved methods. In-vivo imaging of the human choroidal blood vessel network is demonstrated with the proposed method under the phase stable situation. Depth-resolved fine choroidal blood vessel networks are shown. (C) 2011 Optical Society of America

The method of measuring blood flow in photoacoustic microscopy usually relies on ultrasonic transducers in contact fashion, which is not favored in many applications, such as wound areas, burns, and anabrosis. Here we present a noncontact photoacoustic velocity measurement method to quantitatively map transverse blood flow based on the photoacoustic Doppler (PAD) bandwidth broadening method with an all-optical photoacoustic microscopy system. It is validated that the PAD bandwidth broadening is proportional to the transverse flow within a certain range. The transverse flow speed ranging from 0 to 5.5 mm/s, as well as sectional flow images, was obtained in the blood-mimicking flow phantoms. Furthermore, the blood flow image of the mouse ear demonstrates that the all-optical photoacoustic Doppler method can acquire the information of blood flow in vivo, which could significantly broaden the scope of applications for obtaining the blood flow velocity of the microvasculature in biomedicine.

Cancer (malignant tumor) is one of the serious threats to human life, causing 13% of all human deaths. A crucial step in the metastasis cascade of cancer is hematogenous spreading of tumor cells from a primary tumor. Thus, isolation and identification of cells that have detached from the primary tumor and circulating in the bloodstream (circulating tumor cells, CTCs) is considered to be a potential alternation to detect, characterize, and monitor cancer. Current methods for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity. Here, we propose a high throughput 3D structured microfluidic chip integrated with surface plasmon resonance (SPR) sensor to isolate and identify CTCs from peripheral whole blood sample. The microfluidic velocity-field within the channel of the chip is mediated by an array of microposts protruding from upper surface of the channel. The height of microposts is shorter than that of the channel, forming a gap between the microposts and the lower surface of the channel. The lower surface of the channel also acts as the SPR sensor which can be used to identify isolated CTCs. Microfluidic velocity-field under different parameters of the arrayed microposts is studied through numerical simulation based on finite element method. Measurement on one of such fabricated microchips is conducted by our established optical Doppler tomography technique benefiting from its noninvasive, noncontact, and high-resolution spatial-resolved capabilities. Both simulation and measurement of the microfluidic velocity-field within the structured channel demonstrates that it is feasible to introduce fluidic mixing and causes perpendicular flow component to the lower surface of the channel by the 3D structured microposts. Such mixing and approaching capabilities are especially desirable for isolation and identification of CTCs at the coated SPR sensor.

Optical Doppler Tomography (ODT) provides a novel method to measure the blood flow velocity in vessels with the diameter at micrometer scale. Rats with cranial window are used as a model, and the changes in the blood flow velocity of cerebral arterioles in sensory cortex are measured in real time with an established ODT system, under electrical stimulation and drug administration. The results show significant differences in the blood flow velocity between experimental groups and control groups, demonstrating the feasibility of ODT in the cerebral microcirculation study. Compared with the conventional Doppler ultrasound, ODT provides much higher spatial resolution, and thus holds a promising future in the application of the cerebral microcirculation study, especially in the observation of the blood flow velocity in micrometer scale vessels.

This paper presents an optical doppler tomography(ODT) system using a speaker as a method to achieve depth measurement in a flowing sample. The use of the speaker provides easy implementation with a low cost. The nonlinear characteristics of the speaker has hindered its adaptation because it produces inconsistent fringe frequencies at different depths. This paper reports an adaptive algorithm to compensate the nonlinear characteristics, and could, resultantly, acquire the Doppler frequency shift caused by the sample. The experiment utilizes a flowing scattering particle solution in a capillary tube at a certain flow rate. The Doppler frequency profile over the lumen was calculated by using spectrogram method. and we obtained the velocity image of the sample.

We report the design of and results obtained by using a field programmable gate array (FPGA) to digitally process optical Doppler tomography signals. The processor fits into the analog signal path in an existing optical coherence tomography setup. We demonstrate both Doppler frequency and envelope extraction using the Hilbert transform, all in a single FPGA. An FPGA implementation has certain advantages over general purpose digital signal processor (DSP) due to the fact that the processing elements operate in parallel as opposed to the DSP, which is primarily a sequential processor.

© 2016 Optical Society of America. Doppler optical coherence tomography (DOCT) is considered one of the most promising functional imaging modalities for neuro biology research and has demonstrated the ability to quantify cerebral blood flow velocity at a high accuracy. However, the measurement of total absolute blood flow velocity (BFV) of major cerebral arteries is still a difficult problem since it is related to vessel geometry. In this paper, we present a volumetric vessel reconstruction approach that is capable of measuring the absolute BFV distributed along the entire middle cerebral artery (MCA) within a large field-of-view. The Doppler angle at each point of the MCA, representing the vessel geometry, is derived analytically by localizing the artery from pure DOCT images through vessel segmentation and skeletonization. Our approach could achieve automatic quantification of the fully distributed absolute BFV across different vessel branches. Experiments on rodents using swept-source optical coherence tomography showed that our approach was able to reveal the consequences of permanent MCA occlusion with absolute BFV measurement.

There is growing interest in new neuroimage techniques that permit not only high-resolution quantification of cerebral blood flow velocity (CBFv) in capillaries, but also a large field of view to map the CBFv network dynamics. Such image capabilities are of great importance for decoding the functional difference across multiple cortical layers under stimuli. To tackle the limitation of optical penetration depth, we present a new ultrahigh-resolution optical coherence Doppler tomography (μODT) system at 1310 nm and compare it with a prior 800 nm μODT system for mouse brain 3D CBFv imaging. We show that the new 1310 nm μODT allows for dramatically increased depth (∼4 times) of quantitative CBFv imaging to 1.4 mm, thus covering the full thickness of the mouse cortex (i.e., layers I-VI). Interestingly, we show that such a unique 3D CBFv imaging capability allows identification of microcirculatory redistribution across different cortical layers resulting from repeated cocaine exposures.

We describe the application of optical coherence tomography (OCT) for noninvasive analysis of follicular development in mouse ovaries. Ovaries contain many follicles and oocytes. Quantification of follicles at various developmental stages is an indication of the ability of an ovary to provide oocytes capable of fertilization. Three-dimensional structural OCT images identified each developmental stage, from a primary follicle (50 μm in diameter) to an antral follicle (350 μm in diameter), in the ovary of a 25.5-day-old mouse. We discovered time-varying OCT signals at the oocytes that differentiated them from surrounding tissues. These signals were clearly enhanced by interframe intensity-based Doppler OCT techniques. The OCT technology was effective in analyzing the development of follicles and oocytes in ovaries.

We show reconstructed images of realistic phantoms constructed using 3D printing. This approach is versatile, aids in the development of optical imaging methods, and can be used in the calibration of live mouse imaging data.

We introduce a novel contrast mechanism for imaging superparamagnetic iron oxide (SPIO) nanoparticles (average diameter ${\sim}100nm$ ) using magneto-motive optical Doppler tomography (MM-ODT), which combines an externally applied temporally oscillating high-strength magnetic field with ODT to detect the nanoparticles flowing through a glass capillary tube. A solenoid cone-shaped ferrite core extensively increased the magnetic field strength ( $B_{max}=1\;T,\;{\Delta}|B|^2=220T^2/m$ ) at the tip of the core and also focused the magnetic force on targeted samples. Nanoparticle contrast was demonstrated in a capillary tube filled with the SPIO solution by imaging the Doppler frequency shift which was observed independent of the flow rate and direction. Results suggest that MM-ODT may be a promising technique to enhance SPIO nanoparticle contrast for imaging fluid flow.

Noninvasive angiography is demonstrated for the in vivo human eye. Three-dimensional flow imaging has been performed with high-speed spectral-domain optical coherence tomography. Sample motion is compensated by two algorithms. Axial motion between adjacent A-lines within one OCT image is compensated by the Doppler shift due to bulk sample motion. Axial displacements between neighboring images are compensated by a correlation-based algorithm. Three-dimensional vasculature of ocular vessels has been visualized. By integrating volume sets of flow images, two-dimensional images of blood vessels are obtained. Retinal and choroidal blood vessel images are simultaneously obtained by separating the volume set into retinal part and choroidal parts. These are corresponding to fluorescein angiogram and indocyanine angiogram., This paper was published in Optics Express and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-17-7821. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law.

We report on local superficial blood flow monitoring in biological tissue from laser Doppler holographic imaging. In time averaging recording conditions, holography acts as a narrowband bandpass filter, which, combined with a frequency shifted reference beam, permits frequency selective imaging in the radiofrequency range. These Doppler images are acquired with an off axis Mach Zehnder interferometer. Microvascular hemodynamic components mapping is performed in the cerebral cortex of the mouse and the eye fundus of the rat with near-infrared laser light without any exogenous marker. These measures are made from a basic inverse method analysis of local first order optical fluctuation spectra at low radiofrequencies, from 0 Hz to 100 kHz. Local quadratic velocity is derived from Doppler broadenings induced by fluid flows, with elementary diffusing wave spectroscopy formalism in backscattering configuration. We demonstrate quadratic mean velocity assessment in the 0.1 to 10 millimeters per second range in vitro and imaging of superficial blood perfusion with a spatial resolution of about 10 micrometers in rodent models of cortical and retinal blood flow., 15 pages

Frequency domain phase-resolved optical Doppler tomography (ODT) was developed with Doppler variance imaging capability. It is shown that utilizing the frequency domain method, phase-resolved ODT can achieve much higher imaging speed and velocity dynamic range than the time domain method. Structural, Doppler and Doppler variance images of fluid flow through glass channels were quantified and blood flow through vessels were demonstrated in vivo. © 2004 Elsevier B.V. All rights reserved.

Phase-resolved optical Doppler tomography (ODT), an imaging technique based on low coherence interferometry, is presented as a new tool to determine electroosmotic flow (EOF) in microfluidic channel. This is a non-contact technique that not only can image cross-sectional EOF profile but also can determine electroosmotic mobility. The electroosmotic coefficient measured by ODT was verified by conventional current monitor method. Since phase-resolved ODT provides cross-sectional imaging of flow velocity, complicated flow dynamics caused by microscale effects such as electrokinetic effects, can be investigated and quantified using this tool, particularly for turbid media such as biofluids, which cannot be readily investigated using other techniques. © 2003 Published by Elsevier B.V.

An ultra-high-speed spectral domain optical Doppler tomography (SD-ODT) system is used to acquire images of blood flow in a human retina in vivo, at 29,000 depth profiles (A-lines) per second and with data acquisition over 99% of the measurement time. The phase stability of the system is examined and image processing algorithms are presented that allow accurate determination of bi-directional Doppler shifts. Movies are presented of human retinal flow acquired at 29 frames per second with 1000 A-lines per frame over a time period of 3.28 seconds, showing accurate determination of vessel boundaries and time-dependent bi-directional flow dynamics in artery-vein pairs. The ultra-high-speed SD-ODT system allows visualization of the pulsatile nature of retinal blood flow, detects blood flow within the choroid and retinal capillaries, and provides information on the cardiac cycle. In summary, accurate video rate imaging of retinal blood flow dynamics is demonstrated at ocular exposure levels below 600 microW.

Significant improvements are reported in the measurable velocity range and tissue motion artefact rejection of a phase-resolved optical coherence tomography and optical Doppler tomography system. Phase information derived from an in-phase and quadrature demodulator is used to estimate the mean blood flow velocity by the Kasai autocorrelation algorithm. A histogram-based velocity segmentation algorithm is used to determine block tissue movement and remove tissue motion artefacts that can be faster or slower in velocity than that of the microcirculation. The minimum detectable Doppler frequency is about 100 Hz, corresponding to a flow velocity resolution of 30 μm/s with an axial-line scanning frequency of 8.05 kHz and a mean phase change measured over eight sequential scans; the maximum detectable Doppler frequency is ±4 kHz (for bi-directional flow) before phase wrap-around.

Author(s): Viator, John A; Au, Gigi; Paltauf, Guenther; Jacques, Steven L; Prahl, Scott A; Ren, Hongwu; Chen, Zhongping; Nelson, J Stuart | Abstract: Background and objectiveSuccessful laser treatment of port wine stain (PWS) birthmarks requires knowledge of lesion geometry. Laser parameters, such as pulse duration, wavelength, and radiant exposure, and other treatment parameters, such as cryogen spurt duration, need to be optimized according to epidermal melanin content and lesion depth. We designed, constructed, and clinically tested a photoacoustic probe for PWS depth determination.Study design/materials and methodsEnergy from a frequency-doubled, Nd:YAG laser (lambda=532 nm, tau(p)=4 nanoseconds) was coupled into two 1,500 mum optical fibers fitted into an acrylic handpiece containing a piezoelectric acoustic detector. Laser light induced photoacoustic waves in tissue phantoms and a patient's PWS. The photoacoustic propagation time was used to calculate the depth of the embedded absorbers and PWS lesion.ResultsCalculated chromophore depths in tissue phantoms were within 10% of the actual depths of the phantoms. PWS depths were calculated as the sum of the epidermal thickness, determined by optical coherence tomography (OCT), and the epidermal-to-PWS thickness, determined photoacoustically. PWS depths were all in the range of 310-570 microm. The experimentally determined PWS depths were within 20% of those measured by optical Doppler tomography (ODT).ConclusionsPWS lesion depth can be determined by a photoacoustic method that utilizes acoustic propagation time.

Retinal oxygen metabolic rate can be effectively measured by visible-light optical coherence tomography (vis-OCT), which simultaneously quantifies oxygen saturation and blood flow rate in retinal vessels through spectroscopic analysis and Doppler measurement, respectively. Doppler OCT relates phase variation between sequential A-lines to the axial flow velocity of the scattering medium. The detectable phase shift is between -π and π due to its periodicity, which limits the maximum measurable unambiguous velocity without phase unwrapping. Using shorter wavelengths, vis-OCT is more vulnerable to phase ambiguity since flow induced phase variation is linearly related to the center wavenumber of the probing light. We eliminated the need for phase unwrapping using spectroscopic Doppler analysis. We split the whole vis-OCT spectrum into a series of narrow subbands and reconstructed vis-OCT images to extract corresponding Doppler phase shifts in all the subbands. Then, we quantified flow velocity by analyzing subband-dependent phase shift using linear regression. In the phantom experiment, we showed that spectroscopic Doppler analysis extended the measurable absolute phase shift range without conducting phase unwrapping. We also tested this method to quantify retinal blood flow in rodents in vivo.

Background: The purpose of our study is to use a noninvasive tomographic imaging technique with high spatial resolution to characterize and monitor biological tissue responses associated with laser thermal injury. Methods: Optical doppler tomography (ODT) combines laser doppler flowmetry (LDF) with optical coherence tomography (OCT) to obtain high resolution tomographic velocity and structural images of static and moving constituents in highly scattering biological tissues. A SurgiLase XJ150 carbon dioxide (CO2) laser using a continuous mode of 3 watts (W) was used to create first, second or third degree burns on anesthetized Sprague–Dawley rats. Additional parameters for laser thermal injury were assessed as well. Results: The rationale for using ODT in the evaluation of laser thermal injury offers a means of constructing a high resolution tomographic image of the structure and perfusion of laser damaged skin. In the velocity images, the blood flow is coded at 1300 μm/s and 0 velocity, 1000 μm/s and 0 velocity, 700 μm/s and 0 velocity adjacent to the first, second, and third degree injuries, respectively. Conclusion: ODT produces exceptional spatial resolution while having a non-invasive way of measurement, therefore, ODT is an accurate measuring method for high-resolution fluid flow velocity and structural images for biological tissue with laser thermal injury.

We developed an augmented-reality system that combines optical coherence tomography (OCT) with a surgical microscope. By sharing the common optical path in the microscope and OCT, we could simultaneously acquire OCT and microscope views. The system was tested to identify the middle-ear and inner-ear microstructures of a mouse. Considering the probability of clinical application including otorhinolaryngology, diseases such as middle-ear effusion were visualized using in vivo mouse and OCT images simultaneously acquired through the eyepiece of the surgical microscope during surgical manipulation using the proposed system. This system is expected to realize a new practical area of OCT application. (C) 2014 Optical Society of America

We review the principle and some recent applications of Doppler optical coherence tomography (OCT). The advances of the phase-resolved Doppler OCT method are described. Functional OCT algorithms which are based on an extension of the phase-resolved scheme are also introduced. Recent applications of Doppler OCT for quantification of flow, imaging of microvasculature and vocal fold vibration, and optical coherence elastography are briefly discussed. © 2013 Chinese Optics Letters.

The detection of thermoelastic displacement by differential phase optical coherence tomography (DP-OCT) was analytically evaluated for identifying atherosclerotic plaques. Analytical solutions were developed to understand the dynamics of physical distribution of point hear sources during/after laser irradiation on thermoelastic responses of MION-injected tissue. Both analytical and experimental results demonstrated a delayed peak displacement along with slow decay after laser pulse due to heterogeneous distribution of the point heat sources. Detailed description of the heat sources in tissue as well as integration of a scanning mirror can improve computational accuracy as well as clinical applicability of DPOCT for diagnosing vulnerable plaque. (C) 2014 Optical Society of America

Optical Doppler tomography (ODT) is an imaging modality that takes advantage of the short coherence length of a broad-band light sources to perform micrometer-scale, cross-sectional imaging of tissue structure and blood flow dynamics simultaneously. The authors review in this paper the principal of ODT and its applications. Results from in vitro and in vivo model studies demonstrated that ODT can map the blood flow velocity profile with high spatial resolution in scattering medium. ODT detection mechanisms are illustrated using Monte Carlo simulations. The application of ODT to image brain hemodynamics is demonstrated. Finally, the authors discuss the limitations of the current technology and application of a phase resolved technique to improve image speed and quality.

Vascular responses were monitored to understand the role of the microvasculature in tumor destruction as a result of photodynamic therapy (PDT). Rats received an intravenous dose of 2 mg/kg Benzoporphyrin Derivative (BPD), at 20 min, 4 h, or 7 h before laser irradiation. With Photofrin (10 mg/kg), drug-light intervals were 20 min or 8 h. Jejunal blood vessels were exposed to 12 J/cm/sup 2/ at 690 nm (with BPD) or at 630 nm (with Photofrin). Optical Doppler tomography (ODT) was used to evaluate PDT-induced changes in vessel diameter and blood flow. At the shortest drug-light time interval (20 min), BPD-mediated PDT caused transient constriction of arteries, accompanied by decreased blood flow, followed by vasodilation until baseline was reached or overshoot occurred. Veins became occluded with no restoration of the vessel lumen. At longer drug-light intervals, vasoconstriction diminished and venodilation was observed. With Photofrin, vasoconstriction and venodilation increased with the drug-light interval. Application of a higher light dose (48 J/cm/sup 2/) resulted in irreversible hemostasis. ODT can be used to study changes in lumen diameter and blood flow, which are important diagnostic parameters of PDT.

A Monte Carlo model has been developed for optical Doppler tomography (ODT) within the framework of a model for optical coherence tomography (OCT). A phantom situation represented by blood flowing in a horizontal 100 microm diameter vessel placed at 250 microm axial depth in 2% intralipid solution was implemented for the Monte Carlo simulation, and a similar configuration used for experimental ODT measurements in the laboratory. Simulated depth profiles through the centre of the vessel of average Doppler frequency demonstrated an accuracy of 3-4% deviation in frequency values and position localization of flow borders, compared with true values. Stochastic Doppler frequency noise was experimentally observed as a shadowing in regions underneath the vessel and also seen in simulated Doppler frequency depth profiles. By Monte Carlo simulation, this Doppler noise was shown to represent a nearly constant level over an investigated 100 microm interval of depth underneath the vessel. The noise level was essentially independent of the numerical aperture of the detector and angle between the flow velocity and the direction of observation, as long as this angle was larger than 60 degrees. Since this angle determines the magnitude of the Doppler frequency for backscattering from the flow region, this means that the signal-to-noise ratio between Doppler signal from the flow region to Doppler noise from regions underneath the flow is improved by decreasing the angle between the flow direction and direction of observation. Doppler noise values from Monte Carlo simulations were compared with values from statistical analysis.

We developed GPU(Graphics Processing Unit) processing to display correlation mapping OCT images in real-time. A display rate of 91 frames per second for processed images (1024 FFT size × 512 lateral A-scans) was achieved.

Measurement of three-dimensional (3D) absorption distribution in a scattered medium is very useful for biomedical applications. Optical coherent tomography (OCT) is a very powerful tool, but the measurement depth is limited to a few mm due to the multiple scattering. Optical diffused tomography (ODT) is alternative method to measure the absorption distribution in deeper depth than that by OCT. In ODT, numerical way is applied to reconstruct a 3D absorption distribution. For successful reconstruction, it is important to develop a way to extract effectively the information of absorbers in output scattered light.

The significantly less stringent operation of a two-reference swept-source optical coherence tomography (OCT) system for suppressing the mirror image is demonstrated based on the spatially localized image processing method. With this method, the phase difference between the two reference signals is not limited to 90 degrees. Based on the current experimental operation, the mirror image can be effectively suppressed as long as the phase difference is larger than 20 degrees. In other words, the adjustment of the beam splitter orientation for controlling the phase difference becomes much more flexible. Also, based on a phantom experiment, the combination the spatially localized mirror image suppression method with the two-reference OCT operation leads to the implementation of full-range optical Doppler tomography.

The diffraction tomography theorem is adapted to one-dimensional length measurement. The resulting spectral interferometry technique is described and the first length measurements using this technique on a model eye and on a human eye in vivo are presented.

We conducted an initial feasibility study using real-time magneto-motive optical Doppler tomography (MM-ODT) with enhanced contrast to investigate the detection of superparamagnetic iron oxide (SPIO) magnetic nanoparticles implanted into in vivo melanoma tissue. The MM-ODT signals were detected owing to the phase shift of the implanted magnetic nanoparticles, which occurred due to the action of an applied magnetic field. An amplifier circuit-based solenoid was utilized for generating high-intensity oscillating magnetic fields. The MM-ODT system was confirmed as an effective in vivo imaging method for detecting melanoma tissue, with the performance comparable to those of conventional optical coherence tomography and optical Doppler tomography methods. Moreover, the optimal values of the SPIO nanoparticles concentration and solenoid voltage for obtaining the uppermost Doppler velocity were derived as well. To improve the signal processing speed for real-time imaging, we adopted multithread programming techniques and optimized the signal path. The results suggest that this imaging modality can be used as a powerful tool to identify the intracellular and extracellular SPIO nanoparticles in melanoma tissues in vivo.

Ophthalmology Volume 123, Number 3, March 2016 Descemet stripping endothelial keratoplasty. Arch Ophthalmol 3. Kopplin LJ, Przepyszny K, Schmotzer B, et al. Relationship of Fuchs endothelial corneal dystrophy severity to central corneal thickness. Arch Ophthalmol 2012;130:433–9. 4. Wacker K, McLaren JW, Amin SR, et al. Corneal high-order aberrations and backscatter in Fuchs’ endothelial corneal dys- trophy. Ophthalmology 2015;122:1645–52. 5. McLaren JW, Bachman LA, Kane KM, Patel SV. Objective assessment of the corneal endothelium in Fuchs’ endothelial dystrophy. Invest Ophthalmol Vis Sci 2014;55:1184–90. Re: Spaide et al.: Volume-rendering optical coherence tomography angio- graphy of macular telangiectasia type 2 (Ophthalmology 2015;122:2261-9) We agree very much with Spaide et al 1 that a great advantage of optical coherence tomography (OCT) angiography is its 3-dimensional (3D) nature, and that color coding for the depth of vessels is a natural way to represent this 3D information. However, we think it is disingenuous for Dr Spaide to imply that he was the first to adapt this approach from CT and MR literature and apply it to OCT angiography. He wrote: TO THE EDITOR: Instead of imaging the average or densest voxel, volume rendering uses all the voxel values to make an image that retains the sense of depth and is less prone to image artifact. These methods were adapted to OCTA, which does not use dye, by Dr. Spaide. In fact, volume rendering of angiography and color coding of vessel depth had already been used by several groups since the early days of OCT angiography. Barton et al 2 reported volume rendering of skin vasculature detected by Doppler OCT as early as 1999. Zhao et al 3 had described 3D volume-rendered Doppler OCT angi- ography of port wine stain vasculature by 2001. Yasuno et al had described 3D volume-rendered OCT angiography of in vivo human macular and optic nerve head vessels in 2006. 4 Wang et al (Optics Express 2007) had shown 3D volume rendering of mouse cortical vessels. Fingler et al (Optics Express 2009) had demonstrated volume rendering of human retinal microvasculature. Vakoc et al (Nature Medicine 2009) had used color coding to render complex 3D vasculature in tumor and brain layers using OCT angiography. Mariampillai et al (Optics Letters), Srinivasan et al (Optics Letters), and Yu et al (Journal of Biomedical Optics) had all used volume rendering to visualize 3D vascular structures in 2010. Jia et al (Proceedings of the National Academy of Sciences 2015) had used color to separate shallower retinal circulation from deeper choroidal circulation using the en face slab visualization approach, as did Kim et al (Proceedings of the National Academy of Sciences 2013) for visualizing vascular beds in geographic atrophy. And pixel transparency of the retinal slab was adjusted to optimize the visualization of choroidal neovascularization. 5 We list these prior works here so the reader can appreciate the work of pioneers in this field. Z HONGPING C HEN , P H D 1 D AVID H UANG , MD, P H D 2 J OSEPH A. I ZATT , P H D 3 R UIKANG K. W ANG , P H D 4 e24 J OHN S. W ERNER , P H D 5 Y OSHIAKI Y ASUNO , P H D 6 Department of Biomedical Engineering, University of California at Irvine, Irvine, California; 2 Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; 3 Department of Biomedical Engineering, Duke University, Durham, North Carolina; 4 Department of Biomedical Engineering, University of Washington, Seattle, Washington; 5 Department of Ophthalmology, University of California at Davis, Sacramento, California; 6 Institute of Applied Physics, University of Tsukuba, Tsukuba, Japan Financial Disclosure(s): The authors made the following disclosures: Z.C.: Grants e NIH, during the conduct of the study; Personal fees and Other: OCT Medical Imaging Inc., outside the submitted work; Patent pending, a patent issued, a patent licensed, and a patent with royalties paid. D.H.: has a significant financial interest in Carl Zeiss Meditec; Oregon Health & Science University (OHSU) and Dr. Huang has a significant financial interest in Optovue, Inc., a company that may have a commercial interest in the results of this research and technology. These potential conflicts of interest have been reviewed and managed by OHSU. J.A.I.: US Patent No. 6,735,463, “Doppler Flow Imaging Using Optical Coherence Tomography” with royalties paid to Leica Microsystems, Inc. R.K.W.: Grants e National Institutes of Health; Grants and Nonfinancial support e Carl Zeiss Meditec Inc., outside the submitted work. In addition, Dr. Wang has a patent Oregon Health & Science University with royalties paid to Carl Zeiss Meditec, Inc., and a patent University of Washington pending. Y.Y.: Grants e Japan Society for the Promotion of Science, Japan Science and Technology Agency, Topcon Corp., Nidek Inc., Canon, during the conduct of the study; Grants e Tomey Corp., outside the submitted work. Correspondence: David Huang, MD, PhD, Oregon Health & Science University, 3375 SW Terwilliger Boulevard, Portland, OR 97239. E-mail: davidhuang@ alum.mit.edu. References 1. Spaide RF, Klancnik JM Jr, Cooney MJ, et al. Volume- rendering optical coherence tomography angiography of macular telangiectasia type 2. Ophthalmology 2015;122:2261–9. 2. Kehlet Barton J, Izatt JA, Kulkarni MD, et al. Three-dimensional reconstruction of blood vessels from in vivo color Doppler op- tical coherence tomography images. Dermatology 1999;198: 3. Zhao Y, Brecke KM, Ren H, et al. Three-dimensional recon- struction of in vivo blood vessels in human skin using phase- resolved optical Doppler tomography. IEEE J Sel Top Quantum Electron 2001;7:931–5. 4. Makita S, Hong Y, Yamanari M, et al. Optical coherence angiography. Opt Express 2006;14:7821–40. 5. Jia Y, Bailey ST, Hwang TS, et al. Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye. Proc Natl Acad Sci U S A 2015;112:E2395–402. We wish to thank the authors for their letter and also thank them for reading our paper. 1 Volume rendering has been used in representation of both computed tomography and magnetic resonance imaging for more than 3 decades. Thus, the techniques used in our paper are not new. We suggest reading the quoted sentence in the context of the paragraph in which it was embedded. We stated that the previous REPLY

We report a newly developed high speed 1050nm spectral domain optical coherence tomography (SD-OCT) system for imaging posterior segment of human eye. The system is capable of an axial resolution at ~10 µm in air, an imaging depth of 6.1 mm in air, a system sensitivity fall-off at ~6 dB/3mm and an imaging speed of 120,000 A-scans per second. We experimentally demonstrate the system's capability to perform phase-resolved imaging of dynamic blood flow within retina, indicating high phase stability of the SDOCT system. Finally, we show an example that uses this newly developed system to image posterior segment of human eye with a large view of view (10 × 9 mm(2)), providing detailed visualization of microstructural features from anterior retina to posterior choroid. The demonstrated system parameters and imaging performances are comparable to those that a typical 1 µm swept source OCT would deliver for retinal imaging.

Recently, there has been growing interest in using OCT for brain imaging. A feasibility study of OCT for guiding deep brain probes has found that OCT can differentiate the white matter and gray matter because the white matter tends to have a higher peak reflectivity and steeper attenuation rate compared to gray matter. In vivo 3D visualization of the layered organization of a rat olfactory bulb with OCT has been demonstrated. OCT has been used for single myelin fiber imaging in living rodents without labeling. The refractive index in the rat somatosensory cortex has also been measured with OCT. In addition, functional extension of OCT, such as Doppler-OCT (D-OCT), polarization sensitive-OCT (PS-OCT), and phase-resolved-OCT (PR-OCT), can image and quantify physiological parameters in addition to the morphological structure image. Based on the scattering changes during neural activity, OCT has been used to measure the functional activation in neuronal tissues. PS-OCT, which combines polarization sensitive detection with OCT to determine tissue birefringence, has been used for the localization of nerve fiber bundles and the mapping of micrometer-scale fiber pathways in the brain. D-OCT, also named optical Doppler tomography (ODT), combines the Doppler principle with OCT to obtain high resolution tomographic images of moving constituents in highly scattering biological tissues. D-OCT has been successfully used to image cortical blood flow and map the blood vessel network for brain research. In this chapter, the principle and technology of OCT and D-OCT are reviewed and examples of potential applications are described.

We compare, in detail, the phase-resolved color Doppler (PRCD), phase-resolved Doppler variance (PRDV) and intensity-based Doppler variance (IBDV) methods. All the methods are able to quantify flow speed when the flow rate is within a certain range, which is dependent on the adjacent A-line time interval. While PRCD is most sensitive when the flow direction is along the probing beam, PRDV and IBDV can be used to measure the flow when the flow direction is near perpendicular to the probing beam. However, the values of PRDV and IBDV are Doppler angle-dependent when the Doppler angle is above a certain threshold. The sensitivity of all the methods can be improved by increasing the adjacent A-line time interval while still maintaining a high sampling density level. We also demonstrate for the first time, to the best of our knowledge, high resolution inter-frame PRDV method. In applications where mapping vascular network such as angiogram is more important than flow velocity quantification, IBDV and PRDV images show better contrast than PRCD images. The IBDV and PRDV show very similar characteristics and demonstrate comparable results for vasculature mapping. However, the IBDV is less sensitive to bulk motion and with less post-processing steps, which is preferred for fast data processing situations. In vivo imaging of mouse brain with intact skull and human skin with the three methods were demonstrated and the results were compared. The IBDV method was found to be able to obtain high resolution image with a relative simple processing procedure.

We demonstrate an ultrafast displaying Spectral Domain Optical Doppler Tomography system using Graphics Processing Unit (GPU) computing. The calculation of FFT and the Doppler frequency shift is accelerated by the GPU. Our system can display processed OCT and ODT images simultaneously in real time at 120 fps for 1,024 pixels × 512 lateral A-scans. The computing time for the Doppler information was dependent on the size of the moving average window, but with a window size of 32 pixels the ODT computation time is only 8.3 ms, which is comparable to the data acquisition time. Also the phase noise decreases significantly with the window size. Since the performance of a real-time display for OCT/ODT is very important for clinical applications that need immediate diagnosis for screening or biopsy. Intraoperative surgery can take much benefit from the real-time display flow rate information from the technology. Moreover, the GPU is an attractive tool for clinical and commercial systems for functional OCT features as well.

The correct segmentation of blood vessels in optical coherence tomography (OCT) images may be an important requirement for the analysis of intra-retinal layer thickness in human retinal diseases. We developed a shape model based procedure for the automatic segmentation of retinal blood vessels in spectral domain (SD)-OCT scans acquired with the Spectralis OCT system. The segmentation procedure is based on a statistical shape model that has been created through manual segmentation of vessels in a training phase. The actual segmentation procedure is performed after the approximate vessel position has been defined by a shadowgraph that assigns the lateral vessel positions. The active shape model method is subsequently used to segment blood vessel contours in axial direction. The automated segmentation results were validated against the manual segmentation of the same vessels by three expert readers. Manual and automated segmentations of 168 blood vessels from 34 B-scans were analyzed with respect to the deviations in the mean Euclidean distance and surface area. The mean Euclidean distance between the automatically and manually segmented contours (on average 4.0 pixels respectively 20 µm against all three experts) was within the range of the manually marked contours among the three readers (approximately 3.8 pixels respectively 18 µm for all experts). The area deviations between the automated and manual segmentation also lie within the range of the area deviations among the 3 clinical experts. Intra reader variability for the experts was between 0.9 and 0.94. We conclude that the automated segmentation approach is able to segment blood vessels with comparable accuracy as expert readers and will provide a useful tool in vessel analysis of whole C-scans, and in particular in multicenter trials.