Tracking Dynamic Microvascular Changes during Healing after Complete Biopsy Punch on the Mouse Pinna Using Optical Microangiography

Optical microangiography (OMAG) and Doppler optical microangiography (DOMAG) are two non-invasive techniques capable of determining the tissue microstructural content, microvasculature angiography, and blood flow velocity and direction. These techniques were used to visualize the acute and chronic microvascular and tissue responses upon an injury in vivo. A tissue wound was induced using a 0.5 mm biopsy punch on a mouse pinna. The changes in the microangiography, blood flow velocity and direction were quantified for the acute (<30 min) wound response and the changes in the tissue structure and microangiography were determined for the chronic wound response (30 min–60 days). The initial wound triggered recruitment of peripheral capillaries, as well as redirection of main arterial and venous blood flow within 3 min. The complex vascular networks and new vessel formation were quantified during the chronic response using fractal dimension. The highest rate of wound closure occurred between days 8 and 22. The vessel tortuosity increased during this time suggesting angiogenesis. Taken together, these data signify that OMAG has the capability to track acute and chronic changes in blood flow, microangiography and structure during wound healing. The use of OMAG has great potential to improve our understanding of vascular and tissue responses to injury in order to develop more effective therapeutics.     

In vivo blood flow imaging of inflammatory human skin induced by tape stripping using optical microangiography

Vasculature response is a hallmark for most inflammatory skin disorders. Tape stripping on human skin causes a minor inflammation which leads to changes in microvasculature. In this study, optical microangiography (OMAG), noninvasive volumetric microvasculature in vivo imaging method, has been used to track the vascular responses after tape stripping. Vessel density has been quantified and used to correlate with the degree of skin irritation. The proved capability of OMAG technique in visualizing the microvasculature network under inflamed skin condition can play an important role in clinical trials of treatment and diagnosis of inflammatory skin disorders. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Application of Thinned-Skull Cranial Window to Mouse Cerebral Blood Flow Imaging Using Optical Microangiography

In vivo imaging of mouse brain vasculature typically requires applying skull window opening techniques: open-skull cranial window or thinned-skull cranial window. We report non-invasive 3D in vivo cerebral blood flow imaging of C57/BL mouse by the use of ultra-high sensitive optical microangiography (UHS-OMAG) and Doppler optical microangiography (DOMAG) techniques to evaluate two cranial window types based on their procedures and ability to visualize surface pial vessel dynamics. Application of the thinned-skull technique is found to be effective in achieving high quality images for pial vessels for short-term imaging, and has advantages over the open-skull technique in available imaging area, surgical efficiency, and cerebral environment preservation. In summary, thinned-skull cranial window serves as a promising tool in studying hemodynamics in pial microvasculature using OMAG or other OCT blood flow imaging modalities.     

In vivo detection of tumor boundary using ultrahigh‐resolution optical coherence angiography and fluorescence imaging

Accurate detection of early tumor margin is of great preclinical and clinical implications for predicting the survival rate of subjects and assessing the response of tumor microenvironment to chemotherapy or radiation therapy. Here, we report a multimodality optical imaging study on in vivo detection of tumor boundary by analyzing neoangiogenesis of tumor microenvironment (microangiography), microcirculatory blood flow (optical Doppler tomography) and tumor proliferation (green fluorescent protein [GFP] fluorescence). Microangiography demonstrates superior sensitivity (77.7 ± 6.4%) and specificity (98.2 ± 1.7%) over other imaging technologies (eg, optical coherence tomography) for tumor margin detection. Additionally, we report longitudinal in vivo imaging of tumor progression and show that the abrupt tumor cell proliferation did not occur until local capillary density and cerebral blood flow reached their peak approximately 2 weeks after tumor implantation. The unique capability of longitudinal multimodality imaging of tumor angiogenesis may provide new insights in tumor biology and in vivo assessment of the treatment effects on anti‐angiogenesis therapy for brain cancer.     

Monitoring Hypoxia Induced Changes in Cochlear Blood Flow and Hemoglobin Concentration Using a Combined Dual-Wavelength Laser Speckle Contrast Imaging and Doppler Optical Microangiography System

A synchronized dual-wavelength laser speckle contrast imaging (DWLSCI) system and a Doppler optical microangiography (DOMAG) system was developed to determine several ischemic parameters in the cochlea due to a systemic hypoxic challenge. DWLSCI can obtain two-dimensional data, and was used to determine the relative changes in cochlear blood flow, and change in the concentrations of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) in mice. DOMAG can obtain three-dimensional data, and was used to determine the changes in cochlear blood flow with single vessel resolution. It was demonstrated that during a hypoxic challenge there was an increase in the concentrations of Hb, a decrease in the concentrations of HbO and cochlear blood flow, and a slight decrease in the concentration of HbT. Also, the rate of change in the concentrations of Hb and HbO was quantified during and after the hypoxic challenge. The ability to simultaneously measure these ischemic parameters with high spatio-temporal resolution will allow the detailed quantitative analysis of several hearing disorders, and will be useful for diagnosing and developing treatments.     

Endoscopic optical coherence tomography angiography using a forward imaging piezo scanner probe

A forward imaging endoscope for optical coherence tomography angiography (OCTA) featuring a piezoelectric fiber scanner is presented. Imaging is performed with an optical coherence tomography (OCT) system incorporating an akinetic light source with a center wavelength of 1300 nm, bandwidth of 90 nm and A‐line rate of 173 kHz. The endoscope operates in contact mode to avoid motion artifacts, in particular, beneficial for OCTA measurements, and achieves a transversal resolution of 12 μm in air at a rigid probe size of 4 mm in diameter and 11.3 mm in length. A spiral scan pattern is generated at a scanning frequency of 360 Hz to sample a maximum field of view of 1.3 mm. OCT images of a human finger as well as visualization of microvasculature of the human palm are presented both in two and three dimensions. The combination of morphological tissue contrast with qualitative dynamic blood flow information within this endoscopic imaging approach potentially enables improved early diagnostic capabilities of internal organs for diseases such as bladder cancer.      

Responses of peripheral blood flow to acute hypoxia and hyperoxia as measured by optical microangiography

Oxygen availability is regarded as a critical factor to metabolically regulate systemic blood flow. There is a debate as to how peripheral blood flow (PBF) is affected and modulated during hypoxia and hyperoxia; however in vivo evaluating of functional PBF under oxygen-related physiological perturbation remains challenging. Microscopic observation, the current frequently used imaging modality for PBF characterization often involves the use of exogenous contrast agents, which would inevitably perturb the intrinsic physiologic responses of microcirculation being investigated. In this paper, optical micro-angiography (OMAG) was employed that uses intrinsic optical scattering signals backscattered from blood flows for imaging PBF in skeletal muscle challenged by the alteration of oxygen concentration. By utilizing optical reflectance signals, we demonstrated that OMAG is able to show the response of hemodynamic activities upon acute hypoxia and hyperoxia, including the modulation of macrovascular caliber, microvascular density, and flux regulation within different sized vessels within skeletal muscle in mice in vivo. Our results suggest that OMAG is a promising tool for in vivo monitoring of functional macro- or micro-vascular responses within peripheral vascular beds.     

Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy

Using high-resolution intravital charge-coupled device video microscopy, we visualized the epicardial capillary network of the beating canine heart in vivo to elucidate its functional role under control conditions, during reactive hyperemia (RH), and during intracoronary adenosine administration. The pencil-lens video-microscope probe was placed over capillaries fed by the left anterior descending artery in atrioventricular-blocked hearts of open-chest, anesthetized dogs paced at 60-90 beats/min (n = 17). In individual capillaries under control conditions, red blood cell flow was predominant during systole or diastole, indicating that the watershed between diastolic arterial and systolic venous flows is located within the capillaries. Capillary flow increased during RH and reached a peak flow velocity (2.1 +/- 0.6 mm/s), twice as high as control (1.2 +/- 0.5 mm/s), with enhancement of intercapillary cross-connection flow and enlargement of diameter (by 17%). With adenosine, capillary flow velocity significantly increased (1.8 +/- 0.7 mm/s). However, the increase in volumetric capillary flow with adenosine estimated from red blood cell velocity and diameter was less than the increase in arterial flow, whereas that during RH was nearly equivalent to the increase in arterial flow. There was a time lag of approximately 1.5 s for refilling of capillaries during RH, indicating their function as capacitance vessels. In conclusion, the coronary capillary network functions as 1) the major watershed between diastolic-dominant arterial and systolic-dominant venous flows, 2) a capacitor, and 3) a significant local flow amplifier and homogenizer of blood supply during RH, but with adenosine the increase in capillary flow velocity was less than the increase in arterial flow.     

Retinal pulse wave velocity measurement using spectral‐domain optical coherence tomography

The human eyes provide a natural window for noninvasive measurement of the pulse wave velocity (PWV) of small arteries. By measuring the retinal PWV, the stiffness of small arteries can be assessed, which may better detect early vascular diseases. Therefore, retinal PWV measurement has attracted increasing attention. In this study, a jump‐scanning method was proposed for noninvasive measurement of retinal PWV using spectral‐domain optical coherence tomography (SD‐OCT). The jump‐scanning method uses the phase‐resolved Doppler OCT to obtain the pulse shapes. To realize PWV measurement, the jump‐scanning method extracts the transit time of the pulse wave from an original OCT scanning site to another through a transient jump. The measured retinal arterial PWV of a young human subject with normal blood pressure was in the order of 20 to 30 mm/s, which was consistent with previous studies. As a comparison, PWV of 50 mm/s was measured for a young human subject with prehypertension, which was in accordance with the finding of strong association between retinal PWV and blood pressure. In summary, it is believed the proposed jump‐scanning method could benefit the research and diagnosis of vascular diseases through the window of human eyes.      

Shear‐induced diffusion of red blood cells measured with dynamic light scattering‐optical coherence tomography

Quantitative measurements of intravascular microscopic dynamics, such as absolute blood flow velocity, shear stress and the diffusion coefficient of red blood cells (RBCs), are fundamental in understanding the blood flow behavior within the microcirculation, and for understanding why diffuse correlation spectroscopy (DCS) measurements of blood flow are dominantly sensitive to the diffusive motion of RBCs. Dynamic light scattering‐optical coherence tomography (DLS‐OCT) takes the advantages of using DLS to measure particle flow and diffusion within an OCT resolution‐constrained three‐dimensional volume, enabling the simultaneous measurements of absolute RBC velocity and diffusion coefficient with high spatial resolution. In this work, we applied DLS‐OCT to measure both RBC velocity and the shear‐induced diffusion coefficient within penetrating venules of the somatosensory cortex of anesthetized mice. Blood flow laminar profile measurements indicate a blunted laminar flow profile and the degree of blunting decreases with increasing vessel diameter. The measured shear‐induced diffusion coefficient was proportional to the flow shear rate with a magnitude of ~0.1 to 0.5 × 10^?6 mm². These results provide important experimental support for the recent theoretical explanation for why DCS is dominantly sensitive to RBC diffusive motion.      

Blood Flow of the Acral Finger Arterioles in Patients With Type 2 Diabetes by Quality Doppler Profiles

Patients with diabetes mellitus exhibit peripheral arterioles lesions that is associated with reduced blood flow. Here, we intended to assess the acral arterioles lesion in patients with type 2 diabetes based on the rate of blood flow by multigate spectral Doppler ultrasonography. Fifty-two patients with type 2 DM were divided into two groups. Group 1 included 13 men and 12 women with an average age of 60.60 ± 14.03 years and a duration of type 2 diabetes for 2.44 ± 1.50 years. Group 2 included 17 men and 11 women with an average age of 64.25 ± 10.84 years and type 2 diabetes for 12.57 ± 6.26 years. Age-matched control subjects (n = 52) were recruited (30 men and 22 woman, mean age of 61.19 ± 10.38 years). A multigate spectral Doppler algorithm was applied to the acral finger of the thumb of the right hand to test the arteriole diameter and hemodynamic parameters, including diameter of the acral finger arterioles (D), area of the blood flow profile of the acral finger arterioles (A _max) and hemodynamic parameters. Patients with diabetes exhibited a significant reduction in the arteriole diameter (1.63 ± 0.18 and 1.57 ± 0.22 mm, respectively, P < 0.001 for both) compared to control subjects (2.09 ± 0.17 mm). A _max were significantly reduced in patients with diabetes (61.35 ± 10.66 mm²/s for group 1 and 46.50 ± 6.59 mm²/s for group 2, P < 0.001 for both) compared to that in control subjects (77.93 ± 12.37 mm²/s). Furthermore, a significant difference in Amax was found between group 1 and group 2 (P < 0.001). The vascular resistance index (RI) was significantly higher in both patient groups 0.58 ± 0.06 for group 1 (P < 0.001) and 0.64 ± 0.07 for group 2 (P < 0.001) than that in control subjects (0.48 ± 0.04). The RI value of the acral finger arterioles differed significantly between group 1 and group 2 (P < 0.01). Diabetic patients exhibited a weak blood flow in the acral finger arterioles. The multigate spectral Doppler technology can be used to test blood flow in the acral finger arterioles and provide hemodynamic data for systematic analyses of the peripheral arteriole lesions in diabetes.     

Cuffing‐based photoacoustic flowmetry in humans in the optical diffusive regime

Measuring blood flow speed in the optical diffusive regime in humans has been a long standing challenge for photoacoustic tomography. In this work, we proposed a cuffing‐based method to quantify blood flow speed in humans with a handheld photoacoustic probe. By cuffing and releasing the blood vessel, we can measure the blood flow speed downstream. In phantom experiments, we demonstrated that the minimum and maximum measurable flow speeds were 0.035 mm/s and 42 mm/s, respectively. In human experiments, flow speeds were measured in three different blood vessels: a radial artery in the right forearm, a radial artery in the index finger of the right hand, and a radial vein in the right forearm. Taking advantage of the handheld probe, our method can potentially be used to monitor blood flow speed in the clinic and at the bedside.

     

Integrated photoacoustic and diffuse optical tomography system for imaging of human finger joints in vivo

In this study, we developed a dual‐modality tomographic system that integrated photoacoustic imaging (PAI) and diffuse optical tomography (DOT) into a single platform for imaging human finger joints with fine structures and associated optical properties. In PAI, spherical focused transducers were utilized to collect acoustic signals, and the concept of virtual detector was applied in a conventional back‐projection algorithm to improve the image quality. A finite‐element based reconstruction algorithm was employed to quantitatively recover optical property distribution in the objects for DOT. The phantom results indicate that PAI has a maximum lateral resolution of 70 µm in resolving structures of targets. DOT was able to recover both optical absorption and reduced scattering coefficients of targets accurately. To validate the potential of this system in clinical diagnosis of joint diseases, the distal interphalangeal (DIP) joints of 4 healthy female volunteers were imaged. We successfully obtained high‐resolution images of the phalanx and the surrounding soft tissue via PAI, and recovered both optical absorption and reduced scattering coefficients of phalanx using DOT. The in vivo results suggest that this dual‐modality system has the potential for the early diagnosis of joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA).

Integrated PAI/DOT imaging interface (top) and typical reconstruction of structures and associated optical properties of a female finger joint via PAI and DOT (bottom).     

Secondary flow mixing due to biofilm growth in capillaries of varying dimensions

Using a magnetic resonance microscopy (MRM) technique, velocity perturbations due to biofouling in capillaries were detected in 3D velocity maps. The velocity images in each of the three square capillary sizes (2, 0.9, and 0.5 mm i.d.) tested indicate secondary flow in both the x‐ and y‐directions for the biofouled capillaries. Similar flow maps generated in a clean square capillary show only an axial component. Investigation of these secondary flows and their geometric and dynamic similarity is the focus of this study. The results showed significant secondary flows present in the 0.9 mm i.d. capillary, on the scale of 20% of the bulk fluid flow. Since this is the “standard 1 mm” size capillary used in confocal microscopy laboratory bioreactors to investigate biofilm properties, it is important to understand how these enhanced flows impact bioreactor transport. Biotechnol. Bioeng. 2009;103: 353–360. © 2009 Wiley Periodicals, Inc.     

Exaggerated postural vasoconstrictor reflex in Raynaud's phenomenon.

The central and local regulation of capillary blood flow in the finger was studied by the local xenon-133 washout technique in women with primary Raynaud''s phenomenon, men with vibration induced white finger, and their respective sex matched controls. The vasoconstrictor response to venous stasis of 40 mm Hg elicited by local reflex was normal in both types of Raynaud''s phenomenon. Change in posture from lying to sitting induced vasoconstriction in all groups, which was abolished by proximal nervous blockade. The vasoconstrictor response to sitting was augmented in both groups of subjects with Raynaud''s phenomenon compared with their sex matched controls. These results show the existence of central and local postural vasoconstrictor reflexes in normal fingers. In both types of Raynaud''s phenomenon there was hyperreactivity of the central sympathetic nervous system to orthostatic stress and normal function of digital arterioles and postganglionic sympathetic nerve fibres.     

The use of intensity‐based Doppler variance method for single vessel response to functional neurovascular activation

This study presents 1 use of optical coherence tomography (OCT) angiography technique to examine neurovascular coupling effect. Repeated B‐scans OCT recording is performed on the rat somatosensory cortex with cranial window preparation while its contralateral forepaw is electrically stimulated to activate the neurons in rest. We use an intensity‐based Doppler variance (IBDV) algorithm mapped cerebral blood vessels in the cortex, and the temporal alteration in blood perfusion during neurovascular activation is analyzed using the proposed IBDV quantitative parameters. By using principal component analysis‐based Fuzzy C Means clustering method, the stimulus‐evoked vasomotion patterns were classified into 3 categories. We found that the response time of small vessels (resting diameter 14.9 ±6.6 μm), middle vessels (resting diameter 21.1 ±7.9 μm) and large vessels (resting diameter 50.7 ±6.5 μm) to achieve 5% change of vascular dilation after stimulation was 1.5, 2 and 5.5 seconds, respectively. Approximately 5% peak change of relative blood flow (RBF) in both small and middle vessels was observed. The large vessels react slowly and their responses nearly 4 seconds delayed, but no significant change in RBF of the large vessels was seen.      

Portable optical‐resolution photoacoustic microscopy for volumetric imaging of multiscale organisms

Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical‐resolution photoacoustic microscopy (pORPAM) system for 3‐dimensional (3D) imaging of small‐to‐large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer‐associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases.      

Quantitative measurement of blood velocity in zebrafish with optical vector field tomography

Microscopy techniques can readily visualize the finest details of embryo vasculature, but still lack to provide a complete three‐dimensional representation of blood flow parameters. We present an in‐vivo 3D imaging technique, able to reconstruct the blood cell velocity vector over a large volume of zebrafish embryos. This low cost and relatively simple technique is exploited to quantitatively assess blood velocity in the zebrafish tail at different stages of development. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (<10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R²>0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.     

Simultaneous measurement of water flow velocity and solute transport in xylem and phloem of adult plants of Ricinus communis over a daily time course by nuclear magnetic resonance spectrometry

A new method for simultaneously quantifying rates of flow in xylem and phloem using the FLASH imaging capabilities of nuclear magnetic resonance (NMR) spectrometry was applied in this study. The method has a time resolution of up to 4 min (for the xylem) and was used to measure the velocity of flows in phloem and xylem for periods of several hours to days. For the first time, diurnal time course measurements of flow velocities and apparent volume flows in phloem and xylem in the hypocotyl of 40‐d‐old Ricinus communis L were obtained. Additional data on gas exchange and the chemical composition of leaves, xylem and phloem sap were used to assess the role of leaves as sinks for xylem sap and sources for phloem. The velocity in the phloem (0·250 ± 0·004 mm s^?1) was constant over a full day and not notably affected by the light/dark cycle. Sucrose was loaded into the phloem and transported at night, owing to degradation of starch accumulated during the day. Concentrations of solutes in the phloem were generally less during the night than during the day but varied little within either the day or night. In contrast to the phloem, flow velocities in the xylem were about 1·6‐fold higher in the light (0·401 ± 0·004 mm s^?1) than in the dark (0·255 ± 0·003 mm s^?1) and volume flow varied commensurately. Larger delays were observed in changes to xylem flow velocity with variation in light than in gas exchange. The relative rates of solute transport during day and night were estimated on the basis of relative flow and solute concentrations in xylem and phloem. In general, changes in relative flow rates were compensated for by changes in solute concentration during the daily light/dark cycle. However, the major solutes (K⁺, NO₃^?) varied appreciably in relative concentrations. Hence the regulation of loading into transport systems seems to be more important to the overall process of solute transport than do changes in mass flow. Due to transport behaviour, the chemical composition of leaves varied during the day only with regard to starch and soluble carbohydrates.