Toward optical coherence tomography angiography-based biomarkers to assess the safety of peripheral nerve electrostimulation.

OBJECTIVE: Peripheral nerves serve as a link between the central nervous system and its targets. Altering peripheral nerve activity through targeted electrical stimulation is being investigated as a therapy for modulating end organ function. To support rapid advancement in the field, novel approaches to predict and prevent nerve injury resulting from electrical stimulation must be developed to overcome the limitations of traditional histological methods. The present study aims to develop an optical imaging-based approach for real-time assessment of peripheral nerve injury associated with electrical stimulation. APPROACH: We developed an optical coherence tomography (OCT) angiography system and a 3D printed stimulating nerve stabilizer (sNS) to assess the real-time microvascular and blood flow changes associated with electrical stimulation of peripheral nerves. We then compared the microvascular changes with established nerve function analysis and immunohistochemistry to correlate changes with nerve injury. MAIN RESULTS: Electrical stimulation of peripheral nerves has a direct influence on vessel diameter and capillary flow. The stimulation used in this study did not alter motor function significantly, but a delayed onset of mechanical allodynia at lower thresholds was observed using a sensory function test. Immunohistochemical analysis pointed to an increased number of macrophages within nerve fascicles and axon sprouting potentially related to nerve injury. SIGNIFICANCE: This study is the first to demonstrate the ability to image peripheral nerve microvasculature changes during electrical stimulation. This expands the knowledge in the field and can be used to develop potential biomarkers to predict nerve injury resulting from electrical stimulation.

Wide-Field Functional Microscopy of Peripheral Nerve Injury and Regeneration.

Severe peripheral nerve injuries often result in partial repair and lifelong disabilities in patients. New surgical techniques and better graft tissues are being studied to accelerate regeneration and improve functional recovery. Currently, limited tools are available to provide in vivo monitoring of changes in nerve physiology such as myelination and vascularization, and this has impeded the development of new therapeutic options. We have developed a wide-field and label-free functional microscopy platform based on angiographic and vectorial birefringence methods in optical coherence tomography (OCT). By incorporating the directionality of the birefringence, which was neglected in the previously reported polarization-sensitive OCT techniques for nerve imaging, vectorial birefringence contrast reveals internal nerve microanatomy and allows for quantification of local myelination with superior sensitivity. Advanced OCT angiography is applied in parallel to image the three-dimensional vascular networks within the nerve over wide-fields. Furthermore, by combining vectorial birefringence and angiography, intraneural vessels can be discriminated from those of the surrounding tissues. The technique is used to provide longitudinal imaging of myelination and revascularization in the rodent sciatic nerve model, i.e. imaged at certain sequential time-points during regeneration. The animals were exposed to either crush or transection injuries, and in the case of transection, were repaired using an autologous nerve graft or acellular nerve allograft. Such label-free functional imaging by the platform can provide new insights into the mechanisms that limit regeneration and functional recovery, and may ultimately provide intraoperative assessment in human subjects.

High-speed optical coherence tomography by circular interferometric ranging.

Existing three-dimensional optical imaging methods excel in controlled environments but are difficult to deploy over large, irregular and dynamic fields. This has limited imaging in areas such as material inspection and medicine. To better address these applications, we developed methods in optical coherence tomography (OCT) to efficiently interrogate sparse scattering fields, i.e., those in which most locations (voxels) do not generate meaningful signal. Frequency comb sources are used to superimpose reflected signals from equispaced locations through optical subsampling. This results in circular ranging, and reduces the number of measurements required to interrogate large volumetric fields. As a result, signal acquisition barriers that have limited speed and field in OCT are avoided. With a new ultrafast, time-stretched frequency comb laser design operating with 7.6 MHz to 18.9 MHz repetition rates, we achieved imaging of multi-cm3 fields at up to 7.5 volumes per second.

Complex differential variance angiography with noise-bias correction for optical coherence tomography of the retina.

Complex differential variance (CDV) provides phase-sensitive angiographic imaging for optical coherence tomography (OCT) with immunity to phase-instabilities of the imaging system and small-scale axial bulk motion. However, like all angiographic methods, measurement noise can result in erroneous indications of blood flow that confuse the interpretation of angiographic images. In this paper, a modified CDV algorithm that corrects for this noise-bias is presented. This is achieved by normalizing the CDV signal by analytically derived upper and lower limits. The noise-bias corrected CDV algorithm was implemented into an experimental 1 µm wavelength OCT system for retinal imaging that used an eye tracking scanner laser ophthalmoscope at 815 nm for compensation of lateral eye motions. The noise-bias correction improved the CDV imaging of the blood flow in tissue layers with a low signal-to-noise ratio and suppressed false indications of blood flow outside the tissue. In addition, the CDV signal normalization suppressed noise induced by galvanometer scanning errors and small-scale lateral motion. High quality cross-section and motion-corrected en face angiograms of the retina and choroid are presented.

Laser thermal therapy monitoring using complex differential variance in optical coherence tomography.

Conventional thermal therapy monitoring techniques based on temperature are often invasive, limited by point sampling, and are indirect measures of tissue injury, while techniques such as magnetic resonance and ultrasound thermometry are limited by their spatial resolution.  The visualization of the thermal coagulation zone at high spatial resolution is particularly critical to the precise delivery of thermal energy to epithelial lesions. In this work, an integrated thulium laser thermal therapy monitoring system was developed based on complex differential variance (CDV), which enables the 2D visualization of the dynamics of the thermal coagulation process at high spatial and temporal resolution with an optical frequency domain imaging system. With proper calibration to correct for noise, the CDV-based technique was shown to accurately delineate the thermal coagulation zone, which is marked by the transition from high CDV upon heating to a significantly reduced CDV once the tissue is coagulated, in 3 different tissue types ex vivo: skin, retina, and esophagus. The ability to delineate thermal lesions in multiple tissue types at high resolution opens up the possibility of performing microscopic image-guided procedures in a vast array of epithelial applications ranging from dermatology, ophthalmology, to gastroenterology and beyond.

In vivo label-free measurement of lymph flow velocity and volumetric flow rates using Doppler optical coherence tomography.

Direct in vivo imaging of lymph flow is key to understanding lymphatic system function in normal and disease states. Optical microscopy techniques provide the resolution required for these measurements, but existing optical techniques for measuring lymph flow require complex protocols and provide limited temporal resolution. Here, we describe a Doppler optical coherence tomography platform that allows direct, label-free quantification of lymph velocity and volumetric flow rates. We overcome the challenge of very low scattering by employing a Doppler algorithm that operates on low signal-to-noise measurements. We show that this technique can measure lymph velocity at sufficiently high temporal resolution to resolve the dynamic pulsatile flow in collecting lymphatic vessels.

Multi-functional angiographic OFDI using frequency-multiplexed dual-beam illumination.

Detection of blood flow inside the tissue sample can be achieved by measuring the local change of complex signal over time in angiographic optical coherence tomography (OCT). In conventional angiographic OCT, the transverse displacement of the imaging beam during the time interval between a pair of OCT signal measurements must be significantly reduced to minimize the noise due to the beam scanning-induced phase decorrelation at the expense of the imaging speed. Recent introduction of dual-beam scan method either using polarization encoding or two identical imaging systems in spectral-domain (SD) OCT scheme shows potential for high-sensitivity vasculature imaging without suffering from spurious phase noise caused by the beam scanning-induced spatial decorrelation. In this paper, we present multi-functional angiographic optical frequency domain imaging (OFDI) using frequency-multiplexed dual-beam illumination. This frequency multiplexing scheme, utilizing unique features of OFDI, provides spatially separated dual imaging beams occupying distinct electrical frequency bands that can be demultiplexed in the frequency domain processing. We demonstrate the 3D multi-functional imaging of the normal mouse skin in the dorsal skin fold chamber visualizing distinct layer structures from the intensity imaging, information about mechanical integrity from the polarization-sensitive imaging, and depth-resolved microvasculature from the angiographic imaging that are simultaneously acquired and automatically co-registered.

Complex differential variance algorithm for optical coherence tomography angiography.

We describe a complex differential variance (CDV) algorithm for optical coherence tomography based angiography. The algorithm exploits both the intensity and phase changes of optical coherence tomography (OCT) signals from flowing blood to achieve high vascular contrast, and also intrinsically reject undesirable phase signals originating from small displacement axial bulk tissue motion and instrument synchronization errors. We present this algorithm within a broader discussion of the properties of OCT signal dynamics. The performance of the algorithm is compared against two other existing algorithms using both phantom measurements and in vivo data. We show that the algorithm provides better contrast for a given number of measurements and equivalent spatial averaging.

Monte Carlo modeling of angiographic optical coherence tomography.

Optical coherence tomography (OCT) provides both structural and angiographic imaging modes. Because of its unique capabilities, OCT-based angiography has been increasingly adopted into small animal and human subject imaging. To support the development of the signal and image processing algorithms on which OCT-based angiography depends, we describe here a Monte Carlo-based model of the imaging approach. The model supports arbitrary three-dimensional vascular network geometries and incorporates methods to simulate OCT signal temporal decorrelation. With this model, it will be easier to compare the performance of existing and new angiographic signal processing algorithms, and to quantify the accuracy of vascular segmentation algorithms. The quantitative analysis of key algorithms within OCT-based angiography may, in turn, simplify the selection of algorithms in instrument design and accelerate the pace of new algorithm development.