Rheological and flow properties of blood investigated by ultrasound

Ultrasonic waves of 1-15 MHz frequencies easily propagate through soft biological tissues, thus providing qualitative and quantitative information on mechanical and flow properties of blood and red blood cell (RBC) suspensions. Two types of techniques allow to investigate blood behaviors: echographic devices via amplitude detection and Doppler effect based devices via frequency detection of the ultrasonic signal. When ever B mode serves to construct images of tissue slabs from the ultrasonic backscattering coefficient and can give qualitative information on the mechanical properties of blood, A-mode allows to quantify the ultrasonic backscattering coefficient. Ultrasonic Doppler modes also provide both qualitative and quantitative information on blood flow velocity: continuous and pulsed Doppler modes provide curves of blood flow versus time when color Doppler and power Doppler imaging visualize blood flowing in human vessels. Association of echographic and Doppler modes to investigate simultaneously structure and velocity of blood is commercially available. Some examples of results given by such ultrasonic techniques that contribute to characterize, both in vitro and in vivo, structure and flow properties of blood or red blood cell (RBC) suspensions are presented.     

Ultrasound techniques for measurement of blood flow and tissue motion

This article will review the ability of ultrasound techniques to provide 3D information on arterial geometry, blood flow and tissue motion.3D systems. 3D datasets can be obtained by sequential acquisition of 2D slices. Ideally a transducer is required in which there is full control of beam steering within a 3D volume. This requires a 2D array consisting of several thousand elements. Prototype 2D arrays have been built which provide several 3D datasets per second.Blood velocity measurement. Current Doppler systems estimate only the component of velocity in the direction of the Doppler beam. Lack of knowledge of the direction of blood motion and also other effects associated with 'spectral broadening' limit the accuracy of velocity measurement. Improved accuracy can be obtained using vector Doppler systems using 2 or 3 beam directions; this approach is referred to as 'vector Doppler'.Tissue motion. Doppler techniques can also be used to detect tissue motion (Tissue Doppler Imaging or TDI). Motion of the artery wall can be calculated from the TDI images. It is possible to estimate simultaneously motion for adjacent diameters within the longitudinal plane, and to visualise the relative motion at different parts of the wall.     

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

Experimental nerve imaging at 1.5-T

Experimental lesions of the peripheral nerve system can be visualized in vivo by magnetic resonance imaging (MRI). Many studies of the rat peripheral nervous systems were performed on dedicated animal MR scanners with a high magnetic field strength for good spatial resolution. Here, we present an MR protocol to study experimental lesions of the rat nervous system with clinical 1.5-T MR scanners and commercially available coils. Using a three-sequence approach (T1-weighted imaging, fat-saturated T2-weighted imaging and fat-saturated T1-weighted imaging with Gd-DTPA in the same plane), the relevant signal changes of the lesioned nerve can be visualized and separated from other structures, e.g., blood vessels. Furthermore, we give an overview on different types of contrast agents used for peripheral nerve MR imaging and MR findings in selected experimental models of rat peripheral nerve injury.     

Doppler optical coherence tomography for measuring flow in engineered tissue

The engineering of human tissue represents a major paradigm shift in clinical medicine. Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures. However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required. This in turn will require the development of reliable on-line monitoring and control systems. This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship. Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo. Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro. As a base line, the current status of DOCT in vivo is therefore reviewed. This paper also reports the first preliminary use of DOCT in tissue engineering. The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ.     

What measurements are necessary for a comprehensive evaluation of the peripheral arterial circulation?

Several methods are available to detect atherosclerotic lesions with a severe degree of stenosis (>70%), but the diagnosis of atherosclerotic lesions with no stenosis or with a minor degree of stenosis (<20%), is problematic. Hemodynamics associated with stenotic lesions are well described by the relationship of blood pressure and blood flow velocity, both as a function of time and localization (along the length and cross-section of the vessel). The use of this relationship in the clinic is difficult because no precise information is available about the geometry and branching of arteries, blood viscosity, and the velocity distribution over the cross-sectional area of the blood vessel. Besides, the invasiveness of the technique to measure arterial pressure as a function of time and localization does not allow routine application in patients. Because of these limitations, alternative methods have been developed. The degree and extensiveness of atherosclerotic disease can, for instance, be estimated from the changes in maximum blood flow velocity and in velocity profile, i.e., velocity distribution along the cross-section of the vessel. Moreover, the delay between simultaneously recorded arterial blood flow velocity tracings (pulse-wave velocity determination) is used to assess the elastic properties of the vessel. Changes in velocity profile occur at relatively slight degrees of arterial stenosis (around 20%), so that determination of these profiles along diseased arteries may contribute to the early diagnosis of atherosclerotic lesions. In man, transcutaneous information about the maximum and mean blood flow velocities over the cross-sectional area of the artery as an instantaneous function of time as well as the flow pattern can be obtained online with continuous wave Doppler flowmeters, at least when audio spectrum analysis is used as a processing technique. Velocity profiles can be determined with multichannel pulsed Doppler systems if the resolution of the system is adequate and a sufficient number of sample volumes can be obtained, limiting the interpolation between these samples. The on-line recording of velocity profiles can be facilitated by combining the pulsed Doppler device with either a velocity imaging system or a B-mode scan. In systems with a high resolution (sample distance 0.5 mm), one should be able to detect local disturbances in the velocity profile at the site of the lesion (due to local increases in shear stress) and proximal to the lesion (due to reflections), so that lesions with a minor degree of stenosis can be detected. In resistive systems (e.g., internal carotid arteries) in which the relationship between pressure and velocity changes during the cardiac cycle is relatively simple, the elasticity of the arterial wall can be determined by relating the relative diameter changes of the vessel, determined on-line with multichannel pulsed Doppler systems, to the instantaneous velocity pulse. Although the detection of atherosclerotic lesions at an early stage of the disease with sophisticated Doppler devices looks promising, further clinical evaluation is required.     

Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics

Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.     

Multimodality registration without a dedicated multimodality scanner

Multimodality scanners that allow the acquisition of both functional and structural image sets on a single system have recently become available for animal research use. Although the resultant registered functional/structural image sets can greatly enhance the interpretability of the functional data, the cost of multimodality systems can be prohibitive, and they are often limited to two modalities, which generally do not include magnetic resonance imaging. Using a thin plastic wrap to immobilize and fix a mouse or other small animal atop a removable bed, we are able to calculate registrations between all combinations of four different small animal imaging scanners (positron emission tomography, single-photon emission computed tomography, magnetic resonance, and computed tomography [CT]) at our disposal, effectively equivalent to a quadruple-modality scanner. A comparison of serially acquired CT images, with intervening acquisitions on other scanners, demonstrates the ability of the proposed procedures to maintain the rigidity of an anesthetized mouse during transport between scanners. Movement of the bony structures of the mouse was estimated to be 0.62 mm. Soft tissue movement was predominantly the result of the filling (or emptying) of the urinary bladder and thus largely constrained to this region. Phantom studies estimate the registration errors for all registration types to be less than 0.5 mm. Functional images using tracers targeted to known structures verify the accuracy of the functional to structural registrations. The procedures are easy to perform and produce robust and accurate results that rival those of dedicated multimodality scanners, but with more flexible registration combinations and while avoiding the expense and redundancy of multimodality systems.     

Software controlling algorithms for the system performance optimization of confocal laser scanning microscope

The relative slow scanning speed of a galvanometer commonly used in a confocal laser scanning microscopy system can dramatically limit the system performance in scanning speed and image quality, if the data collection is simply synchronized with the galvanometric scanning. Several algorithms for the optimization of the galvanometric CLSM system performance are discussed in this work, with various hardware controlling techniques for the image distortion correction such as pixel delay and interlace line switching; increasing signal-to-noise ratio with data binning; or enhancing the imaging speed with region of interest imaging. Moreover, the pixel number can be effectively increased with Acquire-On-Fly scan, which can be used for the imaging of a large field-of-view with a high resolution.     

The choroid and glaucoma

The recent literature describing the relationship between the choroidal blood flow and glaucoma is reviewed. Color Doppler imaging, fundus pulsation amplitudes, videoangiographic studies and scanning laser Doppler flowmetry are examined.     

Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high‐resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cranial MRI of small rodents using a clinical MR scanner

Increasing numbers of small animal models are in use in the field of neuroscience research. Magnetic resonance imaging (MRI) provides an excellent method for non-invasive imaging of the brain. Using three-dimensional (3D) MR sequences allows lesion volumetry, e.g. for the quantification of tumor size. Specialized small-bore animal MRI scanners are available for high-resolution MRI of small rodents' brain, but major drawbacks of this dedicated equipment are its high costs and thus its limited availability. Therefore, more and more research groups use clinical MR scanners for imaging small animal models. But to achieve a reasonable spatial resolution at an acceptable signal-to-noise ratio with these scanners, some requirements concerning sequence parameters have to be matched. Thus, the aim of this paper was to present in detail a method how to perform MRI of small rodents brain using a standard clinical 1.5 T scanner and clinically available radio frequency coils to keep material costs low and to circumvent the development of custom-made coils.     

Nontraumatic aortic blood flow sensing by use of an ultrasonic esophageal probe.

The measurement of blood velocity fields, volume flow, and arterial wall motion in the descending thoracic aorta provides essential hemodynamic information for both research and clinical diagnosis. The close proximity of the esophagus to the aorta in the dog makes it possible to obtain such data nonsurgically using an ultrasonic esophageal probe; however, the accuracy of such a probe is limited if the angle between the sound beam and the flow axis, known as the Doppler angle, is not precisely known. By use of a pulsed Doppler velocity meter (PUDVM) and a triangulation procedure, accurate empirical measurement of the Doppler angle has been obtained, allowing quantification of blood velocity scans across the aorta. Volume flow is obtained by integration of blood velocity profiles and arterial wall motion is measured with an ultrasonic echo tracking device. Accuracy of the probe was substantiated by comparison with ultrasonic and electromagnetic implanted flow cuff measurements. Use of the probe in measurement of blood velocity, volume flow and arterial wall motion at various locations along the 8- and 10-cm length of the descending thoracic aorta in adult beagle dogs is detailed. The simplicity, accuracy, and nontraumatic aspect of the technique should allow increasing use of such a probe in numerous research and clinical applications.     

Doppler velocity measurements from large and small arteries of mice

With the growth of genetic engineering, mice have become increasingly common as models of human diseases, and this has stimulated the development of techniques to assess the murine cardiovascular system. Our group has developed nonimaging and dedicated Doppler techniques for measuring blood velocity in the large and small peripheral arteries of anesthetized mice. We translated technology originally designed for human vessels for use in smaller mouse vessels at higher heart rates by using higher ultrasonic frequencies, smaller transducers, and higher-speed signal processing. With these methods one can measure cardiac filling and ejection velocities, velocity pulse arrival times for determining pulse wave velocity, peripheral blood velocity and vessel wall motion waveforms, jet velocities for the calculation of the pressure drop across stenoses, and left main coronary velocity for the estimation of coronary flow reserve. These noninvasive methods are convenient and easy to apply, but care must be taken in interpreting measurements due to Doppler sample volume size and angle of incidence. Doppler methods have been used to characterize and evaluate numerous cardiovascular phenotypes in mice and have been particularly useful in evaluating the cardiac and vascular remodeling that occur following transverse aortic constriction. Although duplex ultrasonic echo-Doppler instruments are being applied to mice, dedicated Doppler systems are more suitable for some applications. The magnitudes and waveforms of blood velocities from both cardiac and peripheral sites are similar in mice and humans, such that much of what is learned using Doppler technology in mice may be translated back to humans.     

Improving Image Quality by Accounting for Changes in Water Temperature during a Photoacoustic Tomography Scan

The emerging field of photoacoustic tomography is rapidly evolving with many new system designs and reconstruction algorithms being published. Many systems use water as a coupling medium between the scanned object and the ultrasound transducers. Prior to a scan, the water is heated to body temperature to enable small animal imaging. During the scan, the water heating system of some systems is switched off to minimize the risk of bubble formation, which leads to a gradual decrease in water temperature and hence the speed of sound. In this work, we use a commercially available scanner that follows this procedure, and show that a failure to model intra-scan temperature decreases as small as 1.5°C leads to image artifacts that may be difficult to distinguish from true structures, particularly in complex scenes. We then improve image quality by continuously monitoring the water temperature during the scan and applying variable speed of sound corrections in the image reconstruction algorithm. While upgrading to an air bubble-free heating pump and keeping it running during the scan could also solve the changing temperature problem, we show that a software correction for the temperature changes provides a cost-effective alternative to a hardware upgrade. The efficacy of the software corrections was shown to be consistent across objects of widely varying appearances, namely physical phantoms, ex vivo tissue, and in vivo mouse imaging. To the best of our knowledge, this is the first study to demonstrate the efficacy of modeling temporal variations in the speed of sound during photoacoustic scans, as opposed to spatial variations as focused on by previous studies. Since air bubbles pose a common problem in ultrasonic and photoacoustic imaging systems, our results will be useful to future small animal imaging studies that use scanners with similarly limited heating units.     

基于直线电机的大视场快速光声显微成像系统

光声成像技术是近年来发展的一种新型的无损医学成像技术,它是以脉冲激光作为激发源,以检测的声信号为信息载体,通过相应的图像重建算法重建组织内部结构和功能信息的成像方法。该方法结合了光学成像和声学成像的特点,可提供深层组织高分辨率和高对比度的组织层析图像,在生物医学临床诊断以及在体成像领域具有广泛的应用前景。目前光声成像的扫描方式主要有基于步进电机扫描方式和基于振镜的扫描方式,本文针对目前步进电机扫描速度慢(10 mm×10 mm;0.001帧/s),振镜扫描范围小(1 mm2)的不足,发展了基于直线电机扫描的大视场快速光声显微成像系统。同一条扫描线过程中直线电机速度最高可达200 mm/s。该技术采用逐线采集光声信号的方式,比逐点采集光声信号的步进电机快800倍。该系统对10 mm×10 mm全场扫描的扫描速度为0.8帧/s。最大可扫描视场范围可以达到50 mm×50 mm。大视场快速光声显微成像系统的发展将为生物医学提供新的成像工具。     

基于直线电机连续-步进运动的快速光声显微成像系统

报道了一种利用直线电机连续-步进的扫描方式进行光声显微成像的系统,该系统在运动时走弓字型路线,其中直线电机在X轴方向上连续运动,在Y轴方向上以步进的方式运动,采集卡只在X轴电机运动的过程中连续采集。该成像系统较之前振镜扫描的方式扫描的范围更大,可达到厘米尺度范围内的生物组织光声成像;较之前的步进电机逐点扫描的方式扫描速度明显提高。同时本文采用电机带动光和超声换能器一同扫描的方式,较光和超声换能器不动电机带动样品扫描的方式更灵活。另外利用包埋碳丝的模拟样品和在体小鼠耳朵血管来验证系统的成像能力。实验结果表明,这种快速光声显微成像方法及其系统可以实现在体组织的高分辨率成像,有望成为一种无创、实时的光声显微镜应用于生物医学当中。     

Two‐photon focal modulation microscopy for high‐resolution imaging in deep tissue

Two‐photon microscopy (2PM) is one of the most widely used tools for in vivo deep tissue imaging. However, the spatial resolution and penetration depth are still limited due to the strong scattering background. Here we demonstrate a two‐photon focal modulation microscopy. By utilizing the modulation and demodulation techniques, background rejection capability is enhanced, thus spatial resolution and imaging penetration depth are improved. Compared with 2PM, the transverse resolution is increased by 70%, while the axial resolution is increased to 2‐fold. Furthermore, when applied in conventional 2PM mode, it can achieve inertial‐free scanning in either transverse or axial direction with in principle unlimited scanning speed. Finally, we applied 2PFMM in thick scattering samples to further examine the imaging performance. The results show that the signal‐to‐background ratio of 2PFMM can be improved up to five times of 2PM at the depth of 500 μm. Fluorescent imaging in the mouse brain tissue. 3D Thy1‐GFP hippocampal neurons imaged by (A) 2PM compared with (B) 2PFMM; (C‐H) xy maximum‐intensity projection imaged by 2PM compared with 2PFMM. Scale bar 50 μm.      

A deep‐learning‐based approach for noise reduction in high‐speed optical coherence Doppler tomography

Optical coherence Doppler tomography (ODT) increasingly attracts attention because of its unprecedented advantages with respect to high contrast, capillary‐level resolution and flow speed quantification. However, the trade‐off between the signal‐to‐noise ratio of ODT images and A‐scan sampling density significantly slows down the imaging speed, constraining its clinical applications. To accelerate ODT imaging, a deep‐learning‐based approach is proposed to suppress the overwhelming phase noise from low‐sampling density. To handle the issue of limited paired training datasets, a generative adversarial network is performed to implicitly learn the distribution underlying Doppler phase noise and to generate the synthetic data. Then a 3D based convolutional neural network is trained and applied for the image denoising. We demonstrate this approach outperforms traditional denoise methods in noise reduction and image details preservation, enabling high speed ODT imaging with low A‐scan sampling density.