Comparison of laser speckle and laser Doppler perfusion imaging: Measurement in human skin and rabbit articular tissue

Laser Doppler perfusion imaging (LDI) is currently used in a variety of clinical applications, however, LDI instruments produce images of low resolution and have long scan times. A new optical perfusion imager using a laser speckle measurement technique and its use for in vivo blood flow measurements are described. Measurements of human skin and surgically exposed rabbit tissue made using this instrument were compared with a commercial laser Doppler perfusion imaging instrument. Results from blood flow measurements showed that the laser speckle imager measured an 11–67% decrease in blood flow under arterial occlusion. Under similar conditions, the laser Doppler imager measured blood flow decreases of 21–63%. In comparison with LDI, it was observed that the higher temporal resolution of the laser speckle imager was more sensitive to measuring the hyperaemic response immediately following occlusion. This in vivo study demonstrated some of the several advantages laser speckle imaging has over conventional LDI, making the new instrument more versatile in a clinical environment.     

Real-time in vivo color Doppler optical coherence tomography

Color Doppler optical coherence tomography (CDOCT) is a functional extension of optical coherence tomography (OCT) that can image flow in turbid media. We have developed a CDOCT system capable of imaging flow in real time. Doppler processing of the analog signal is accomplished in hardware in the time domain using a novel autocorrelation technique. This Doppler processing method is compatible with a high speed OCT system capable of imaging in real time. Using this system, we demonstrate cross-sectional imaging of bidirectional flow with CDOCT at four frames per second in a tissue-simulating phantom consisting of intralipid solution flowing in glass capillaries. As a demonstration of real-time imaging of blood flow in vivo we imaged pulsatible blood flow in a rat femoral artery at eight frames per second. Issues of velocity sensitivity, imaging speed, and range of velocity measurement are discussed, as well as potential applications of real-time CDOCT.     

Linear response range characterization and in vivo application of laser speckle imaging of blood flow dynamics

Noninvasive blood flow imaging can provide critical information on the state of biological tissue and the efficacy of approaches to treat disease. With laser speckle imaging (LSI), relative changes in blood flow are typically reported, with the assumption that the measured values are on a linear scale. A linear relationship between the measured and actual flow rate values has been suggested. The actual flow rate range, over which this linear relationship is valid, is unknown. Herein we report the linear response range and velocity dynamic range (VDR) of our LSI instrument at two relevant camera integration times. For integration times of 1 and 10 ms, the best case VDR was 80 and 60 dB, respectively, and the worst case VDR was 20 and 50 dB. The best case VDR values were similar to those reported in the literature for optical Doppler tomography. We also demonstrate the potential of LSI for monitoring blood flow dynamics in the rodent dorsal skinfold chamber model. These findings imply that LSI can provide accurate wide-field maps of microvascular blood flow rate dynamics and highlight heterogeneities in flow response to the application of exogenous agents.     

Peri-infarct temporal changes in intrinsic optical signal during spreading depression in focal ischemic rat cortex

The changes of intrinsic optical signals (IOS) are one of the important parameters of spreading depression (SD). The relationship between cerebral blood flow (CBF) and IOS can provide useful information for understanding the role of SD in neurological disorders. Here, we combined laser speckle imaging (LSI), intrinsic optical signal imaging (IOSI), and electrophysiological recording techniques to study the effect of CBF before the occurrence of SD on the spatiotemporal characteristics of IOS related to SD in a ministroke model. Four kinds of temporal pattern of changes in IOS were observed at cortical locations with different level of the CBF before the occurrence of SD. The results indicate that in the surrounding of micro-infarcts, SD-associated IOS vary as a function of blood flow rate, suggesting that the characteristics of IOS during SD might reflect blood flow rates.     

Optical coherence tomography of malignancy in hamster cheek pouches

Optical coherence tomography (OCT)/optical Doppler tomography (ODT) provides real-time in vivo high-resolution (10-microm) imaging of tissues and real-time spatially resolved blood flow in microvasculature. Hamster cheek pouches with induced dysplasia and malignancies were imaged with OCT/ODT to assess the potential for application to airway malignancy. In 22 Golden Syrian hamsters, 0.5% 9,10-dimethyl-1,2-benzanthracene induces carcinogenesis over 10 weeks in right side cheek pouches; the left side three served as controls. The cheek pouches are imaged in vivo prior to sacrifice, and in vitro after excision, using a prototype 1310-nm broadband superluminescent diode based OCT/ODT device. Images are compared to standard histopathology. OCT imaging offers good resolution of the hamster cheek pouches to depths of 1 to 3 mm and paralleled histologic images. The feasibility of high-resolution functional imaging is demonstrated in this hamster cheek pouch tumor model. ODT accurately detects vascular change associated with carcinogenesis.     

Functional imaging of dye concentration in tissue phantoms by spectroscopic optical coherence tomography

We present functional imaging of the concentration of a photodynamic therapy (PDT)-related dye in scattering tissue phantoms based on spatially resolved measurements of optical properties through spectroscopic optical coherence tomography (OCT). Expressions for the OCT signal are developed, enabling estimation of depth-resolved sample optical properties. Based on these expressions, we discuss speckle statistics and speckle correlations of the OCT signal. Speckle noise reduction is performed by spatial filtering and is used to improve accuracy in the estimated optical properties at the expense of spatial resolution. An analytic expression for the precision in the estimated optical properties is derived. This expression shows that axial filtering, and thereby a reduction of axial resolution, gives a larger improvement in precision compared to the same filtering and reduction in the transversal resolution. It also shows that imaging with a shorter coherence length, or a larger numerical aperture, improves precision when the filter length determines the spatial resolution. Good agreement is obtained between experimentally determined and theoretically predicted variance in the estimated attenuation coefficients and dye concentration. Finally, we present guidelines for spectroscopic OCT systems for concentration imaging and discuss application of the method to more realistic phantoms and tissue.     

Monitoring of drug and stimulation induced cerebral blood flow velocity changes in rat sensory cortex using spectral domain Doppler optical coherence tomography

Doppler optical coherence tomography (DOCT) provides a novel method to measure blood flow velocity in vessels with diameter at micrometer scale. In this study, a developed spectral domain DOCT system is applied to monitor cerebral blood flow velocity changes in a rat. An animal model with a cranial window is used, and by application of a drug, light, and electric stimulations, changes in blood flow velocity of the pial artery in sensory cortex are measured in real time. The results show significant differences in blood flow velocity before and after drug administration or light and electric stimulations, demonstrating the feasibility of DOCT in cerebral microcirculation study. Given its noninvasive nature, high spatial resolution, high velocity sensitivity, and high imaging speed, DOCT shows great promise in brain research by imaging blood flow changes at micrometer scale vessels, which helps to understand the pathogenesis of cerebral diseases and neurodegenerative diseases.     

Heart wall velocimetry and exogenous contrast-based cardiac flow imaging in Drosophila melanogaster using Doppler optical coherence tomography

Drosophila melanogaster (fruit fly) is a central organism in biology and is becoming increasingly important in the cardiovascular sciences. Prior work in optical imaging of the D. melanogaster heart has focused on static and dynamic structural anatomy. In the study, it is demonstrated that Doppler optical coherence tomography can quantify dynamic heart wall velocity and hemolymph flow in adult D. melanogaster. Since hemolymph is optically transparent, a novel exogenous contrast technique is demonstrated to increase the backscatter-based intracardiac Doppler flow signal. The results presented here open up new possibilities for functional cardiovascular phenotyping of normal and mutant D. melanogaster.     

Digital signal processor-based real-time optical Doppler tomography system

We present a real-time data-processing and display unit based on a custom-designed digital signal processor (DSP) module for imaging tissue structure and Doppler blood flow. The DSP module is incorporated into a conventional optical coherence tomography system. We also demonstrate the flexibility of embedding advanced Doppler processing algorithms in the DSP module. Two advanced velocity estimation algorithms previously introduced by us are incorporated in this DSP module. Experiments on Intralipid flow demonstrate that a pulsatile flow of several hundred pulses per minute can be faithfully captured in M-scan mode by this DSP system. In vivo imaging of a rat's abdominal blood flow is also presented.     

Modified laser speckle imaging method with improved spatial resolution

A two-dimensional map of blood flow is crucial for physiological studies. We present a modified laser speckle imaging method (LSI) that is based on the temporal statistics of a time-integrated speckle. A model experiment was performed for the validation of this technique. The spatial and temporal resolutions of this method were studied in theory and compared with current laser speckle contrast analysis (LASCA); the comparison indicates that the spatial resolution of the modified LSI is five times higher than that of current LASCA. Cerebral blood flow under different temperatures was investigated by our modified LSI. Compared with the results obtained by LASCA, the blood flow map obtained by the modified LSI possessed higher spatial resolution and provided additional information about changes in blood perfusion in small blood vessels. These results suggest that this is a suitable method for imaging the full field of blood flow without scanning and provides much higher spatial resolution than that of current LASCA and other laser Doppler perfusion imaging methods.     

In vivo, label-free, three-dimensional quantitative imaging of kidney microcirculation using Doppler optical coherence tomography

Doppler optical coherence tomography (DOCT) is a functional extension of optical coherence tomography (OCT) and is currently being employed in several clinical arenas to quantify blood flow in vivo. In this study, the objective was to investigate the feasibility of DOCT to image kidney microcirculation, specifically, glomerular blood flow. DOCT is able to capture three-dimensional (3D) data sets consisting of a series of cross-sectional images in real time, which enables label-free and non-destructive quantification of glomerular blood flow. The kidneys of adult, male Munich-Wistar rats were exposed through laparotomy procedure after being anesthetized. Following exposure of the kidney beneath the DOCT microscope, glomerular blood flow was observed. The effects of acute mannitol and angiotensin II infusion were also observed. Glomerular blood flow was quantified for the induced physiological states and compared with baseline measurements. Glomerular volume, cumulative Doppler volume, and Doppler flow range parameters were computed from 3D OCT/DOCT data sets. Glomerular size was determined from OCT, and DOCT readily revealed glomerular blood flow. After infusion of mannitol, a significant increase in blood flow was observed and quantified, and following infusion of angiontensin II, a significant decrease in blood flow was observed and quantified. Also, blood flow histograms were produced to illustrate differences in blood flow rate and blood volume among the induced physiological states. We demonstrated 3D DOCT imaging of rat kidney microcirculation in the glomerulus in vivo. Dynamic changes in blood flow were detected under altered physiological conditions demonstrating the real-time imaging capability of DOCT. This method holds promise to allow non-invasive imaging of kidney blood flow for transplant graft evaluation or monitoring of altered-renal hemodynamics related to disease progression.     

Photothermal coagulation of blood vessels: a comparison of high-speed optical coherence tomography and numerical modelling.

Optical-thermal models that can accurately predict temperature rise and damage in blood vessels and surrounding tissue may be used to improve the treatment of vascular disorders. Verification of these models has been hampered by the lack of time- and depth-resolved experimental data. In this preliminary study, an optical coherence tomography system operating at 4-30 frames per second was used to visualize laser irradiation of cutaneous (hamster dorsal skin flap) blood vessels. An argon laser was utilized with the following parameters: pulse duration 0.1-2.0 s, spot size 0.1-1.0 mm, power 100-400 mW. Video microscopy images were obtained before and after irradiations, and optical-thermal modelling was performed on two irradiation cases. Time-resolved optical coherence tomography and still images were compared with predictions of temperature rise and damage using Monte Carlo and finite difference techniques. In general, predicted damage agreed with the actual blood vessel and surrounding tissue coagulation seen in images. However, limitations of current optical-thermal models were identified, such as the inability to model the dynamic changes in blood vessel diameter that were seen in the optical coherence tomography images.     

Doppler optical coherence imaging of converging flow

The experimental methods of Doppler optical coherence tomography are applied for two-dimensional flow mapping of highly scattering fluid in flow with complex geometry. Converging flow (die entry) is used to demonstrate non-invasive methods to map varying velocity profiles before and after the entry. Complex geometry flow is scanned with approximately 10 x 10 x 10 microm3 spatial resolution. Structural images of the phantom and specific velocity images are demonstrated. A variety of velocity profiles have been obtained before and after the entry. Concave, blunted, parabolic and triangular profiles are obtained at different distances after the entry. Application of the technique to the study of blood circulation is discussed.     

Measuring hemodynamics in the developing heart tube with four-dimensional gated Doppler optical coherence tomography

Hemodynamics is thought to play a major role in heart development, yet tools to quantitatively assess hemodynamics in the embryo are sorely lacking. The especially challenging analysis of hemodynamics in the early embryo requires new technology. Small changes in blood flow could indicate when anomalies are initiated even before structural changes can be detected. Furthermore, small changes in the early embryo that affect blood flow could lead to profound abnormalities at later stages. We present a demonstration of 4-D Doppler optical coherence tomography (OCT) imaging of structure and flow, and present several new hemodynamic measurements on embryonic avian hearts at early stages prior to the formation of the four chambers. Using 4-D data, pulsed Doppler measurements could accurately be attained in the inflow and outflow of the heart tube. Also, by employing an en-face slice from the 4-D Doppler image set, measurements of stroke volume and cardiac output are obtained without the need to determine absolute velocity. Finally, an image plane orthogonal to the blood flow is used to determine shear stress by calculating the velocity gradient normal to the endocardium. Hemodynamic measurements will be crucial to identifying genetic and environmental factors that lead to congenital heart defects.     

Optical coherence photoacoustic microscopy: accomplishing optical coherence tomography and photoacoustic microscopy with a single light source

We developed optical coherence photoacoustic microscopy (OC-PAM) to demonstrate that the functions of optical coherence tomography (OCT) and photoacoustic microscopy (PAM) can be achieved simultaneously by using a single illuminating light source. We used a pulsed broadband laser centered at 580 nm and detected the absorbed photons through photoacoustic detection and the back-scattered photons with an interferometer. In OC-PAM, each laser pulse generates both one OCT A-line and one PAM A-line simultaneously; as a result, the two imaging modalities are intrinsically co-registered in the lateral directions. In vivo images of the mouse ear were acquired to demonstrate the capabilities of OC-PAM.     

Measuring red blood cell flow dynamics in a glass capillary using Doppler optical coherence tomography and Doppler amplitude optical coherence tomography

Blood, being a suspension of deformable red cells suspended in plasma, displays flow dynamics considerably more complicated than those of an ideal Newtonian fluid. Flow dynamics in blood capillaries of a few hundred micrometers in diameter are investigated using Doppler optical coherence tomography (DOCT) and Doppler amplitude optical coherence tomography (DAOCT), a novel extension of DOCT. Velocity profiles and concentration distributions of normal and rigidified in vitro red blood cell suspensions are shown to vary as functions of mean flow velocity, cell concentration, and cell rigidity. Deviation from the parabolic velocity profile expected for Pouseille flow is observed for both rigid and normal cells at low flow rates. Axial red cell migration both toward and away from the tube axis is observed for both rigid and normal cells as a function of flow velocity. Good agreement is found between our measurements, and theoretical expectations.     

Frequency domain multiplexing for speckle reduction in optical coherence tomography

Quantitative analysis of optical coherence tomography data can be strongly hampered by speckle. Here, we introduce a new method to reduce speckle, which leverages from Fourier-domain configurations and operates on individual axial scans. By subdividing the digitized spectrum into a number of distinct narrower windows, each with a different center frequency, several independent speckle patterns result. These can be averaged to yield a lower-resolution image with strongly reduced speckle. The full resolution image remains available for human interpretation; the low resolution version can be used for parametric imaging or quantitative analysis. We demonstrate this technique using intravascular optical frequency domain imaging data acquired in vivo.     

Real-time spectral domain Doppler optical coherence tomography and investigation of human retinal vessel autoregulation

Investigation of the autoregulatory mechanism of human retinal perfusion is conducted with a real-time spectral domain Doppler optical coherence tomography (SDOCT) system. Volumetric, time-sequential, and Doppler flow imaging are performed in the inferior arcade region on normal healthy subjects breathing normal room air and 100% oxygen. The real-time Doppler SDOCT system displays fully processed, high-resolution [512 (axial) x 1000 (lateral) pixels] B scans at 17 frames/sec in volumetric and time-sequential imaging modes, and also displays fully processed overlaid color Doppler flow images comprising 512 (axial) x 500 (lateral) pixels at 6 frames/sec. Data acquired following 5 min of 100% oxygen inhalation is compared with that acquired 5 min postinhalation for four healthy subjects. The average vessel constriction across the population is -16+/-26% after oxygen inhalation with a dilation of 36+/-54% after a return to room air. The flow decreases by -6+/-20% in response to oxygen and in turn increases by 21+/-28% as flow returns to normal in response to room air. These trends are in agreement with those previously reported using laser Doppler velocimetry to study retinal vessel autoregulation. Doppler flow repeatability data are presented to address the high standard deviations in the measurements.     

Influence of tissue optical properties on laser Doppler perfusion imaging, accounting for photon penetration depth and the laser speckle phenomenon

The influence of tissue optical properties on laser Doppler perfusion imaging (LDPI) is not well understood. We address this problem by quantifying the dependence of the signal response to tissue optical properties based on speckles or coherence areas and on photon statistics. We investigate the effect in vivo, showing the amplitude of photocurrent fluctuations in normal skin and port-wine stain with a range of beam diameters, and its relation to the speckle size variation difference between these two tissues. For the case of a low concentration of moving particles moving within a static turbid medium, a model is described and applied to predict the influence of speckles on the overall and depth sensitivity of LDPI, for a range of scattering levels and absorption levels. The results show that the speckle related effects on overall and depth sensitivity are large and that the depth sensitivity is highly likely to be misinterpreted without taking the speckle phenomenon into account.     

Laser speckle contrast imaging of cerebral blood flow in freely moving animals

We designed a miniature laser speckle imager that weighs ～20 g and is 3.1-cm high for full-field high-resolution imaging of cerebral blood flow (CBF) in freely moving animals. Coherent laser light illuminates the cortex through a multimode optical fiber bundle fixed onto the supporting frame of the imager. The reflected lights are then collected by a miniature macrolens system and imaged by a high-resolution CMOS camera at a high frame rate (50 fps). Using this miniature imager, we achieve high spatiotemporal resolution laser speckle contrast imaging of CBF in freely moving animals in real time.