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.

Phase-stable Doppler OCT at 19 MHz using a stretched-pulse mode-locked laser.

We present a swept-wavelength optical coherence tomography (OCT) system with a 19 MHz laser source and electronic phase-locking of the source, acquisition clock, and beam scanning mirrors. The laser is based on stretched-pulse active mode-locking using an electro-optic modulator. Beam scanning in the fast axis uses a resonant micro-electromechanical systems (MEMS) -based mirror at ~23.8 kHz. Acquisition is performed at 1.78 Gigasamples per second using an external fixed clock. Phase sensitive imaging without need for k-clocking, A-line triggers, or phase-calibration methods is demonstrated. The system was used to demonstrate inter-frame and inter-volume Doppler imaging in the mouse ear and brain at 4D acquisition rates of 1, 30, 60 and 100 volumes/sec (V-scans/s). Angiography based on inter-frame and inter-volume methods are presented. The platform offers extremely fast and phase-stable measurements that can be used in preclinical angiographic and Doppler investigations of perfusion dynamics.

Methicillin-resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema-fluid accumulation in tissue caused by impaired lymphatic vessel function-is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and the cause of lymphatic vessel dysfunction is not known. Intravital imaging in mice revealed an acute reduction in both lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation with MRSA-conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)-controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild-type MRSA. Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.

Lymphatic function measurements influenced by contrast agent volume and body position.

Several imaging modalities have been used to assess lymphatic function, including fluorescence microscopy, near-infrared fluorescence (NIRF) imaging, and Doppler optical coherence tomography (DOCT). They vary in how the mouse is positioned, the invasiveness of the experimental setup, and the volume of contrast agent injected. Here, we present how each of these experimental parameters affects functional measurements of collecting lymphatic vessels. First, fluorescence microscopy showed that supine mice have a statistically lower contraction frequency compared with mice sitting upright. To assess the effect of different injection volumes on these endpoints, mice were injected with 4, 10, or 20 µl of dye. The lowest frequencies were observed after 20-µl injections. Interestingly, lymph-flow DOCT revealed that although there was lower contraction frequency in mice injected with 20 µl versus 4 µl, mice showed a higher volumetric flow with a 20-µl injection. This indicates that contraction frequency alone is not sufficient to understand lymphatic transport. Finally, NIRF revealed that removing the skin reduced contraction frequency. Therefore, this study reveals how sensitive these techniques are to mouse position, removal of skin, and dye volume. Care should be taken when comparing results obtained under different experimental conditions.

Simultaneous measurements of lymphatic vessel contraction, flow and valve dynamics in multiple lymphangions using optical coherence tomography.

Lymphatic dysfunction is involved in many diseases including lymphedema, hypertension, autoimmune responses, graft rejection, atherosclerosis, microbial infections, cancer and cancer metastasis. Expanding our knowledge of lymphatic system function can lead to a better understanding of these disease processes and improve treatment options. Here, optical coherence tomography (OCT) methods were used to reveal intraluminal valve dynamics in 3 dimensions, and measure lymph flow and vessel contraction simultaneously in 3 neighboring lymphangions of the afferent collecting lymphatic vessels to the popliteal lymph node in mice. Flow measurements were based on Doppler OCT techniques in combination with exogenous lymph labeling by Intralipid. Through these imaging methods, it is possible to study lymphatic function and pumping more comprehensively. These capabilities can lead to a better understanding of the regulation and dysregulation of lymphatic vessels in health and disease. The image depicts the dynamic measurements of lymphatic valves, lymphatic vessels cross-sectional area and lymph velocity simultaneously measured in vivo with optical coherence tomography.

Murine chronic lymph node window for longitudinal intravital lymph node imaging.

Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice. The CLNW is custom-made from titanium and incorporates a standard coverslip. It allows stable longitudinal imaging without the need for serial surgeries while preserving lymph node blood and lymph flow. We also describe how to build and use an imaging stage specifically designed for the CLNW to prevent (large) rotational changes as well as respiratory movement during imaging. The entire procedure takes approximately half an hour per mouse, and subsequently allows for longitudinal intravital imaging of the murine lymph node and surrounding structures for up to 14 d. Small-animal surgery experience is required to successfully carry out the protocol.

Lymph node effective vascular permeability and chemotherapy uptake.

OBJECTIVE: Lymph node metastases are a poor prognostic factor. Additionally, responses of lymph node metastasis to therapy can be different from the primary tumor. Investigating the physiologic lymph node blood vasculature might give insight into the ability of systemic drugs to penetrate the lymph node, and thus into the differential effect of therapy between lymph node metastasis and primary tumors. Here, we measured effective vascular permeability of lymph node blood vessels and attempted to increase chemotherapy penetration by increasing effective vascular permeability. METHODS: We developed a novel three-dimensional method to measure effective vascular permeability in murine lymph nodes in vivo. VEGF-A was systemically administered to increase effective vascular permeability. Validated high-performance liquid chromatography protocols were used to measure chemotherapeutic drug concentrations in untreated and VEGF-A-treated lymph nodes, liver, spleen, brain, and blood. RESULTS: VEGF-A-treated lymph node blood vessel effective vascular permeability (mean 3.83 × 10-7  cm/s) was significantly higher than untreated lymph nodes (mean 9.87 × 10-8  cm/s). No difference was found in lymph node drug accumulation in untreated versus VEGF-A-treated mice. CONCLUSIONS: Lymph node effective vascular permeability can be increased (~fourfold) by VEGF-A. However, no significant increase in chemotherapy uptake was measured by pretreatment with VEGF-A.

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.

Line-field parallel swept source MHz OCT for structural and functional retinal imaging.

We demonstrate three-dimensional structural and functional retinal imaging with line-field parallel swept source imaging (LPSI) at acquisition speeds of up to 1 MHz equivalent A-scan rate with sensitivity better than 93.5 dB at a central wavelength of 840 nm. The results demonstrate competitive sensitivity, speed, image contrast and penetration depth when compared to conventional point scanning OCT. LPSI allows high-speed retinal imaging of function and morphology with commercially available components. We further demonstrate a method that mitigates the effect of the lateral Gaussian intensity distribution across the line focus and demonstrate and discuss the feasibility of high-speed optical angiography for visualization of the retinal microcirculation.

Doppler optical coherence tomography.

Optical Coherence Tomography (OCT) has revolutionized ophthalmology. Since its introduction in the early 1990s it has continuously improved in terms of speed, resolution and sensitivity. The technique has also seen a variety of extensions aiming to assess functional aspects of the tissue in addition to morphology. One of these approaches is Doppler OCT (DOCT), which aims to visualize and quantify blood flow. Such extensions were already implemented in time domain systems, but have gained importance with the introduction of Fourier domain OCT. Nowadays phase-sensitive detection techniques are most widely used to extract blood velocity and blood flow from tissues. A common problem with the technique is that the Doppler angle is not known and several approaches have been realized to obtain absolute velocity and flow data from the retina. Additional studies are required to elucidate which of these techniques is most promising. In the recent years, however, several groups have shown that data can be obtained with high validity and reproducibility. In addition, several groups have published values for total retinal blood flow. Another promising application relates to non-invasive angiography. As compared to standard techniques such as fluorescein and indocyanine-green angiography the technique offers two major advantages: no dye is required and depth resolution is required is provided. As such Doppler OCT has the potential to improve our abilities to diagnose and monitor ocular vascular diseases.

Angle independent flow assessment with bidirectional Doppler optical coherence tomography.

Bidirectional Doppler optical coherence tomography is a stable and accurate method to extract the absolute velocity of vessels close to perpendicular to the optical axis by illuminating the sample under two different angles. However it requires knowledge of the vessel angle in the en face plane. In this Letter, we demonstrate that a direct calculation of the flow out of bidirectional Doppler cross sections perpendicular to the illumination plane is independent of that angle and of the Doppler angle, thereby improving the accuracy and flexibility of that technique. We validate our approach with an in vitro experiment and in vivo measurements of a human retinal vessel and discuss the practical limitations of this approach. The method yields accurate flow values for most vascular plexuses without precise knowledge of the vessel orientation. The precision gradually decreases for larger en face angles.

Dove prism based rotating dual beam bidirectional Doppler OCT.

Traditional Doppler OCT is highly sensitive to motion artifacts due to the dependence on the Doppler angle. This limits its accuracy in clinical practice. To overcome this limitation, we use a bidirectional dual beam technique equipped with a novel rotating scanning scheme employing a Dove prism. The volume is probed from two distinct illumination directions with variable controlled incidence plane, allowing for reconstruction of the true flow velocity at arbitrary vessel orientations. The principle is implemented with Swept Source OCT at 1060nm with 100,000 A-Scans/s. We apply the system to resolve pulsatile retinal absolute blood velocity by performing segment scans around the optic nerve head and circumpapillary scan time series.

Intrasweep phase-sensitive optical coherence tomography for noncontact optical photoacoustic imaging.

We introduce a method to extract the photoacoustic (PA) signal from the phase time evolution of an optical coherence tomography (OCT) swept source spectral sweep. This all-optical detection is achieved in a noncontact fashion directly on the sample surface by using its specular reflection. High-speed measurement and referencing allow for close to shot noise limited phase-sensitive detection. It offers a simple way to perform OCT and PA imaging by sharing the same system components.

In situ structural and microangiographic assessment of human skin lesions with high-speed OCT.

We demonstrate noninvasive structural and microvascular contrast imaging of different human skin diseases in vivo using an intensity difference analysis of OCT tomograms. The high-speed swept source OCT system operates at 1310 nm with 220 kHz A-scan rate. It provides an extended focus by employing a Bessel beam. The studied lesions were two cases of dermatitis and two cases of basal cell carcinoma. The lesions show characteristic vascular patterns that are significantly different from healthy skin. In case of inflammation, vessels are dilated and perfusion is increased. In case of basal cell carcinoma, the angiogram shows a denser network of unorganized vessels with large vessels close to the skin surface. Those results indicate that assessing vascular changes yields complementary information with important insight into the metabolic demand.

Ultrahigh-speed non-invasive widefield angiography.

Retinal and choroidal vascular imaging is an important diagnostic benefit for ocular diseases such as age-related macular degeneration. The current gold standard for vessel visualization is fluorescence angiography. We present a potential non-invasive alternative to image blood vessels based on functional Fourier domain optical coherence tomography (OCT). For OCT to compete with the field of view and resolution of angiography while maintaining motion artifacts to a minimum, ultrahigh-speed imaging has to be introduced. We employ Fourier domain mode locking swept source technology that offers high quality imaging at an A-scan rate of up to 1.68 MHz. We present retinal angiogram over ∼48 deg acquired in a few seconds in a single recording without the need of image stitching. OCT at 1060 nm allows for high penetration in the choroid and efficient separate characterization of the retinal and choroidal vascularization.

Extended focus high-speed swept source OCT with self-reconstructive illumination.

We present a Bessel beam illumination FDOCT setup using a FDML Swept Source at 1300 nm with up to 440 kHz A-scan rate, and discuss its advantages for structural and functional imaging of highly scattering samples. An extended focus is achieved due to the Bessel beam that preserves its lateral extend over a large depth range. Furthermore, Bessel beams exhibit a self-reconstruction property that allows imaging even behind obstacles such as hairs on skin. Decoupling the illumination from the gaussian detection increases the global sensitivity and enables dark field imaging. Dark field imaging is useful to avoid strong reflexes from the sample surface that adversely affect the sensitivity due to the limited dynamic range of high speed 8 bit acquisition cards. In addition the possibility of contrasting capillaries with high sensitivity is shown, using inter-B-scan speckle variance analysis. We demonstrate intrinsic advantages of the extended focus configuration, in particular the reduction of the phase decorrelation effect below vessels leading to improved axial vessel definition.

Imaging of the parafoveal capillary network and its integrity analysis using fractal dimension.

Using a spectral domain OCT system, equipped with a broadband Ti:sapphire laser, we imaged the human retina with 5 µm x 1.3 µm transverse and axial resolution at acquisition rate of 100 kHz. Such imaging speed significantly reduces motion artifacts. Combined with the ultra-high resolution, this allows observing microscopic retinal details with high axial definition without the help of adaptive optics. In this work we apply our system to image the parafoveal capillary network. We demonstrate how already on the intensity level the parafoveal capillaries can be segmented by a simple structural high pass filtering algorithm. This data is then used to quantitatively characterize the capillary network of healthy and diseased eyes. We propose to use the fractal dimension as index for capillary integrity of pathologic disorders.

Vectorial reconstruction of retinal blood flow in three dimensions measured with high resolution resonant Doppler Fourier domain optical coherence tomography.

Resonant Doppler Fourier domain optical coherence tomography (FDOCT) is a functional imaging tool for extracting tissue flow. The method is based on the effect of interference fringe blurring in spectrometer-based FDOCT, where the path difference between structure and reference changes during camera integration. If the reference path length is changed in resonance with the Doppler frequency of the sample flow, the signals of resting structures will be suppressed, whereas the signals of blood flow are enhanced. This allows for an easy extraction of vascularization structure. Conventional flow velocity analysis extracts only the axial flow component, which strongly depends on the orientation of the vessel with respect to the incident light. We introduce an algorithm to extract the vessel geometry within the 3-D data volume. The algorithm calculates the angular correction according to the local gradients of the vessel orientations. We apply the algorithm on a measured 3-D resonant Doppler dataset. For validation of the reproducibility, we compare two independently obtained 3-D flow maps of the same volunteer and region.

Resonant Doppler flow imaging and optical vivisection of retinal blood vessels.

For Fourier domain optical coherence tomography any sample movement during camera integration causes blurring of interference fringes and as such reduction of sensitivity for flow detection. The proposed method overcomes this problem by phase-matching a reference signal to the sample motion. The interference fringes corresponding to flow signal will appear frozen across the detector whereas those of static sample structures will be blurred resulting in enhanced contrast for blood vessels. An electro-optic phase modulator in the reference arm, driven with specific phase cycles locked to the detection frequency, allows not only for qualitative but also for quantitative flow detection already from the relative signal intensities. First applications to extract in-vivo retinal flow and to visualize 3D vascularization, i.e. optical vivisection, are presented.