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Background Vasomotion is the spontaneous oscillation in vascular tone in the microcirculation and is believed to be a physiological mechanism facilitating the transport of blood gases and nutrients to and from tissues. So far, Laser Doppler flowmetry has constituted the gold standard for in vivo vasomotion analysis. Materials and methods We applied vasomotion analysis to speed-resolved perfusion, oxygen saturation, red blood cell tissue (RBC) tissue fraction, and average vessel diameter from five healthy individuals at rest measured by the newly developed Periflux 6000 EPOS system over 10 minutes. Magnitude scalogram and the time-averaged wavelet spectra were divided into frequency intervals reflecting endothelial, neurogenic, myogenic, respiratory, and cardiac function. Results Recurrent high-intensity periods of the myogenic, neurogenic, and endothelial frequency intervals were found. The neurogenic activity was considerably more pronounced for the oxygen saturation, RBC tissue fraction, and vessel diameter signals, than for the perfusion signals. In a correlation analysis we found that changes in perfusion in the myogenic, neurogenic, and endothelial frequency intervals precede changes in the other signals. Furthermore, changes in average vessel diameter were in general negatively correlated to the other signals in the same frequency intervals, indicating the importance of capillary recruitment. Conclusion We conclude that vasomotion can be observed in signals reflecting speed resolved perfusion, oxygen saturation, RBC tissue fraction, and vessel diameter. The new parameters enable new aspects of the microcirculation to be observed.

Significance: Developing algorithms for estimating blood oxygenation from snapshot multi-spectral imaging (MSI) data is challenging due to the complexity of sensor characteristics and photon transport modeling in tissue. We circumvent this using a method where artificial neural networks (ANNs) are trained on in vivo MSI data with target values from a point-measuring reference method. Aim: To develop and evaluate a methodology where a snapshot filter mosaic camera is utilized for imaging skin hemoglobin oxygen saturation (S-O2), using ANNs. Approach: MSI data were acquired during occlusion provocations. ANNs were trained to estimate S-O2 with MSI data as input, targeting data from a validated probe-based reference system. Performance of ANNs with different properties and training data sets was compared. Results: The method enables spatially resolved estimation of skin tissue S-O2. Results are comparable to those acquired using a Monte-Carlo-based approach when relevant training data are used. Conclusions: Training an ANN on in vivo MSI data covering a wide range of target values acquired during an occlusion protocol enable real-time estimation of S-O2 maps. Data from the probe-based reference system can be used as target despite differences in sampling depth and measurement position. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Funding Agencies|VINNOVA Grants via the Swelife and MedTech4Health programsVinnova [2016-02211, 2017-01435, 2019-01522]

Significance: Hemoglobin oxygen saturation and red blood cell (RBC) tissue fraction are important parameters when assessing microvascular status. Functional information can be attained using temporally resolved measurements performed during stimulus–response protocols. Pointwise assessments can currently be conducted with probe-based systems. However, snapshot multispectral imaging (MSI) can be used for spatial–temporal measurements. Aim: To validate if hemoglobin oxygen saturation and RBC tissue fraction can be quantified using a snapshot MSI system and an inverse Monte Carlo algorithm. Approach: Skin tissue measurements from the MSI system were compared to those from a validated probe-based system during arterial and venous occlusion provocation on 24 subjects in the wavelength interval 450 to 650 nm, to evaluate a wide range of hemoglobin oxygen saturation and RBC tissue fraction levels. Results: Arterial occlusion results show a mean linear regression R2 = 0.958 for hemoglobin oxygen saturation. Comparing relative RBC tissue fraction during venous occlusion results in R2 = 0.925. The MSI system shows larger dynamic changes than the reference system, which might be explained by a deeper sampling including more capacitance vessels. Conclusions: The snapshot MSI system estimates hemoglobin oxygen saturation and RBC tissue fraction in skin microcirculation showing a high correlation (R2 > 0.9 in most subjects) with those measured by the reference method. Funding: Swedens Innovation Agency VINNOVA via the programs Swelife and MedTech4Health [2017-01435, 2019-01522]; CENIIT research organization within Linkoping University [11.02]

Chronic limb-threatening ischemia (CLTI) has a major impact on patient's lives and is associated with a heavy health care burden with high morbidity and mortality. Treatment by endovascular intervention is mostly based on macrocirculatory information from angiography and does not consider the microcirculation. Despite successful endovascular intervention according to angiographic criteria, a proportion of patients fail to heal ischemic lesions. This might be due to impaired microvascular perfusion and variations in the supply to different angiosomes. Non-invasive optical techniques for microcirculatory perfusion and oxygen saturation imaging have the potential to provide the interventionist with additional information in real-time, supporting clinical decisions during the intervention. This study presents a novel multimodal imaging system, based on multi-exposure laser speckle contrast imaging and multi-spectral imaging, for continuous use during endovascular intervention. The results during intervention display spatiotemporal changes in the microcirculation compatible with expected physiological reactions during balloon dilation, with initially induced ischemia followed by a restored perfusion, and local administration of a vasodilator inducing hyperemia. We also present perioperative and postoperative follow-up measurements with a pulsatile microcirculation perfusion. Finally, cases of spatial heterogeneity in the observed oxygen saturation and perfusion are discussed. In conclusion, this technical feasibility study shows the potential of the methodology to characterize changes in microcirculation before, during, and after endovascular intervention. Funding: Swedish Research CouncilSwedish Research CouncilEuropean Commission [2014-6141]; Swedens Innovation Agency VINNOVAVinnova [2017-01435, 2019-01522]

The objective of this study was to explore the associations between skin microcirculatory function and established cardiovascular risk factors in a large Swedish cohort. As part of the Swedish CArdioPulmonary bioImage Study (SCAPIS), microcirculatory data were acquired at Linkoping University hospital, Linkoping, Sweden during 2016–2017. The subjects, aged 50–64 years, were randomly selected from the national population register. Microcirculatory reactivity was assessed using a 5-min arterial occlusion-release protocol. Comprehensive skin microcirculatory data were continuously acquired by using a fiberoptic probe placed on the lower right arm. After exclusion of missing data (208), 1557 subjects were remaining. Among the parameters, skin microcirculatory peak oxygen saturation after occlusion release, had the strongest relationship to the cardiovascular risk factors. The linear associations between peak oxygen saturation and cardiovascular risk factors were analyzed adjusted for age and sex. We found a negative association with peak oxygen saturation (standardized regression coefficient) for blood pressure (systolic −0.05 (95% CI: −0.10;-0.003) and diastolic −0.05 (−0.10; −0.003)), BMI −0.18 (−0.23; −0.13), waist circumference (males −0.20 (−0.32; −0.16), females −0.18 (−0.25; −0.11)), prevalent diabetes −0.31 (−0.49; −0.12), hypertension −0.30 (−0.42; −0.18), hyperlipidemia −0.24 (−0.40; −0.09), fasting glucose level −0.06 (−0.12; −0.01), HbA1c −0.07 (−0.12; −0.02), triglyceride level −0.09 (−0.14; −0.04), hsCRP −0.12 (−0.17; −0.07), and current smoker versus never smoked −0.50 (−0.67; −0.34). A positive association with peak oxygen saturation was found for cholesterol level 0.05 (0.005; 0.11) and HDL 0.11 (0.06; 0.17). This is the first study showing that post-ischemic skin microvascular peak oxygen saturation is associated with virtually all established cardiovascular risk factors in a population-based middle-aged cohort.

Significance: Diffuse reflectance spectroscopy (DRS) is frequently used to assess oxygen saturation and hemoglobin concentration in living tissue. Methods solving the inverse problem may include time-consuming nonlinear optimization or artificial neural networks (ANN) determining the absorption coefficient one wavelength at a time. Aim: To present an ANN-based method that directly outputs the oxygen saturation and the hemoglobin concentration using the shape of the measured spectra as input. Approach: A probe-based DRS setup with dual source-detector separations in the visible wavelength range was used. ANNs were trained on spectra generated from a three-layer tissue model with oxygen saturation and hemoglobin concentration as target. Results: Modeled evaluation data with realistic measurement noise showed an absolute root-mean-square (RMS) deviation of 5.1% units for oxygen saturation estimation. The relative RMS deviation for hemoglobin concentration was 13%. This accuracy is at least twice as good as our previous nonlinear optimization method. On blood-intralipid phantoms, the RMS deviation from the oxygen saturation derived from partial oxygen pressure measurements was 5.3% and 1.6% in two separate measurement series. Results during brachial occlusion showed expected patterns. Conclusions: The presented method, directly assessing oxygen saturation and hemoglobin concentration, is fast, accurate, and robust to noise. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Funding Agencies|Swedish Research CouncilSwedish Research Council [2014-6141]; Swedens innovation agency VINNOVA via the program MedTech4Health [2016-02211]; Swedens innovation agency VINNOVA via the program Swelife and MedTech4Health [2017-01435]

This talk highlights a novel instrument for real-time video-rate imaging of microcirculation blood flow. The instrument utilizes multi-exposure laser speckle contrast imaging (MELSCI) with a high-speed camera, custom integrated electronics, and machine learning for massive computational bandwidth. This instrument enables, for the first time, continuous in vivo imaging of blood flow at frame rates sufficiently high to track dynamic changes, with perfusion estimates strongly correlated with that of laser Doppler flowmetry (LDF). This is an important step enabling a better understanding of the role of the microcirculation in several cardiovascular diseases. A pilot study on healthy subjects is presented.

Noninvasive in vivo imaging of microcirculatory blood flow or perfusion (average speed times concentration of moving red blood cells) is of interest in several clinical applications, including monitoring of burn wounds1,2 and investigation of peripheral arterial disease,3 as well as several others.4 One technique that has gained a lot of focus in the last decades is laser speckle contrast imaging [LSCI; sometimes laser speckle contrast analysis (LASCA)] in which tissue is illuminated with a laser and the resulting speckle pattern is detected with a camera. Light that scatters when interacting with moving particles in the tissue will obtain a Doppler frequency shift depending on the velocity of the particles. The interference of light with different frequencies will give rise to fluctuations in the speckle pattern formed on the imaging sensor. The movement of the speckles on the sensor will cause an image blur that increases with exposure time. The local spatial statistics of this blur can be related to the movement of particles in the tissue, as is done in LSCI.5 LSCI has been increasingly used over laser Doppler imaging mainly due to its simple setup and fast acquisition and data processing. However, conventional LSCI has drawbacks such as a nonlinear response to perfusion, dependency to static scattering contrast, and a high variability in the presence of noise.5,6 Recent work has gone into calibrating and correcting for these issues to make LSCI a more accurate technique.7 Despite this, while it is generally accepted that LSCI is related to microcirculatory perfusion, the relationship is complex and a direct mapping is still not known.4,8 For the older laser Doppler flowmetry (LDF) technique, the relation to microcircular perfusion is better understood, where it is, for example, possible to theoretically show that the perfusion estimate is linearly related to the flow speed.9,10 To address the nonlinearity of LSCI to perfusion, Parthasarathy et al.11 proposed a new setup using multiple exposure times. The technique, called multi-exposure laser speckle contrast imaging (MELSCI, sometimes MESI), obtains information about the speckle motion blur at various exposures, enabling more advanced models to be used when estimating perfusion from the contrast.5,12 Several approaches to capture multi-exposure images have been proposed. The initial approach by Parthasarathy et al.11 was to use a time-modulated laser to achieve multi-exposure images. By capturing all images with a fixed exposure time but only illuminating the tissue during parts of that time, multi-exposures can be generated. However, a problem with this approach is that images with different exposure times are separated in time since they are captured sequentially. Dragojevic et al.13 suggested another approach that addressed this drawback. This was based on a high-speed camera, only capturing images at the shortest exposure time required, and in postprocessing adding successive images together to create longer exposure times. These synthetic exposure times are valid if the interframe delay in the camera is negligible. This method is faster and more accurate than previous methods due to all exposure times essentially being captured simultaneously, but it produces immense amounts of data that must be transferred and processed. At the time, this was, therefore, limited to an offline technique. Hence, MELSCI has so far been held back by technical limitations in both the imaging setup and the computation time of the models. To address this problem of a data and processing bottleneck, we have previously presented a system for synthetic MELSCI,14 based on a high-speed 1000-frames per second (fps) 1-megapixel camera directly interfaced to a field programmable gate array (FPGA) performing the multi-exposure contrast algorithm outlined by Dragojevic13 in real-time. Processed contrast images were sent to a computer, massively reducing the data throughput requirements. This system could produce multi-exposure contrast images at 15.6 fps, using all available frames from the 1000-fps camera, without any loss of data. However, due to insufficient processing power, the system was not able to transfer the processed images to the computer fast enough, and thus could not provide a real-time video-rate output. Furthermore, while the calculation of multi-exposure contrast images was fast enough, at the time there was no multi-exposure perfusion algorithm fast enough to keep up with the continuous stream of contrast images. The model proposed by Parthasarathy et al.11 and later refined by Kazmi et al.15 requires nonlinear fitting to extract perfusion-related parameters from the multi-exposure contrast. This process must be performed individually in each pixel for full field perfusion images, which is not feasible in real-time with satisfactory frame rates.6 We have previously presented a method using machine learning and simulated data from thousands of Monte Carlo tissue models from which we train an artificial neural network (ANN) to translate multi-exposure contrast to laser Doppler perfusion. We showed that this technique not only achieves a model-inference time of

The PeriFlux 6000 EPOS system combines diffuse reflectance spectroscopy (DRS) and laser Doppler flowmetry (LDF) for the assessment of oxygen saturation (expressed in percentage), red blood cell (RBC) tissue fraction (expressed as volume fraction, %RBC), and perfusion (%RBC × mm / s) in the microcirculation. It also allows the possibility of separating the perfusion into three speed regions (0 to 1, 1 to 10, and10 mm / s). We evaluate the speed-resolved perfusion components, i.e., the relative amount of perfusion within each speed region, using a blood-flow phantom. Human blood was pumped through microtubes with an inner diameter of 0.15 mm. Measured DRS and LDF spectra were compared to Monte Carlo-simulated spectra in an optimization routine, giving the best-fit parameters describing the measured spectra. The root-mean-square error for each of the three speed components (0 to 1, 1 to 10, and10 mm / s, respectively) when describing the blood-flow speed in the microtubes was 2.9%, 8.1%, and 7.7%. The presented results show that the system can accurately discriminate blood perfusion originating from different blood-flow speeds, which may enable improved measurement of healthy and dysfunctional microcirculatory flow.

Laser speckle contrast imaging (LSCI) enables video rate imaging of blood flow. However, its relation to tissue blood perfusion is nonlinear and depends strongly on exposure time. By contrast, the perfusion estimate from the slower laser Doppler flowmetry (LDF) technique has a relationship to blood perfusion that is better understood. Multiexposure LSCI (MELSCI) enables a perfusion estimate closer to the actual perfusion than that using a single exposure time. We present and evaluate a method that utilizes contrasts from seven exposure times between 1 and 64 ms to calculate a perfusion estimate that resembles the perfusion estimate from LDF. The method is based on artificial neural networks (ANN) for fast and accurate processing of MELSCI contrasts to perfusion. The networks are trained using modeling of Doppler histograms and speckle contrasts from tissue models. The importance of accounting for noise is demonstrated. Results show that by using ANN, MELSCI data can be processed to LDF perfusion with high accuracy, with a correlation coefficient R = 1.000 for noise-free data, R = 0.993 when a moderate degree of noise is present, and R = 0.995 for in vivo data from an occlusion-release experiment.

Laser speckle-based techniques are frequently used to assess microcirculatory blood flow. Perfusion estimates are calculated either by analyzing the speckle fluctuations over time as in laser Doppler flowmetry (LDF), or by analyzing the speckle contrast as in laser speckle contrast imaging (LSCI). The perfusion estimates depend on the amount of blood and its speed distribution. However, the perfusion estimates are commonly given in arbitrary units as they are nonlinear and depend on the magnitude and the spatial distribution of the optical properties in the tissue under investigation. We describe how the spatial confinement of blood to vessels, called the vessel packaging effect, can be modeled in LDF and LSCI, which affect the Doppler power spectra and speckle contrast, and the underlying bio-optical mechanisms for these effects. As an example, the perfusion estimate is reduced by 25% for LDF and often more than 50% for LSCI when blood is located in vessels with an average diameter of 40 μm, instead of being homogeneously distributed within the tissue. This significant effect can be compensated for only with knowledge of the average diameter of the vessels in the tissue.

Skin and kidney microvascular functions may be affected independently in diabetes mellitus. We investigated skin microcirculatory function in 79 subjects with diabetes type 2, where 41 had microalbuminuria and 38 not, and in 41 age-matched controls. The oxygen saturation, fraction of red blood cells and speed-resolved microcirculatory perfusion (% red blood cells × mm/s) divided into three speed regions: 0–1, 1–10 and above 10 mm/s, were assessed during baseline and after local heating of the foot with a new device integrating diffuse reflectance spectroscopy and laser Doppler flowmetry. Arterial stiffness was assessed as carotid-femoral pulse wave velocity. Subjects with diabetes and microalbuminuria had significantly higher carotid-femoral pulse wave velocity compared to subjects without microalbuminuria and to controls. The perfusion for speeds 0–1 mm/s and red blood cell tissue fraction were reduced in subjects with diabetes at baseline and after heating, independent of microalbuminuria. These parameters were correlated to HbA1c. In conclusion, the reduced nutritive perfusion and red blood cell tissue fraction in type 2 diabetes were related to long-term glucose control but independent of microvascular changes in the kidneys and large-vessel stiffness. This may be due to different pathogenic pathways in the development of nephropathy, large-vessel stiffness and cutaneous microvascular impairment.

A multiple exposure laser speckle contrast imaging (MELSCI) setup for visualizing blood perfusion was developed using a field programmable gate array (FPGA), connected to a 1000 frames per second (fps) 1-megapixel camera sensor. Multiple exposure time images at 1, 2, 4, 8, 16, 32 and 64 milliseconds were calculated by cumulative summation of 64 consecutive snapshot images. The local contrast was calculated for all exposure times using regions of 4 × 4 pixels. Averaging of multiple contrast images from the 64-millisecond acquisition was done to improve the signal-to-noise ratio. The results show that with an effective implementation of the algorithm on an FPGA, contrast images at all exposure times can be calculated in only 28 milliseconds. The algorithm was applied to data recorded during a 5 minutes finger occlusion. Expected contrast changes were found during occlusion and the following hyperemia in the occluded finger, while unprovoked fingers showed constant contrast during the experiment. The developed setup is capable of massive data processing on an FPGA that enables processing of MELSCI data in 15.6 fps (1000/64 milliseconds). It also leads to improved frame rates, enhanced image quality and enables the calculation of improved microcirculatory perfusion estimates compared to single exposure time systems.

Hyperspectral imaging (HSI) can estimate the spatial distribution of skin blood oxygenation, using visible to near-infrared light. HSI oximeters often use a liquid-crystal tunable filter, an acousto-optic tunable filter or mechanically adjustable filter wheels, which has too long response/switching times to monitor tissue hemodynamics. This work aims to evaluate a multispectral snapshot imaging system to estimate skin blood volume and oxygen saturation with high temporal and spatial resolution. We use a snapshot imager, the xiSpec camera (MQ022HG-IM-SM4X4-VIS, XIMEA (R)), having 16 wavelength-specific Fabry-Perot filters overlaid on the custom CMOS-chip. The spectral distribution of the bands is however substantially overlapping, which needs to be taken into account for an accurate analysis. An inverse Monte Carlo analysis is performed using a two-layered skin tissue model, defined by epidermal thickness, haemoglobin concentration and oxygen saturation, melanin concentration and spectrally dependent reduced-scattering coefficient, all parameters relevant for human skin. The analysis takes into account the spectral detector response of the xiSpec camera. At each spatial location in the field-of-view, we compare the simulated output to the detected diffusively backscattered spectra to find the best fit. The imager is evaluated for spatial and temporal variations during arterial and venous occlusion protocols applied to the forearm. Estimated blood volume changes and oxygenation maps at 512x272 pixels show values that are comparable to reference measurements performed in contact with the skin tissue. We conclude that the snapshot xiSpec camera, paired with an inverse Monte Carlo algorithm, permits us to use this sensor for spatial and temporal measurement of varying physiological parameters, such as skin tissue blood volume and oxygenation. Funding Agencies|Centre for Industrial Information Technology (CENIIT) at Linkoping University; SEMEOTICONS (SEMEiotic Oriented Technology for Individuals CardiOmetabolic risk self-assessmeNt and Self-monitoring) [611516]

Endothelial function is important for regulating peripheral blood flow to meet varying metabolic demands and can be measured indirectly during vascular provocations. In this study, we compared the PAT finger response (EndoPAT) after a 5-minutes arterial occlusion to that from forearm skin comprehensive microcirculation analysis (EPOS).Measurements in 16 subjects with varying cardiovascular risk factors were carried out concurrently with both methods during arterial occlusion, while forearm skin was also evaluated during local heating.Peak values for EPOS skin PerfWe have demonstrated for the first time a significant relationship between forearm skin microcirculatory perfusion and oxygen saturation and finger PAT. Both local heating and reactive hyperemia are useful skin provocations. Further studies are needed to understand the precise regulation mechanisms of blood flow and oxygenation during these tests.

A new method for estimating the measurement depth and volume in laser Doppler flowmetry (LDF) is presented. The method is based on Monte Carlo simulations of light propagation in tissue. The contribution from each individual Doppler shift is calculated and thereby multiple Doppler shifts are handled correctly. Different LDF setups for both probe based (0.0, 0.25, 0.5, and 1.2 mm source-detector separation) and imaging systems (0.5 and 2.0 mm beam diameter) are considered, at the wavelengths 543 nm, 633 nm, and 780 nm. Non-linear speckle pattern effects are accounted for in the imaging system setups. The effects of tissue optical properties, blood concentration, and blood oxygen saturation are evaluated using both homogeneous tissue models and a layered skin model. The results show that the effect on the measurement depth of changing tissue properties is comparable to the effect of changing the system setup, e.g. source-detector separation and wavelength. Skin pigmentation was found to have a negligible effect on the measurement depth. Examples of measurement depths are (values are given for a probe based system with 0.25 mm source-detector separation and an imaging system with a 0.5 mm beam diameter, respectively, both operating at 780 nm): muscle - 0.55/0.79 mm; liver - 0.40/0.53 mm; gray matter - 0.48/0.68 mm; white matter - 0.20/0.20 mm; index finger pulp - 0.41/0.53 mm; forearm skin - 0.53/0.56 mm; heat provoked forearm skin - 0.66/0.67 mm. Original Publication:Ingemar Fredriksson, Marcus Larsson and Tomas Strömberg, Measurement depth and volume in laser Doppler flowmetry, 2009, Microvascular Research, (78), 1, 4-13.http://dx.doi.org/10.1016/j.mvr.2009.02.008Copyright: Elsevier Science B.V., Amsterdamhttp://www.elsevier.com/

We have developed a new fiber-optic system that combines diffuse reflectance spectroscopy (DRS) and laser Doppler Flowmetry (LDF) for a multi-modal assessment of the microcirculation. Quantitative data is achieved with an inverse Monte Carlo algorithm based on an individually adaptive skin model. The output parameters are calculated from the model and given in absolute units: hemoglobin oxygen saturation (%), red blood cell (RBC) tissue fraction (%), and the speed resolved RBC perfusion separated into three speed regions; 0–1 mm/s, 1–10 mm/s and above 10 mm/s (% mm/s). The aim was to explore microcirculatory parameters using the new optical method, integrating DRS and LDF in a joint skin model, during local heating of the dorsal foot and venous and arterial occlusion of the forearm in 23 healthy subjects (age 20–28 years). There were differences in the three speed regions in regard to blood flow changes due to local heating, where perfusion for high speeds increased the most. There was also a high correlation between changes in oxygenation and changes in perfusion for higher speeds. Oxygen saturation at baseline was 44% on foot, increasing to 83% at plateau after heating. The larger increase in perfusion for higher speeds than for lower speeds together with the oxygenation increase during thermal provocation, shows a local thermoregulatory blood flow in presumably arteriolar dermal vessels. In conclusion, there are improved possibilities to assess microcirculation using integrated DRS and LDF in a joint skin model by enabling both oxygenation and speed resolved blood flow assessment simultaneously and in the same skin site. Output parameters in absolute units may also yield new insights about the microcirculatory system. Funding text: VINNOVA (Swedens innovation agency); Perimed AB through the SamBIO research collaboration program [2008-00149]; Research&Grow program (VINNOVA) [2011-03074]; NovaMedTech - European Union Regional Development Fund [68737, 160382]

By using a combined inverse model for diffuse reflectance spectroscopy (DRS) and laser Doppler flowmetry (LDF) the tissue fraction of red blood cells (RBCs), their oxygenation and speed-resolved perfusion are estimated in absolute units. DRS spectra (450 to 850 nm) are measured at two source-detector distances; 0.4 and 1.2 mm. LDF spectra are measured at 1.2 mm, integrated in the same fiber-optic probe. Inverse Monte Carlo technique and an adaptive tissue model is used to quantify the microcirculatory parameters. Measurements were done during venous occlusion of the tissue. The model fitting yields a good spectral fit for the two DRS spectra and the LDF spectrum. The physiological responses regarding for example which speed regions respond to provocations follows a priori expectations. The combined model gives quantitative measures of RBC tissue fraction, oxygenation and speed resolved perfusion from the same sampling volume which gives new opportunities to interpret data.

Laser Doppler flowmetry (LDF) and laser speckle contrast analysis (LASCA) both utilize the spatiotemporalproperties of laser speckle patterns to assess microcirculatory blood flow in tissue. Although the techniquesanalyze the speckle pattern differently, there is a close relationship between them. We present atheoretical overview describing how the LDF power spectrum and the LASCA contrast can be calculatedfrom each other, and how both these can be calculated from an optical Doppler spectrum containing variousdegrees of Doppler shifted light. The theoretical relationships are further demonstrated using time-resolvedspeckle simulations. A wide range of Monte Carlo simulated tissue models is then used to show how perfusionestimates for LDF and LASCA are affected by changes in blood concentration and speed distribution, as well asby geometrical and optical properties. We conclude that perfusion estimates from conventional single exposuretime LASCA are in general more sensitive to changes in optical and geometrical properties and are less accuratein the prediction of real perfusion changes, especially speed changes. Since there is a theoretical one-to-onerelationship between Doppler power spectrum and contrast, one can conclude that those drawbacks with theLASCA technique can be overcome using a multiple exposure time setup. Funding agencies: Swedish Research Council [2014-6141]; CENIIT research organization within Linkoping University [11.02]

When laser light illuminates a diffuse object, it produces a random interference effect known as a speckle pattern. If there is movement in the object, the speckles fluctuate in intensity. These fluctuations can provide infor- mation about the movement. A simple way of accessing this information is to image the speckle pattern with an exposure time longer than the shortest speckle fluctuation time scale—the fluctuations cause a blurring of the speckle, leading to a reduction in the local speckle contrast. Thus, velocity distributions are coded as speckle con- trast variations. The same information can be obtained by using the Doppler effect, but producing a two-dimen- sional Doppler map requires either scanning of the laser beam or imaging with a high-speed camera: laser speckle contrast imaging (LSCI) avoids the need to scan and can be performed with a normal CCD- or CMOS-camera. LSCI is used primarily to map flow systems, especially blood flow. The development of LSCI is reviewed and its lim- itations and problems are investigated. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.

Laser Doppler flowmetry (LDF) can be used for assessing the microcirculatory perfusion. However, conventional LDF (cLDF) gives only a relative perfusion estimate for an unknown measurement volume, with no information about the blood flow speed distribution. To overcome these limitations, a model-based analysis method for quantitative LDF (qLDF) is proposed. The method uses inverse Monte Carlo technique with an adaptive three-layer skin model. By analyzing the optimal model where measured and simulated LDF spectra detected at two different source-detector separations match, the absolute microcirculatory perfusion for a specified speed region in a predefined volume is determined. qLDF displayed errors12% when evaluated using simulations of physiologically relevant variations in the layer structure, in the optical properties of static tissue, and in blood absorption. Inhomogeneous models containing small blood vessels, hair, and sweat glands displayed errors5%. Evaluation models containing single larger blood vessels displayed significant errors but could be dismissed by residual analysis. In vivo measurements using local heat provocation displayed a higher perfusion increase with qLDF than cLDF, due to nonlinear effects in the latter. The qLDF showed that the perfusion increase occurred due to an increased amount of red blood cells with a speed1 mm∕s.

OBJECTIVE-To compare the microcirculatory velocity distribution in type 2 diabetic patients and nondiabetic control subjects at baseline and after local heating. RESEARCH DESIGN AND METHODS-The skin blood flow response to local heating (44 degrees C for 20 mm) was assessed in 28 diabetic patients and 29 control subjects using a new velocity-resolved quantitative laser Doppler flowmetry technique (qLDF). The qLDF estimates erythrocyte (RBC) perfusion (velocity X concentration), in a physiologically relevant unit (grams RBC per 100 g tissue X millimeters per second) in a fixed output volume, separated into three velocity regions: v less than1 mm/s, v 1-10 mm/s, and v greater than10 mm/s. RESULTS-The increased blood flow occurs in vessels with a velocity greater than1 mm/s. A significantly lower response in qLDF total perfusion was found in diabetic patients than in control subjects after heat provocation because of less high-velocity blood flow (v greater than10 mm/s). The RBC concentration in diabetic patients increased sevenfold for v between 1 and 10 mm/s, and 15-fold for v greater than10 mm/s, whereas no significant increase was found for v less than1 mm/s. The mean velocity increased from 0.94 to 7.3 mm/s in diabetic patients and from 0.83 to 9.7 mm/s in control subjects. CONCLUSIONS-The perfusion increase occurs in larger shunting vessels and not as an increase in capillary flow. Baseline diabetic patient data indicated a redistribution of flow to higher velocity regions, associated with longer duration of diabetes. A lower perfusion was associated with a higher BMI and a lower toe-to-brachial systolic blood pressure ratio. This is an author-created, uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association (ADA), publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version is online at http://diabetes.diabetesjournals.org.:Ingemar Fredriksson, Marcus Larsson, Fredrik Nyström, Toste Länne, Carl Johan Östgren and Tomas Strömberg, Reduced Arteriovenous Shunting Capacity After Local Heating and Redistribution of Baseline Skin Blood Flow in Type 2 Diabetes Assessed With Velocity-Resolved Quantitative Laser Doppler Flowmetry, 2010, Diabetes, (59), 7, 1578-1584.http://dx.doi.org/10.2337/db10-0080Copyright: American Diabetes Association Inc / American Diabetes Association; 1999http://www.diabetes.org/

An optical microvascular skin model, valid at 780 nm, was developed. The model consisted of six layers with individual optical properties, and variable thicknesses and blood concentrations at three different blood flow velocities. Monte Carlo simulations were used to evaluate the impact of various model parameters on the traditional Laser Doppler flowmetry (LDF) measures. A set of reference Doppler power spectra was generated by simulating 7,000 configurations, varying the thickness and blood concentrations. Simulated spectra, at two different source detector separations, were compared with in vivo recorded spectra, using a non-linear search algorithm for minimizing the deviation between simulated and measured spectra. The model was validated by inspecting the thickness and blood concentrations which generated the best fit. These four parameters followed a priori expectations for the measurement situations, and the simulated spectra agreed well with the measured spectra for both detector separations. Average estimated dermal blood concentration was 0.08% at rest and 0.63% during heat provocation (44°C) on the volar side of the forearm, and 1.2% at rest on the finger pulp. The model is crucial for developing a technique for velocity-resolved absolute LDF measurements with known sampling volume, and can also be useful for other bio-optical modalities. Ingemar Fredriksson, Marcus Larsson and Tomas Strömberg, Optical microcirculatory skin model: Assessed by Monte Carlo simulations paired with in vivo laser Doppler flowmetry, 2008, Journal of Biomedical Optics, (13), 1, 14015. http://dx.doi.org/10.1117/1.2854691. Copyright 2008 Society of Photo-Optical Instrumentation Engineers. This paper was published in Journal of Biomedical Optics and is made available as an electronic reprint with permission of SPIE. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.

Hepatocyte growth factor (HGF) is a heparin-binding protein with mitogenic, motogenic and morphogenic activities for various cell types. The regenerative properties of HGF have been the object of several animal and in vitro studies in recent years.To investigate the physiological and therapeutic effects of HGF on chronic leg ulcers.HGF in gel form was locally applied, once daily for 7 days, to 15 of 19 chronic leg ulcers in 11 elderly patients. All patients had previously been treated by conventional methods and their leg ulcers had been in stable conditions for between 1 and 14 years. Any signs of allergy, discomfort or pain were reported daily. Microcirculation perfusion in the ulcers, compared to the intact contiguous skin, was determined by laser Doppler at the beginning of the study, after 1 week and again after 3 months (in seven patients). Ulcer size and characteristics were also documented.It was observed that microcirculatory perfusion, which might reflect the angiogenic effect of HGF, was statistically significantly correlated (r = 0.94, p0.002) to ulcer area reduction in the treated ulcers. Excellent (84-100% area reduction) or partial healing (58-59%) was seen in eight out of 11 patients. No control group was included in this pilot study, which must be completed by proper control studies.This study suggests that HGF may heal chronic leg ulcers, possibly by improving the microcirculation. Proper control studies need to be performed.

Microvascular assessment would benefit from co-registration of blood flow and hemoglobin oxygenation dynamics during stimulus response tests. We used a fiber-optic probe for simultaneous recording of white light diffuse reflectance (DRS; 475-850 nm) and laser Doppler flowmetry (LDF; 780 nm) spectra at two source-detector distances (0.4 and 1.2 mm). An inverse Monte Carlo algorithm, based on a multiparameter three-layer adaptive skin model, was used for analyzing DRS data. LDF spectra were conventionally processed for perfusion. The system was evaluated on volar forearm recordings of 33 healthy subjects during a 5-min systolic occlusion protocol. The calibration scheme and the optimal adaptive skin model fitted DRS spectra at both distances within 10%. During occlusion, perfusion decreased within 5 s while oxygenation decreased slowly (mean time constant 61 s; dissociation of oxygen from hemoglobin). After occlusion release, perfusion and oxygenation increased within 3 s (inflow of oxygenized blood). The increased perfusion was due to increased blood tissue fraction and speed. The supranormal hemoglobin oxygenation indicates a blood flow in excess of metabolic demands. In conclusion, by integrating DRS and LDF in a fiber-optic probe, a powerful tool for assessment of blood flow and oxygenation in the same microvascular bed has been presented.

Analgesized skin, when subjected to heat stimuli, responds by increasing skin perfusion. This response does not originate from increased perfusion in superficial capillaries, but rather in the deeper lying vessels. The aim of this study was to assess changes in blood chromophore content, measured by reflection spectroscopy, in relation to the perfusion increase, especially regarding the chromophores oxyhemoglobin and deoxyhemoglobin. Eleven normal subjects were treated with analgesic cream (EMLA) and placebo for 20, 40, 60, 120, and 180 min. Individual reactions to local heating were classified as responses if the change in reflection data or the change in perfusion, as measured by laser Doppler blood flowmetry, exceeded 2 standard deviations of normal variation. The increase in blood perfusion or in blood content gave rise to an increased absorption, interpreted as an increase due mainly to the chromophore oxyhemoglobin. The number of responses increased with increased treatment time for EMLA-treated areas. In general, there was a good agreement between both methods; 44 of 55 classifications coincided for the two methods used. In conclusion, analgesized forearm skin, which had been exposed to local heating, responded with an elevated perfusion consisting of oxygenated blood. This strengthens the hypothesis that the flow increase occurs through dilatation of larger deeper lying skin vessels and not in the capillaries.

The tissue microcirculation, as measured by laser Doppler flowmetry (LDF), comprises both capillary, arterial and venous blood flow. With the classical LDF approach, it has been impossible to differentiate between different vascular compartments. We suggest an alternative LDF algorithm that estimates at least three concentration measures of flowing red blood cells (RBCs), each associated with a predefined, physiologically relevant, absolute velocity in mm/s. As the RBC flow velocity depends on the dimension of the blood vessel, this approach might enable a microcirculatory flow differentiation. The LDF concentration estimates are derived by fitting predefined Monte Carlo simulated, single velocity, spectra to a measured, multiple velocity LDF spectrum. Validation measurements, using both single and double-tube flow phantoms perfused with a microsphere solution, showed that it is possible to estimate velocity and concentration changes, and to differentiate between flows with different velocities. The presented theory was also applied to RBC flow measurements. A Gegenbauer kernel phase function (αgk = 1:05; ggk = 0:93), with an anisotropy factor of 0.987 at 786 nm, was found suitable for modelling Doppler scattering by red blood cells diluted in physiological saline. The method was developed for low concentrations of RBCs, but can in theory be extended to cover multiple Doppler scattering. Copyright 2006 Society of Photo-Optical Instrumentation Engineers. This paper was published in the Journal of Biomedical Optics, (11), 14024 and is made available as an electronic reprint with permission of SPIE. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited. Marcus Larsson and Tomas Strömberg, Towards a velocity resolved microvascular blood flow measure by decomposition of the laser Doppler spectrum, 2006, Journal of Biomdeical Optics, (11), 14024. http://dx.doi.org/10.1117/1.2166378.

Laser Doppler flowmetry (LDF) is based on the principle that a Doppler shift occurs when coherent light is scattered by a moving object, i.e. red blood cell (RBC). The magnitude of these frequency shifts affects the optical beating that occurs w hen shifted and non-shifted light is mixed. Based on the optical beating, an LDF perfusion measure is calculated. However, the measure is not only sensitive to the RBC velocity and concentration, but also to the photon path Jength in tissue and the scattering characteristics of the RBC. The Jatter two are both govemed by the optical properties (OP), attributes that differ both within and between individuals.The aim of this thesis was to evaluate how the RBC and tissue OP affect the LDF perfusion measure, and to propose methods that partly correct for these errors. Phantom measurements and Monte Carlo simulations showed that the LDF perfusion was significantly affected by variations in OP relevant to skin, especially when comparing individual readings. Simulations revealed that the variations in OP affected the LDF perfusion and the photon path length in a similar manner. This suggests that a path length normalised measure would decrease the OP induced variations, possibly enabling accurate intra and inter-individual comparisons of LDF perfusion measures in different organs.A path length estimation technique, based on spatially diffuse reflectance, is proposed and evaluated. Monte Carlo simulations showed that the algorithm predicted the photon path length with an rms error of less than 5%. In vivo measurement (11 subjects) displayed a longer estimated path length (~35%) for the fingertip compared to the forearm. Comparing individual measurements from similar locations, variations up to 40% (max/min) were found. These findings clearly indicate the need for a path length normalization when comparing LDF readings.The LDF Doppler spectrum is govemed by the RBC velocity distribution and its phase function. In this thesis, an approach is presented where a measured LDF Doppler spectrum is decomposed using a number of theoretical, single-velocity spectra. As a result, a velocity-resolved perfusion measure is achieved. As the blood flow velocity depends on the dimension of the blood vessel, this approach has the potential to differentiate between arteriole/ venule and capillary activity. In addition, the path length estimation technique and the RBC scattering theory, presented in this thesis, provides a promising step towards an absolute perfusion measure.