Dynamic evaluation of blood flow microcirculation by combined use of the laser Doppler flowmetry and high‐speed videocapillaroscopy methods

The dynamic light scattering methods are widely used in biomedical diagnostics involving evaluation of blood flow. However, there exist some difficulties in quantitative interpretation of backscattered light signals from the viewpoint of diagnostic information. This study considers the application of the high‐speed videocapillaroscopy (VCS) method that provides the direct measurement of the red blood cells (RBCs) velocity into a capillary. The VCS signal presents true oscillation nature of backscattered light caused by moving RBCs. Thus, the VCS signal can be assigned as a reference one with respect to more complicated signals like in laser Doppler flowmetry (LDF). An essential correlation between blood flow velocity oscillations in a separate human capillary and the integral perfusion estimate obtained by the LDF method has been found. The observation of blood flow by the VCS method during upper arm occlusion has shown emergence of the reverse blood flow effect in capillaries that corresponds to the biological zero signal in the LDF. The reverse blood flow effect has to be taken into account in interpretation of LDF signals.      

Evaluation of laser-Doppler flowmetry as a measure of tissue blood flow

In this study the technique of laser-Doppler flowmetry was evaluated for the measurement of tissue blood flow by comparing laser-Doppler flow (LDF) signal in the renal cortex, gracilis muscle, and cremaster muscle of anesthetized rats to whole-organ blood flow measured with an electromagnetic flowmeter or radioactive microspheres. In vitro, LDF signal was closely correlated (r = 0.99) to changes in erythrocyte velocity generated with a rotating wheel. Although individual LDF readings varied in situ, mean LDF signal calculated from multiple readings on the tissue surface were significantly correlated (r = 0.74-0.95) with tissue blood flows measured at various perfusion pressures. However, significant differences in the slope of the LDF signal vs. blood flow relationship were observed in different tissues and with different methods of measurement in the same tissue. This study indicates that mean laser-Doppler flow signal provides a good estimate of tissue blood flow, provided a sufficient number of points is scanned. However, there appears to be no universal calibration factor for the method.     

Laser Doppler fluxmetry

BACKGROUND: Laser Doppler fluxmetry (LDF) is an extraordinary sensitive noninvasive method of examination. It can be used for monitoring changes in the cutaneous peripheral microcirculation(15). It uses a monochromatic low-energy laser beam. This beam penetrates the tissue and, depending on individual tissue penetration, it is reflected, recorded by a sensitive sensor and subsequently analyzed using the Doppler. Laser Doppler fluxmetry detects movement of cells in the peripheral circulation and microcirculation. METHODS: The light from the laser source is delivered via optical fibers to the tissue. In the tissue the light collides with moving blood elements; after a collision, the wave length of the light changes--this phenomenon is called Doppler shift. Our own method of measurement has already been fully developed. We have a group of patients with physiological findings and also groups of patients with vasoneurosis, patients with type 1 diabetes mellitus, vasculitis patients, and other patient groups. We examine the blood flow on the dorsal side of fingers or toes. There are many protocols for laser Doppler examination. For all the patient groups we used a protocol evaluating an algorithm, developed in the training department of the Perimed company, using provocation tests for the assessment of the vascular wall function in addition to records at rest. USE OF LASER DOPPLER: We have developed a method of use of laser Doppler examination in about 8 years. We have examined several groups of patients. First a patient group with physiological findings was examined, and subsequently groups of patients with vasculitis, vasoneurosis, and a group of patients with type l diabetes mellitus. Recently, monitoring has been performed in patients after cardioversion in chronic atrial fibrillation with sinus rhythm restitution, and also data obtained before and after a varicose vein operation on lower limbs have been compared. CONCLUSION: Clear diagnostic criteria for this method do not exist so far. For the time being this is a rather theoretical method that we used even in practice for the above mentioned diseases. The method provides enough data even for more detailed analyses. The information value of the curve arises after statistical data analysis with the t-test.     

Movement correction method for laser speckle contrast imaging of cerebral blood flow in cranial windows in rodents

Laser speckle contrast imaging (LSCI) is used in clinical research to dynamically image blood flow. One drawback is its susceptibility to movement artifacts. We demonstrate a new, simple method to correct motion artifacts in LSCI signals measured in awake mice with cranial windows during sensory stimulation. The principle is to identify a region in the image in which speckle contrast (SC) is independent of blood flow and only varies with animal movement, then to regress out this signal from the data. We show that (1) the regressed signal correlates well with mouse head movement, (2) the corrected signal correlates better with independently measured blood volume and (3) it has a (59 ± 6)% higher signal-to-noise ratio. Compared to three alternative correction methods, ours has the best performance. Regressing out flow-independent global variations in SC is a simple and accessible way to improve the quality of LSCI measurements.     

A multipixel diffuse correlation spectroscopy system based on a single photon avalanche diode array

The autocorrelation of laser speckles from coherent near infrared light is used for noninvasive estimates of relative changes in blood perfusion in techniques such as laser Doppler flowmetry (LDF) and diffuse correlation spectroscopy (DCS). In this study, a 2D array of single photon avalanche diodes (SPADs) was used to combine the strengths of multiple detectors in LDF with high light sensitivity in DCS. The system was tested on milk phantoms with varying detector fiber diameter (200 and 600 μm), source‐detector fiber separation (4.6‐10.2 mm), fiber‐SPAD distance (2.5‐36.5 mm), contiguous measurement time per repetition for the autocorrelation (1‐33 ms) and temperature (15.6‐46.7°C). An in vivo blood occlusion test was also performed. The multipixel approach improved signal‐to‐noise ratio (SNR) and, in our setup, the use of a multimode detector fiber was beneficial for SNR. In conclusion, the multipixel system works, but improvements and further studies regarding, for example, the data acquisition and optimal settings are still needed.      

Development of a polarization imaging method to detect paraffin-embedded pathology tissues before applying other techniques

The present article describes the development of a technique, applied to paraffin-embedded tissues, which uses three different wavelengths of monochromatic light (λ1 = 445 nm, λ2 = 540 nm and λ3 = 660 nm) for the measures of the degree of polarization, degree of linear polarization, degree of circular polarization and birefringence, all obtained from measurements of Stokes parameters by using polarized light. The goal of this study was to detect changes in developing embryonic mouse eye when pregnant mice fed diets without folic acid for variable periods compared with a healthy control group. We present a biomedical diagnostic technique based on polarized light detection applied to paraffin-embedded tissues to visualize the structural damage to aid us in the diagnosis before applying other techniques. Through this method, we can visualize and identify which parts of the tissue were altered with respect to the control group.     

Respiratory‐induced vasoconstriction measured by light transmission and by laser Doppler signal

Changes in finger tissue blood volume (TBV) measured by light transmission and in laser Doppler flow (LDF) were obtained during long breathing (of 12 s period) and associated with the respiratory phases, inspiration and expiration. For fifteen out of sixteen subjects TBV and LDF started to decrease 0–2 s after the start of expiration and increased during inspiration but the start of increase occurred before the start of inspiration, showing that the respiratory‐induced changes in TBV and LDF are mainly associated with the expiration. Decrease of TBV and LDF after expiration was also found during the inspiratory gasps (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Exogenous zinc improves blood fluidity but has no effect on the mechanisms of vascular response to acetylcholine iontophoresis in humans

Recent findings in cellular signaling function of zinc through the mobilization intracellular calcium or by inducing ATP release suggest that extracellular zinc plays an important role in many physiological functions. However, such an extracellular signaling action of zinc for most cells is not known. Therefore, we investigated whether zinc plays any role in endothe-lium-dependent acetylcholine (ACh)-induced vasodilatation in microvascular beds. Transdermal iontophoresis was used to transport ACh through the forearm skin and cutaneous perfusion was measured using a laser Doppler flowmeter (LDF). Experiments were repeated using (1) zinc instead of ACh to test the effect of zinc ions alone and (2) concomitant iontophoresis of ACh and zinc to explore the effect of zinc on ACh-induced vasodilatation. Although zinc augments blood flow, curve-fitting to LDF signals indicate that zinc has no effect on the neural and endothelial component of ACh-induced vasodilatation. Additionally, no effect of Zn²⁺ on blood flow was found during its iontophoresis alone. Therefore, it is suggested from the Fourier analysis of LDF signals that the Zn⁺ might influence blood fluidity by its action on red blood cells deformability/aggregability during a high-blood-flow condition, which might, in turn, decrease blood viscosity and improve blood flow in vivo.     

Heterogeneity of laser Doppler flowmetry in perfused muscle indicative of nutritive and nonnutritive flow

Laser Doppler flowmetry (LDF) signal responses have been compared with metabolic changes using both a surface macroprobe and randomly placed implantable microprobes in muscles of the constant-flow-perfused rat hindlimb. Changes in response to total flow and to vasoconstrictors that are known to increase (norepinephrine, NE) or decrease (serotonin, 5-HT) hindlimb oxygen uptake were assessed. The surface macroprobe (anterior end of biceps femoris) identified only one type of LDF response characterized by increased signal in response to NE and decreased signal in response to 5-HT. Implanted microprobes (tibialis, gastrocnemius, vastus, or bicep femoris) identified sites that gave three LDF responses of differing character. These responses were where the LDF signal increased with NE and decreased with 5-HT (56.7%), where the LDF signal decreased with NE and increased with 5-HT (16.5%), or where there was no net response to either vasoconstrictor (24.7%). The data are consistent with discrete regions of nutritive and nonnutritive flow in muscle where flow in each as controlled by vasoconstrictors relates directly to the metabolic behavior of the tissue.     

Laser-Doppler and plethysmographic skin blood flow during exercise and during acute heat stress in the sauna

Forearm skin blood flow was measured in six male subjects by laser-Doppler flowmetry (LDF) and venous occlusion plethysmography (VOP) during constant-load (125-200 W) upright bicycle exercise in a warm environment (X + SD, ta 34.6 +/- 0.2 degrees C) and during a 15 min sauna bath (ta 69.0 +/- 2.8 degrees C). During the sauna test the LDF values correlated well with the VOP measurements in the initial phase of active cutaneous vasodilation, after which the LDF values almost leveled off in spite of a steady increase in VOP measurements. During the exercise the mean VOP and LDF values rose in parallel with each other to steady state levels. The relationship between the results of the two methods proved to be nonlinear. It was concluded that different parameters were measured by VOP and LDF. The latter measured mainly the integrated velocity of blood flow in the outermost cutaneous tissue, and this velocity seemed to be partly dependent on the level of the arterial inflow (VOP), but also on the prevailing pressure-flow and pressure-volume relations in the cutaneous vascular bed.     

Histological classification of atherosclerotic arteries using high-speed confocal Raman microscopy with machine learning

Confocal Raman microscopy is a useful tool to observe composition and constitution of label-free samples at high spatial resolution. However, accurate characterization of microstructure of tissue and its application in diagnostic imaging are challenging due to weak Raman scattering signal and complex chemical composition of tissue. We have developed a method to improve imaging speed, diffraction efficiency, and spectral resolution of confocal Raman microscopy. In addition to the novel imaging technique, the machine learning method enables confocal Raman microscopy to visualize accurate histology of tissue sections. Here, we have demonstrated the performance of the proposed method by measuring histological classification of atherosclerotic arteries and compared the histological confocal Raman images with the conventional staining method. Our new confocal Raman microscopy enables us to comprehend the structure and biochemical composition of tissue and diagnose the buildup of atherosclerotic plaques in the arterial wall without labeling.     

Removing dynamic distortions from laser speckle flowgraphy using Eigen-decomposition and spatial filtering

Laser speckle flowgraphy (LSFG) has been widely used in the investigation of blood flows in ophthalmology. However, the dynamic changes of the ocular optics can impose artificial contrasts to the LSFG, corrupting the detection of both retinal vasculature and blood pulsation at the posterior segment of the human eye. In this study, we propose to use Eigen-decomposition method to separate the spatially and temporally varying speckle patterns from the static tissues. Spatial filtering is further applied to remove the distortion-correlated modulation of the speckle patterns. We experimentally show that with the proposed method, the integrity of blood vessels is significantly improved and the distortions in pulse waveforms can be well corrected.     

In vivo sensing of cutaneous edema: A comparative study of diffuse reflectance,Raman spectroscopy and multispectral imaging

Quantitative noninvasive assessment of water content in tissues is important for biomedicine. Optical spectroscopy is potentially capable of solving this problem; however, its applicability for clinical diagnostics remains questionable. The presented study compares diffuse reflectance spectroscopy, Raman spectroscopy and multispectral imaging in the characterization of cutaneous edema. The source-detector geometries for each method are selected based on Monte Carlo simulations results to detect the signal from the dermis. Then, the kinetics of the edema development is studied for two models. All methods demonstrate synchronous trends for histamine-induced edema: The water content reaches a maximum of 1 hour after histamine application and then gradually decreases. For the venous occlusion, a 51% increase in water content is observed with Raman spectroscopy. The differences in water content estimation by three methods are explained based on the light propagation model. The obtained results are essential for introducing quantitative optical water measurement technology to the clinics.     

Race‐specific differences in the phase coherence between blood flow and oxygenation: A simultaneous NIRS,white light spectroscopy and LDF study

Race‐specific differences in the level of glycated hemoglobin are well known. However, these differences were detected by invasive measurement of mean oxygenation, and their understanding remains far from complete. Given that oxygen is delivered to the cells by hemoglobin through the cardiovascular system, a possible approach is to investigate the phase coherence between blood flow and oxygen transportation. Here we introduce a noninvasive optical method based on simultaneous recordings using NIRS, white light spectroscopy and LDF, combined with wavelet‐based phase coherence analysis. Signals were recorded simultaneously for individuals in two groups of healthy subjects, 16 from Sub‐Saharan Africa (BA group) and 16 Europeans (CA group). It was found that the power of myogenic oscillations in oxygenated and de‐oxygenated hemoglobin is higher in the BA group, but that the phase coherence between blood flow and oxygen saturation, or blood flow and hemoglobin concentrations is higher in the CA group     

Measurement of airway wall blood flow in sheep by laser-Doppler flowmetry: interpretation and problems

We have used laser-Doppler flowmetry (LDF), a technique that detects movement of erythrocytes, to measure tracheal and bronchial wall blood flow in anesthetized open-chest sheep. LDF derives continuous measurements noninvasively, although fiber-optic bronchoscopy is necessary to introduce the LDF probe into the airways. The response of the LDF flow signals at four regions of the airway walls to varying bronchial arterial flow rates was examined in both live and dead sheep by cannulation and subsequent perfusion of the common bronchial artery at different flow rates by use of a roller pump. In the live sheep, variations in bronchial arterial blood flow resulted in variations in LDF signals in the principal bronchus and in lobar and segmental bronchi but not in the trachea. In the dead sheep, variations in bronchial arterial blood flow resulted in variations in LDF signals in all four regions. Within regions, the average response of the LDF signals to varying bronchial blood flow rates was approximately linear in both live and dead sheep, but considerable site-to-site variation in response was observed. In the live sheep, significant LDF signals were observed when the bronchial arterial flow was set to zero and when the bronchial artery was perfused with dextran solution, which would in theory be expected to produce no LDF signal. A small LDF signal was also detected under zero flow conditions in the dead sheep. These observations suggest that the LDF technique, in addition to detecting blood flow from the bronchial artery also detects background noise and/or collateral circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairments of cerebral blood flow microcirculation in rats brought on by cardiac cessation and respiratory arrest

The impairments of cerebral blood flow microcirculation brought on by cardiac and respiratory arrest were assessed with multi-modal diagnostic facilities, utilising laser speckle contrast imaging, fluorescence spectroscopy and diffuse reflectance spectroscopy. The results of laser speckle contrast imaging show a notable reduction of cerebral blood flow in small and medium size vessels during a few minutes of respiratory arrest, while the same effect was observed in large sinuses and their branches during the circulatory cessation. Concurrently, the redox ratio assessed with fluorescence spectroscopy indicates progressing hypoxia, NADH accumulation and increase of FAD consumption. The results of diffuse reflectance spectra measurements display a more rapid grow of the perfusion of deoxygenated blood in case of circulatory impairment. In addition, consequent histopathological analysis performed by using new tissue staining procedure developed in-house. It shows notably higher reduction of size of the neurons due to their wrinkling within brain tissues influenced by circulation impair. Whereas, the brain tissues altered with the respiratory arrest demonstrate focal perivascular oedema and mild hypoxic changes of neuronal morphology. Thus, the study suggests that consequences of a cessation of cerebral blood flow become more dramatic and dangerous compare to respiratory arrest.     

激光低损伤加热疗法的理论分析

激光低损伤加热疗法将激光照射与表面冷却相结合,能实现只让病变组织细胞凝固,而体表健康组织则不受损伤,它是治疗靠近体表肿瘤的一种有希望的方法。为了进一步认识这种治疗方法,本文提出了一个适用于激光低损伤加热疗法的数值计算模型,模拟了热疗过程中的光传输和热传递,得到组织内部的瞬态温度分布。在此基础上,详细讨论了散射系数、吸收系数、各向异性系数等光学参数,激光加热功率、照射半径、表面对流换热系数等热学参数。以及血液灌注率和新陈代谢等生理参数对这种低损伤加热疗法的影响。结果发现,只要通过适当调节吸收系数、散射系数和照射功率等参数就可实现对加热区域位置和范围的控制。     

Cholinergic mechanisms of cutaneous active vasodilation during heat stress in cystic fibrosis.

To test the hypothesis that cutaneous active vasodilation in heat stress is mediated by a redundant cholinergic cotransmitter system, we examined the effects of atropine on skin blood flow (SkBF) increases during heat stress in persons with (CF) and without cystic fibrosis (non-CF). Vasoactive intestinal peptide (VIP) has been implicated as a mediator of cutaneous vasodilation in heat stress. VIP-containing cutaneous neurons are sparse in CF, yet SkBF increases during heat stress are normal. In CF, augmented ACh release or muscarinic receptor sensitivity could compensate for decreased VIP; if so, active vasodilation would be attenuated by atropine in CF relative to non-CF. Atropine was administered into skin by iontophoresis in seven CF and seven matched non-CF subjects. SkBF was monitored by laser-Doppler flowmetry (LDF) at atropine treated and untreated sites. Blood pressure [mean arterial pressure (MAP)] was monitored (Finapres), and cutaneous vascular conductance was calculated (CVC = LDF/MAP). The protocol began with a normothermic period followed by a 3-min cold stress and 30-45 min of heat stress. Finally, LDF sites were warmed to 42 degrees C to effect maximal vasodilation. CVC was normalized to its site-specific maximum. During heat stress, CVC increased in both CF and non-CF (P < 0.01). CVC increases were attenuated by atropine in both groups (P < 0.01); however, the responses did not differ between groups (P = 0.99). We conclude that in CF there is not greater dependence on redundant cholinergic mechanisms for cutaneous active vasodilation than in non-CF.     

Treatment of Raynaud's phenomenon with ketanserin in patients with connective tissue disorders.

The serotonin receptor blocker ketanserin was given orally in a double blind crossover study to 10 patients with connective tissue disorders and Raynaud''s phenomenon. Eight of the 10 patients improved clinically on ketanserin and none on placebo. Digital blood flow was assessed with laser Doppler flowmetry (LDF), photoplethysmography, and skin temperature measurements. Laser Doppler flowmetry was the most useful method, showing a significant reduction in recovery time after a standard cold provocation. Although the resting flow was not significantly improved, digital ulcers healed in four out of five patients, providing evidence of increased nutritive flow. The results of this study suggest that orally administered ketanserin may be an effective and well tolerated treatment for Raynaud''s phenomenon associated with connective tissue disorders, especially scleroderma.     

Skin blood flow influences near-infrared spectroscopy-derived measurements of tissue oxygenation during heat stress.

Near-infrared (NIR) spectroscopy is a noninvasive optical technique that is increasingly used to assess muscle oxygenation during exercise with the assumption that the contribution of skin blood flow to the NIR signal is minor or nonexistent. We tested this assumption in humans by monitoring forearm tissue oxygenation during selective cutaneous vasodilation induced by locally applied heat (n = 6) or indirect whole body heating (i.e., heating subject but not area surrounding NIR probes; n = 8). Neither perturbation has been shown to cause a measurable change in muscle blood flow or metabolism. Local heating (approximately 41 degrees C) caused large increases in the NIR-derived tissue oxygenation signal [before heating = 0.82 +/- 0.89 optical density (OD), after heating = 18.21 +/- 2.44 OD; P < 0.001]. Similarly, whole body heating (increase internal temperature 0.9 degrees C) also caused large increases in the tissue oxygenation signal (before heating = -0.31 +/- 1.47 OD, after heating = 12.48 +/- 1.82 OD; P < 0.001). These increases in the tissue oxygenation signal were closely correlated with increases in skin blood flow during both local heating (mean r = 0.95 +/- 0.02) and whole body heating (mean r = 0.89 +/- 0.04). These data suggest that the contribution of skin blood flow to NIR measurements of tissue oxygenation can be significant, potentially confounding interpretation of the NIR-derived signal during conditions where both skin and muscle blood flows are elevated concomitantly (e.g., high-intensity and/or prolonged exercise).