Multimodal evaluation of the effects of low-intensity ultrasound on cerebral blood flow after traumatic brain injury in mice.

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide, and destruction of the cerebrovascular system is a major factor in the cascade of secondary injuries caused by TBI. Laser speckle imaging (LSCI)has high sensitivity in detecting cerebral blood flow. LSCI can visually show that transcranial focused ultrasound stimulation (tFUS) treatment stimulates angiogenesis and increases blood flow. To study the effect of tFUS on promoting angiogenesis in Controlled Cortical impact (CCI) model. tFUS was administered daily for 10 min and for 14 consecutive days after TBI. Cerebral blood flow was measured by LSCI at 1, 3, 7 and 14 days after trauma. Functional outcomes were assessed using LSCI and neurological severity score (NSS). After the last test, Nissl staining and vascular endothelial growth factor (VEGF) were used to assess neuropathology. TBI can cause the destruction of cerebrovascular system. Blood flow was significantly increased in TBI treated with tFUS. LSCI, behavioral and histological findings suggest that tFUS treatment can promote angiogenesis after TBI.

Pudendal, but not tibial, nerve stimulation modulates vulvar blood perfusion in anesthetized rodents.

INTRODUCTION AND HYPOTHESIS: Preclinical studies have shown that neuromodulation can increase vaginal blood perfusion, but the effect on vulvar blood perfusion is unknown. We hypothesized that pudendal and tibial nerve stimulation could evoke an increase in vulvar blood perfusion. METHODS: We used female Sprague-Dawley rats for non-survival procedures under urethane anesthesia. We measured perineal blood perfusion in response to 20-minute periods of pudendal and tibial nerve stimulation using laser speckle contrast imaging (LSCI). After a thoracic-level spinalization and a rest period, we repeated each stimulation trial. We calculated average blood perfusion before, during, and after stimulation for three perineal regions (vulva, anus, and inner thigh), for each nerve target and spinal cord condition. RESULTS: We observed a significant increase in vulvar, anal, and inner thigh blood perfusion during pudendal nerve stimulation in spinally intact and spinalized rats. Tibial nerve stimulation had no effect on perineal blood perfusion for both spinally intact and spinalized rats. CONCLUSIONS: This is the first study to examine vulvar hemodynamics with LSCI in response to nerve stimulation. This study demonstrates that pudendal nerve stimulation modulates vulvar blood perfusion, indicating the potential of pudendal neuromodulation to improve genital blood flow as a treatment for women with sexual dysfunction. This study provides further support for neuromodulation as a treatment for women with sexual arousal disorders. Studies in unanesthetized animal models of genital arousal disorders are needed to obtain further insights into the mechanisms of neural control over genital hemodynamics.

Progressive Observation of Covid-19 Vaccination Effects on Skin-Cellular Structures by Use of Intelligent Laser Speckle Classification (ILSC) Technique.

The recent pandemic showed that the current global research strategies on vaccine development in an emergency period necessitates more optimized supplementary techniques to observe instant progressive vaccines' subtle effects on human metabolisms to make better and speedy evolutionary health assessments. To fill this gap, we have followed a multi-disciplinary approach exploiting AI, laser-optics, and specific imaging methods. The proposed technique can make progressive observations on Covid-19 Astra Zeneca vaccination effects on skin cellular network by use of the well-established technique-Intelligent Laser Speckle Classification (ILSC), as Covid-19 is a skin-affecting systemic disease. The method also managed to distinguish between three different subject groups via their laser speckle skin image samplings, grouped as early-vaccinated, late-vaccinated and non-vaccinated participants. The results have proven that the ILSC technique, in association with the parametrically optimised Bayesian network, can classify hidden skin changes of vaccinated and non-vaccinated individuals up to 90% accuracy and is also capable of detecting instant progressive developments pertaining to skin cellular properties. The proposed method has also proven that the continuous Covid-19 vaccine effect on the sub-skin layers can be observable by high frequency and speedy non-invasive data collection in real-time with high reliability.

The application of SFDI and LSI system to evaluate the blood perfusion in skin flap mouse model.

The aim of this study is to evaluate whether the blood perfusion to tissues for detecting ischemic necrosis can be quantitatively monitored by spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) in a skin flap mouse model. Skin flaps were made on Institute of Cancer Research (ICR) mice. Using SFDI and LSI, the following parameters were estimated: oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), total hemoglobin (THb), tissue oxygen saturation (StO2), and speckle flow index (SFI). Histologically, epithelium thickness, collagen deposition, and blood vessel count of skin flap tissues were analyzed. Then, the correlation of SFDI and histological results was assessed by application of Spearman rank correlation method. As the result, the number of blood vessels and the percentage of collagen areas showed significant difference between the necrotic tissue group and the non-necrotic one. Especially, the necrotic tissue had a complete epithelial loss and loses its normal structure. We identified that SFDI/LSI parameters were significantly different between non-necrotic and necrotic tissue groups. Especially, all SFDI and LSI parameters measured on the 1st day after surgery showed significant difference between necrotic tissue and non-necrotic tissue. In addition, the number of blood vessel and percentage of collagen area were positively correlated with HbO2 and StO2 among SFDI/LSI parameters. Meanwhile, the number of blood vessel and percentage of collagen area showed the negative correlation with Hb. By applying SFDI and LSI simultaneously to the skin flap, we could quantitatively monitor the blood perfusion and the tissue condition which can help us to detect ischemic necrosis objectively in early stage.

Multi-wavelength, handheld laser speckle imaging for skin evaluation.

OBJECTIVE: A handheld device was developed and qualified for in vivo human skin evaluation using laser speckle imaging technology. METHODS: Each laser speckle device prototype allows the choice of up to three different laser wavelengths in the range of 400 nm to 800 nm in total. Speckle pattern analysis gives various speckle parameters, for example, speckle contrast, speckle size, speckle modulation or fractal dimension. The developed laser speckle device prototypes were evaluated investigating three skin issues. RESULTS: We receive reproducible results from the speckle imaging device. For skin ageing, we found significant changes within three age groups. The effect of a methyl nicotinate treatment was clearly visible and quantifiable using a moorFLPI device as well as our speckle imaging device. In terms of basal cell carcinoma diagnosis, we found significant differences between normal and diseased skin, even though the number of samples was limited. CONCLUSION: As shown with first application examples, it was possible to demonstrate the potential of the method for skin evaluation in vivo.

Improving imaging depth by dynamic laser speckle imaging and topical optical clearing for in vivo blood flow monitoring.

Laser speckle contrast imaging (LSI) is a promising non-invasive full-field blood flow monitoring technique. However, it is still far from clinic practice due to insufficient contrast-to-noise ratio (CNR) and limited detection depth. In this study, an in vivo laser speckle imaging visualization system was constructed to observe the blood circulation on a dorsal skin. A dynamic laser speckle imaging (dLSI) scheme, other than traditional laser speckle contrast analysis method, was applied to separate the dynamic light scattering from the static one to increase the image CNR. Based on the theoretical optimization for dLSI, at least two pixels are required for speckle pattern sampling and a spatial window size of 7 × 7 was optimal to balance the spatial resolution and statistical accuracy. The in vivo experiment observation shows that the CNR is improved 8.4 times by dLSI. The blood vessels were more pronounced, and more capillaries can be observed than in traditional laser speckle contrast images. Topical optical clearing technique by thiazone was combined with dLSI to increase the sampling depth from 700 to 1000 µm.

Extraction of the Structural Properties of Skin Tissue via Diffuse Reflectance Spectroscopy: An Inverse Methodology.

For the laser treatment of vascular dermatosis, the blood vessel morphology and depth in skin tissue is essential to achieve personalized intelligent therapy. The morphology can be obtained from the laser speckle imaging, and vessel depth was extracted by an inverse methodology based on diffuse reflectance spectrum. With optimized spot size of 0.5 mm and known optical properties, the proposed method was experimentally validated via the spectral measurement of microcapillary with known size and depth embedded in an epoxy resin-based skin phantom. Results prove that vessel depth can be extracted with an average relative error of 5%, thereby providing the foundation for a personalized, precise, and intelligent laser treatment of vascular dermatosis.

Peri-Infarct Hot-Zones Have Higher Susceptibility to Optogenetic Functional Activation-Induced Spreading Depolarizations.

BACKGROUND AND PURPOSE: Spreading depolarizations (SDs) are recurrent and ostensibly spontaneous depolarization waves that may contribute to infarct progression after stroke. Somatosensory activation of the metastable peri-infarct tissue triggers peri-infarct SDs at a high rate. METHODS: We directly measured the functional activation threshold to trigger SDs in peri-infarct hot zones using optogenetic stimulation after distal middle cerebral artery occlusion in Thy1-ChR2-YFP mice. RESULTS: Optogenetic activation of peri-infarct tissue triggered SDs at a strikingly high rate (64%) compared with contralateral homotopic cortex (8%; P=0.004). Laser speckle perfusion imaging identified a residual blood flow of 31±2% of baseline marking the metastable tissue with a propensity to develop SDs. CONCLUSIONS: Our data reveal a spatially distinct increase in SD susceptibility in peri-infarct tissue where physiological levels of functional activation are capable of triggering SDs. Given the potentially deleterious effects of peri-infarct SDs, the effect of sensory overstimulation in hyperacute stroke should be examined more carefully.

Noninvasive assessment of bowel blood perfusion using intraoperative laser speckle flowgraphy.

PURPOSE: Laser speckle flowgraphy (LSFG) is a noninvasive method for quantitative evaluation of blood flow using the mean blur rate (MBR) as the blood flow index. We investigated whether LSFG can intraoperatively detect the demarcation line after vessel dissection and reduce the incidence of anastomotic leakage (AL). METHODS: This study included 36 patients who underwent left-sided colorectal surgery. First, we compared the demarcation line (determined by LSFG) with the transection line (TL) at which the marginal vessels were divided. We then measured the MBR on both sides of the TL to determine where the MBR changed significantly. We investigated the presence or absence of significant differences between the MBR on the proximal side and that on the distal side of the TL. Finally, we retrospectively compared the patient characteristics and AL rates in the LSFG group (n = 36) and control group (n = 87). RESULTS: In total, 58.3% (21/36) of the demarcation lines determined by LSFG matched the TL. The median distance between the demarcation line determined by LSFG and the TL was 0.0 mm (0.0-12.1 mm). The MBR sharply decreased at the TL in 80.6% (29/36) of cases. The median MBR was significantly lower on the distal than proximal side. The AL rate was not significantly lower in the LSFG group than in the control group. CONCLUSION: LSFG accurately detected the demarcation line during surgery. However, LSFG did not reduce the incidence of AL.

Optical Access to Arteriovenous Cerebral Microcirculation Through a Transparent Cranial Implant.

BACKGROUND AND OBJECTIVE: Microcirculation plays a critical role in physiologic processes and several disease states. Laser speckle imaging (LSI) is a full-field, real-time imaging technique capable of mapping microvessel networks and providing relative flow velocity within the vessels. In this study, we demonstrate that LSI combine with multispectral reflectance imaging (MSRI), which allows for distinction between veins and arteries in the vascular flow maps produced by LSI. We apply this combined technique to mouse cerebral vascular network in vivo, comparing imaging through the skull, to the dura mater and brain directly through a craniectomy, and through a transparent cranial "Window to the Brain" (WttB) implant. STUDY DESIGN/MATERIALS AND METHODS: The WttB implant used in this study is made of a nanocrystalline Yttria-Stabilized-Zirconia ceramic. MSRI was conducted using white-light illumination and filtering the reflected light for 560, 570, 580, 590, 600, and 610 nm. LSI was conducted using an 810 nm continuous wave near-infrared laser with incident power of 100 mW, and the reflected speckle pattern was captured by a complementary metal-oxide-semiconductor (CMOS) camera. RESULTS: Seven vessel branches were analyzed and comparison was made between imaging through the skull, craniectomy, and WttB implant. Through the skull, MSRI did not detect any vessels, and LSI could not image microvessels. Imaging through the WttB implant, MSRI was able to identify veins versus arteries, and LSI was able to image microvessels with only slightly higher signal-to-noise ratio and lower sharpness than imaging the brain through a craniectomy. CONCLUSIONS: This study demonstrates the ability to perform MSRI-LSI across a transparent cranial implant, to allow for cerebral vascular networks to be mapped, including microvessels. These images contain additional information such as vein-artery separation and relative blood flow velocities, information which is of value scientifically and medically. The WttB implant provides substantial improvements over imaging through the murine cranial bone, where microvessels are not visible and MSRI cannot be performed. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Hemodynamic effects of intraoperative anesthetics administration in photothrombotic stroke model: a study using laser speckle imaging.

BACKGROUND: Previous neuroimaging studies have shown the hemodynamic effect of either preconditioning or postconditioning anesthesia in ischemic stroke model. However, the anesthetic effect in hemodynamics during and immediately after the stroke modeling surgery remains unknown due to the lack of appropriate anesthesia-free stroke model and intraoperative imaging technology. In the present study, we utilized our recently developed photothrombotic model of focal cerebral ischemia in conscious and freely moving rats, and investigated transient hemodynamic changes with or without isoflurane administration. Laser speckle imaging was applied to acquire real-time two-dimensional full-field cerebral blood flow (CBF) information throughout the surgical operations and early after. RESULTS: Significantly larger CBF reduction area was observed in conscious rats from 8 min immediately after the onset of stroke modeling, compared with anesthetized rats. Stroke rats without isoflurane administration also showed larger lesion volume identified by magnetic resonance imaging 3 h post occlusion (58.9%), higher neurological severity score 24 h post occlusion (28.3%), and larger infarct volume from 2,3,5-triphenyltetrazolium chloride staining 24 h post occlusion (46.9%). CONCLUSIONS: Our results demonstrated that the hemodynamic features were affected by anesthetics at as early as during the stroke induction. Also, our findings about the neuroprotection of intraoperative anesthetics administration bring additional insights into understanding the translational difficulty in stroke research.

Quantitative long-term measurements of burns in a rat model using Spatial Frequency Domain Imaging (SFDI) and Laser Speckle Imaging (LSI).

BACKGROUND AND OJECTIVES: The current standard for diagnosis of burn severity and subsequent wound healing is through clinical examination, which is highly subjective. Several new technologies are shifting focus to burn care in an attempt to help quantify not only burn depth but also the progress of healing. While accurate early assessment of partial thickness burns is critical for dictating the course of treatment, the ability to quantitatively monitor wound status over time is critical for understanding treatment efficacy. SFDI and LSI are both non-invasive imaging modalities that have been shown to have great diagnostic value for burn severity, but have yet to be tested over the course of wound healing. METHODS: In this study, a hairless rat model (n = 6, 300-450 g) was used with a four pronged comb to create four identical partial thickness burns (superficial n = 3 and deep n = 3) that were used to monitor wound healing over a 28 days period. Weekly biopsies were taken for histological analysis to verify wound progression. Both SFDI and LSI were performed weekly to track the evolution of hemodynamic (blood flow and oxygen saturation) and structural (reduced scattering coefficient) properties for the burns. RESULTS: LSI showed significant changes in blood flow from baseline to 220% in superficial and 165% in deep burns by day 7. In superficial burns, blood flow returned to baseline levels by day 28, but not for deep burns where blood flow remained elevated. Smaller increases in blood flow were also observed in the surrounding tissue over the same time period. Oxygen saturation values measured with SFDI showed a progressive increase from baseline values of 66-74% in superficial burns and 72% in deep burns by day 28. Additionally, SFDI showed significant decreases in the reduced scattering coefficient shortly after the burns were created. The scattering coefficient progressively decreased in the wound area, but returned towards baseline conditions at the end of the 28 days period. Scattering changes in the surrounding tissue remained constant despite the presence of hemodynamic changes. CONCLUSIONS: Here, we show that LSI and SFDI are capable of monitoring changes in hemodynamic and scattering properties in burn wounds over a 28 days period. These results highlight the potential insights that can be gained by using non-invasive imaging technologies to study wound healing. Further development of these technologies could be revolutionary for wound monitoring and studying the efficacy of different treatments. Lasers Surg. Med. 49:293-304, 2017. © 2017 Wiley Periodicals, Inc.

Imaging functional blood vessels by the laser speckle imaging (LSI) technique using Q-statistics of the generalized differences algorithm.

In this work, we report about q statistics concept to improve the performance of generalized differences algorithm based on intensity histogram for imaging functional blood vessel structures in a rodent window chamber of a mice. The method uses the dynamic speckle signals obtained by transilluminating the rodent window chamber to create activity maps of vasculatures. The proposed method of generalized differences with q statistics (GDq) is very sensitive to the values of defined parameters such as: camera exposure time, the q value and the camera frame number. Appropriate choice of q values enhances the visibility (contrast) of functional blood vessels but at the same time without sacrificing the spatial resolution, which is of utmost importance for in-vivo vascular imaging.

Observation of vasculature alternation by intense pulsed light combined with physicochemical methods.

Intense pulsed light (IPL) with low energy insufficient to completely destroy a vasculature was applied to rabbit ears to investigate vasculature alteration. Glycerol was combined with IPL to enhance the transfer efficacy of IPL energy. Both trans-illumination and laser speckle contrast images were obtained and analyzed after treatment. The application of IPL and glycerol combination induced vasodilation and improvement in blood flow. Moreover, such phenomenon was maintained over time. IPL may be applied to treat blood circulatory diseases by inducing vasodilation and to improve blood flow.

Endothelial dysfunction abrogates the efficacy of normobaric hyperoxia in stroke.

Hyperoxia has been uniformly efficacious in experimental focal cerebral ischemia. However, pilot clinical trials have showed mixed results slowing its translation in patient care. To explain the discordance between experimental and clinical outcomes, we tested the impact of endothelial dysfunction, exceedingly common in stroke patients but under-represented in experimental studies, on the neuroprotective efficacy of normobaric hyperoxia. We used hyperlipidemic apolipoprotein E knock-out and endothelial nitric oxide synthase knock-out mice as models of endothelial dysfunction, and examined the effects of normobaric hyperoxia on tissue perfusion and oxygenation using high-resolution combined laser speckle and multispectral reflectance imaging during distal middle cerebral artery occlusion. In normal wild-type mice, normobaric hyperoxia rapidly and significantly improved tissue perfusion and oxygenation, suppressed peri-infarct depolarizations, reduced infarct volumes, and improved neurological function. In contrast, normobaric hyperoxia worsened perfusion in ischemic brain and failed to reduce infarct volumes or improve neurological function in mice with endothelial dysfunction. These data suggest that the beneficial effects of hyperoxia on ischemic tissue oxygenation, perfusion, and outcome are critically dependent on endothelial nitric oxide synthase function. Therefore, vascular risk factors associated with endothelial dysfunction may predict normobaric hyperoxia nonresponders in ischemic stroke. These data may have implications for myocardial and systemic circulation as well.

Targeted narrowband intense pulsed light on cutaneous vasculature.

BACKGROUND AND OBJECTIVES: Laser based therapies are the standard treatment protocol for port wine stain in the United States, but complete removal is infrequently achieved. Intense pulsed light (IPL) offers a broadband light spectrum approach as a viable treatment alternative. Previous studies suggest that IPL can be more effective in treatment of port wine stain by utilizing multiple wavelengths to selectively target different peaks in oxy- and deoxy-hemoglobin. Our study objectives were to (i) determine a characteristic radiant exposure able to achieve persistent vascular shutdown with narrowband IPL irradiation, (ii) determine the degree to which narrowband IPL irradiation can achieve persistent vascular shutdown, and (iii) compare the effectiveness of narrowband IPL radiation to single wavelength pulsed dye laser (PDL) irradiation in achieving persistent vascular shutdown. STUDY DESIGN/MATERIALS AND METHODS: We utlized either single pulse or double, stacked pulses in narrowband IPL experiments, with the IPL operating over a 500-600 nm wavelength range on the rodent dorsal window chamber model. We compared the results from our narrowband IPL experiments to acquired PDL data from a previous study and determined that narrowband IPL treatments can also produce persistent vascular shutdown. We ran Monte Carlo simulations to investigate the relationship between absorbed energy, wavelength, and penetration depth. RESULTS: For single and double pulse narrowband IPL irradiation we observed (i) little to no change in blood flow, resulting in no persistent vascular shutdown, (ii) marked acute disruption in blood flow and vascular structure, followed by partial to full recovery of blood flow, also resulting in no persistent vascular shutdown, and (iii) immediate changes in blood flow and vascular structure, resulting in prolonged and complete vascular shutdown. Monte Carlo modeling resulted in a 53.2% and 69.0% higher absorbed energy distribution in the top half and the total simulated vessel when comparing the composite narrowband IPL to the 595 nm (PDL), respectively. CONCLUSIONS: Our data collectively demonstrate the potential to achieve removal of vascular lesions using a 500-600 nm range. Additionally, the narrowband IPL was tuned to optimize a specific wavelength range that can be used to treat PWS, whereas the PDL can only operate at one discrete wavelength.

Device for simultaneous positron emission tomography, laser speckle imaging and RGB reflectometry: validation and application to cortical spreading depression and brain ischemia in rats.

Brain function critically relies on the supply with energy substrates (oxygen and glucose) via blood flow. Alterations in energy demand as during neuronal activation induce dynamic changes in substrate fluxes and blood flow. To study the complex system that regulates cerebral metabolism requires the combination of methods for the simultaneous assessment of multiple parameters. We developed a multimodal imaging device to combine positron emission tomography (PET) with laser speckle imaging (LSI) and RGB reflectometry (RGBR). Depending on the radiotracer, PET provides 3-dimensional quantitative information of specific molecular processes, while LSI and RGBR measure cerebral blood flow (CBF) and hemoglobin oxygenation at high temporal and spatial resolution. We first tested the functional capability of each modality within our system and showed that interference between the modalities is negligible. We then cross-calibrated the system by simultaneously measuring absolute CBF using (15)O-H2O PET (CBF(PET)) and the inverse correlation time (ICT), the LSI surrogate for CBF. ICT and CBF(PET) correlated in multiple measurements in individuals as well as across different animals (R(2)=0.87, n=44 measurements) indicating that ICT can be used for absolute quantitative assessment of CBF. To demonstrate the potential of the combined system, we applied it to cortical spreading depression (CSD), a wave of transient cellular depolarization that served here as a model system for neurovascular and neurometabolic coupling. We analyzed time courses of hemoglobin oxygenation and CBF alterations coupled to CSD, and simultaneously measured regional uptake of (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) used as a radiotracer for regional glucose metabolism, in response to a single CSD and to a cluster of CSD waves. With this unique combination, we characterized the changes in cerebral metabolic rate of oxygen (CMRO2) in real-time and showed a correlation between (18)F-FDG uptake and the number of CSD waves that passed the local tissue. Finally, we examined CSD spontaneously occurring during focal ischemia also referred to as peri-infarct depolarization (PID). In the vicinity of the ischemic territory, we observed PIDs that were characterized by reduced CMRO2 and increased oxygen extraction fraction (OEF), indicating a limitation of oxygen supply. Simultaneously measured PET showed an increased (18)F-FDG uptake in these regions. Our combined system proved to be a novel tool for the simultaneous study of dynamic spatiotemporal alterations of cortical blood flow, oxygen metabolism and glucose consumption under normal and pathologic conditions.

Optimized parametric skin modelling for diagnosis of skin abnormalities by combining light back-scatter and laser speckle imaging.

BACKGROUND/PURPOSE: Optical and parametric skin imaging methods which can efficiently identify invisible sub-skin features or subtle changes in skin layers are very important for accurate optical skin modelling. In this study, a hybrid method is introduced that helps develop a parametric optical skin model by utilizing interdisciplinary techniques including light back-scatter analysis, laser speckle imaging, image-texture analysis and Bayesian inference methods. The model aims to detect subtle skin changes and hence very early signs of skin abnormalities/diseases. METHODS: Light back-scatter and laser speckle image textural analysis are applied onto the normal and abnormal skin regions (lesions) to generate set of attributes/parameters. These are then optimized by Bayesian inference method in order to build an optimized parametric model. RESULTS: The attributes selected by Bayesian inference method in the optimization stage were used to build an optimized model and then successfully verified. It was clearly proven that Bayesian inference based optimization process yields good results to build an optimized skin model. CONCLUSION: The outcome of this study clearly shows the applicability of this hybrid method in the analysis of skin features and is therefore expected to lead development of non-invasive and low-cost instrument for early detection of skin changes.

Multimodal optical imaging can reveal changes in microcirculation and tissue oxygenation during skin wound healing.

BACKGROUND AND OBJECTIVE: Faster and better wound healing is a longstanding goal. Blood flow, angiogenesis, and tissue oxygenation are important parameters in evaluating the healing process. Optical microangiography (OMAG) allows 3D imaging of tissue vasculature and can provide quantitative blood flow information down to the capillary level of resolution. Dual wavelength laser speckle imaging (DW-LSI) can measure tissue oxygenation status. MATERIALS AND METHODS: Cutaneous wound healing of a mouse ear model using a multimodal imaging system that combines OMAG with DWLSI was studied. RESULTS: A complete microvasculature map of the ear in vivo was obtained. The imaging system revealed both hemodynamic and metabolic changes during acute stage wound healing. Blood flow velocity, blood flow direction, as well as changes in concentration of oxygenated hemoglobin (ΔHbO) and deoxygenated hemoglobin (ΔHb) were measured and quantified. In addition, capillary recruitment and angiogenesis were visualized during the chronic stage of repairing. CONCLUSIONS: The combination of DW-LSI and OMAG imaging technique may be a powerful tool to visualize and understand microvascular, hemodynamic, and metabolic changes during cutaneous wound healing.

Accurate estimation of the inhibition zone of antibiotics based on laser speckle imaging and multiple random speckle illumination.

The antimicrobial susceptibility test (AST) plays a crucial role in selecting appropriate antibiotics for the treatment of bacterial infections in patients. The diffusion disk method is widely adopted AST method due to its simplicity, cost-effectiveness, and flexibility. It assesses antibiotic efficacy by measuring the size of the inhibition zone where bacterial growth is suppressed. Quantification of the zone diameter is typically achieved using tools such as rulers, calipers, or automated zone readers, as the inhibition zone is visually discernible. However, challenges arise due to inaccuracies stemming from human errors or image processing of intensity-based images. Here, we proposed a bacterial activity-based AST using laser speckle imaging (LSI) with multiple speckle illumination. LSI measures a speckle pattern produced by interferences of scattered light from the sample; therefore, LSI enables the detection of variation or movement within the sample such as bacterial activity. We found that LSI with multiple speckle illuminations provides consistent and uniform analysis of measured time-varying speckle images. Furthermore, our proposed method effectively identified the boundary of the inhibition zone using the k-means clustering algorithm, exploiting a result of speckle pattern analysis as features. Collectively, the proposed method offers a versatile analytical tool in the diffusion disk method.