Hansford C. Hendargo

Doppler velocity detection limitations in spectrometer-based versus swept-source optical coherence tomography

Recent advances in Doppler techniques have enabled high sensitivity imaging of biological flow to measure blood velocities and vascular perfusion. Here we compare spectrometer-based and wavelength-swept Doppler OCT implementations theoretically and experimentally, characterizing the lower and upper observable velocity limits in each configuration. We specifically characterize the washout limit for Doppler OCT, the velocity at which signal degradation results in loss of flow information, which is valid for both quantitative and qualitative flow imaging techniques. We also clearly differentiate the washout effect from the separate phenomenon of phase wrapping. We demonstrate that the maximum detectable Doppler velocity is determined by the fringe washout limit and not phase wrapping. Both theory and experimental results from phantom flow data and retinal blood flow data demonstrate the superiority of the swept-source technique for imaging vessels with high flow rates.

Automated non-rigid registration and mosaicing for robust imaging of distinct retinal capillary beds using speckle variance optical coherence tomography

Variance processing methods in Fourier domain optical coherence tomography (FD-OCT) have enabled depth-resolved visualization of the capillary beds in the retina due to the development of imaging systems capable of acquiring A-scan data in the 100 kHz regime. However, acquisition of volumetric variance data sets still requires several seconds of acquisition time, even with high speed systems. Movement of the subject during this time span is sufficient to corrupt visualization of the vasculature. We demonstrate a method to eliminate motion artifacts in speckle variance FD-OCT images of the retinal vasculature by creating a composite image from multiple volumes of data acquired sequentially. Slight changes in the orientation of the subject’s eye relative to the optical system between acquired volumes may result in non-rigid warping of the image. Thus, we use a B-spline based free form deformation method to automatically register variance images from multiple volumes to obtain a motion-free composite image of the retinal vessels. We extend this technique to automatically mosaic individual vascular images into a widefield image of the retinal vasculature.

Combined hyperspectral and spectral domain optical coherence tomography microscope for noninvasive hemodynamic imaging

We have combined hyperspectral imaging with spectral domain optical coherence tomography (SDOCT) to noninvasively image changes in hemoglobin saturation, blood flow, microvessel morphology, and sheer rate on the vessel wall with tumor growth. Changes in these hemodynamic variables were measured over 24 h in dorsal skin fold window chamber tumors. There was a strong correlation between volumetric flow and hemoglobin saturation (ρ=0.89, p=9×10−6, N=15) and a moderate correlation between shear rate on the vessel wall and hemoglobin saturation (ρ=0.56, p=0.03, N=15).

Synthetic wavelength based phase unwrapping in spectral domain optical coherence tomography

Phase sensing implementations of spectral domain optical coherence tomography (SDOCT) have demonstrated the ability to measure nanometer-scale temporal and spatial profiles of samples. However, the phase information suffers from a 2pi ambiguity that limits observations of larger sample displacements to lengths less than half the source center wavelength. We introduce a synthetic wavelength phase unwrapping technique in SDOCT that uses spectral windowing and corrects the 2pi ambiguity, providing accurate measurements of sample motion with information gained from standard SDOCT processing. We demonstrate this technique by using a common path implementation of SDOCT and correctly measure phase profiles from a phantom phase object and human epithelial cheek cells which produce multiple wrapping artifacts. Using a synthetic wavelength for phase unwrapping could prove useful in Doppler or other phase based implementations of OCT.

Synthetic wavelength-based phase unwrapping in Fourier domain optical coherence tomography

Phase-sensitive adjuncts to optical coherence tomography (OCT) including Doppler and polarization-sensitive implementations allow for quantitative depth-resolved measurements of sample structure and dynamics including fluid flows and orientation of birefringent structures. The development of Fourier-domain OCT (FDOCT), particularly spectrometer-based spectral-domain systems with no moving parts (spectral-domain OCT or SDOCT), have greatly enhanced the phase stability of OCT systems particularly when implemented in a common-path geometry. The latter combination has given rise to a new class of nm-scale sensitive quantitative phase microscopies we have termed spectral domain phase microscopy. However, the phase information in all of these techniques suffers from a 2π ambiguity that limits resolvable pathlength differences to less than half the source center wavelength. This is problematic for situations such as cellular imaging, Doppler velocimetry, or polarization sensitive applications where it may be necessary to monitor sample profiles, displacements, phase differences, or refractive index variations which vary rapidly in space or time. A technique previously introduced in phase shifting interferometry uses phase information from multiple wavelengths to overcome this limitation. We show that by appropriate spectral windowing of the broadband light source already used in OCT, particularly by reshaping the source spectrum about two different center wavelengths, the resulting phase variation may be cast in terms of a much longer synthetic wavelength chosen to span the phase variation of interest. We show theoretically that the optimal choice of synthetic wavelength depends upon a tradeoff between the minimum resolvable phase and the length of unambiguous phase measurement. We demonstrate this technique using a broadband source centered at 790 nm by correctly reconstructing the phase profile from a phantom sample containing multiple 2π wrapping artifacts at the center wavelength and compare our result with atomic force microscopy.

Luminescent Difluoroboron β-Diketonate PEG-PLA Oxygen Nanosensors for Tumor Imaging

Surface modification of nanoparticles and biosensors is a dynamic, expanding area of research for targeted delivery in vivo. For more efficient delivery, surfaces are PEGylated to impart stealth properties, long circulation, and enable enhanced permeability and retention (EPR) in tumor tissues. Previously, BF2 dbm(I)PLA was proven to be a good oxygen nanosensor material for tumor hypoxia imaging in vivo, though particles were applied directly to the tumor and surrounding region. Further surface modification is needed for this dual-emissive oxygen sensitive material for effective intravenous (IV) administration and passive and active delivery to tumors. In this paper, an efficient synthesis of a new dual-emissive material BF2 dbm(I)PLA-mPEG is presented and in vitro stability studies are conducted. It is found that fabricated nanoparticles are stable for 24 weeks as a suspension, while after 25 weeks the nanoparticles swell and both dye and polymer degradation escalates. Preliminary studies show BF2 dbm(I)PLA-mPEG nanoparticle accumulation in a window chamber mammary tumor 24 h after IV injection into mice (C57Bl/6 strain) enabling tumor oxygen imaging.

Snap-shot multispectral imaging of vascular dynamics in a mouse window-chamber model

Understanding tumor vascular dynamics through parameters such as blood flow and oxygenation can yield insight into tumor biology and therapeutic response. Hyperspectral microscopy enables optical detection of hemoglobin saturation or blood velocity by either acquiring multiple images that are spectrally distinct or by rapid acquisition at a single wavelength over time. However, the serial acquisition of spectral images over time prevents the ability to monitor rapid changes in vascular dynamics and cannot monitor concurrent changes in oxygenation and flow rate. Here, we introduce snap shot-multispectral imaging (SS-MSI) for use in imaging the microvasculature in mouse dorsal-window chambers. By spatially multiplexing spectral information into a single-image capture, simultaneous acquisition of dynamic hemoglobin saturation and blood flow over time is achieved down to the capillary level and provides an improved optical tool for monitoring rapid in vivo vascular dynamics.

Combined hyperspectral and spectral domain optical coherence tomography microscope for non-invasive hemodynamic imaging

We have combined hyperspectral imaging with spectral domain optical coherence tomography (SDOCT) to non-invasively image changes in hemoglobin saturation, blood flow, microvessel morphology and sheer rate on the vessel wall with tumor growth. Changes in these hemodynamic variables were measured over 24 hours in dorsal skin fold window chamber tumors. There was a strong correlation between volumetric flow and hemoglobin saturation (ρ = 0.89, p = 9 × 10-6, N = 15), and a moderate correlation between shear rate on the vessel wall and hemoglobin saturation (ρ = 0.56, p = 0.03, N=15).

Dual-depth SSOCT for simultaneous complex resolved anterior segment and conventional retinal imaging

We present a novel optical coherence tomography (OCT) system design that employs coherence revival-based heterodyning and polarization encoding to simultaneously image the ocular anterior segment and the retina. Coherence revival heterodyning allows for multiple depths within a sample to be simultaneously imaged and frequency encoded by carefully controlling the optical pathlength of each sample path. A polarization-encoded sample arm was used to direct orthogonal polarizations to the anterior segment and retina. This design is a significant step toward realizing whole-eye OCT, which would enable customized ray-traced modeling of patient eyes to improve refractive surgical interventions, as well as the elimination of optical artifacts in retinal OCT diagnostics. We demonstrated the feasibility of this system by acquiring images of the anterior segments and retinas of healthy human volunteers.

Doppler velocity detection limitations in spectrometer and swept-source Fourier-domain optical coherence tomography

Recent advances in Doppler and variance techniques have enabled high sensitivity imaging in regions of biological flow to measure blood velocities and vascular perfusion. In recent years, the sensitivity and imaging speed benefits of Fourier domain OCT have become apparent. Spectrometer-based and wavelength-swept implementations have both undergone rapid development. Comparative analysis of the potential benefits and limitations for the various configurations would be useful for matching technology capabilities to specific clinical problems. Here we take a first step in such a comparative analysis by presenting theoretical predictions and experimental results characterizing the lower and upper observable velocity limits in spectrometer-based versus swept-source Doppler OCT. Furthermore, we characterize the washout limit, the velocity at which signal degradation results in loss of flow information. We present comparative results from phantom flow data as well as retinal data obtained with a commercial spectrometer OCT system and a custom high-speed swept-source retinal OCT system.