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.     

Full-field optical multi-functional angiography based on endogenous hemodynamic characteristics

Blood flow functional imaging is widely applied in biological research to provide vascular morphological and statistical parameters. It relies on the absorption difference and is, therefore, easily affected by complex biological structures, and it cannot accommodate abundant functional information. We propose a full-field multi-functional angiography method to classify arteriovenous vessels and to display flow velocity and vascular diameter distribution simultaneously. Unlike previous methods, an under-sampled laser Doppler acquisition mode is used to record the low-coherence speckle, and multi-functional angiography is achieved by modulating the endogenous hemodynamic characteristics from low-coherence speckle. To demonstrate the combination of classified angiography, blood flow velocity measurement, and vascular diameter measurement realized using our method, we performed experiments on the flow phantom and living chicken embryos and generated multi-functional angiograms. The proposed method can be used as a label-free multi-functional angiography technique in which red blood cells provide a strong endogenous source of naturally hemodynamic characteristics.     

Osteoporosis evaluation through full developed speckle imaging

Osteoporosis is a disease characterized by bone mineral density reduction, weakening the bone structure. Its diagnosis is performed using ionizing radiation, increasing health risk. Optical techniques are safer, due to non‐ionizing radiation use, but limited to the analyses of bone tissue. This limitation may be circumvented in the oral cavity. In this work we explored the use of laser speckle imaging (LSI) to differentiate the sound and osteoporotic maxilla and mandible bones in an in vitro model. Osteoporosis lesions were simulated with acid attack. The samples were evaluated by optical profilometry and LSI, using a custom software. Two image parameters were evaluated, speckle contrast ration and patches ratio. With the speckle contrast ratio, it was possible to differentiate sound from osteoporotic tissue. From speckle patches ratio it was observed a negative correlation with the roughness parameter. LSI is a promissory technique for assessment of osteoporosis lesions on alveolar bone.     

Development of an imaging device for label-free parathyroid gland identification and vascularity assessment

During thyroid surgeries, it is important for surgeons to accurately identify healthy parathyroid glands and assess their vascularity to preserve their function postoperatively, thus preventing hypoparathyroidism and hypocalcemia. Near infrared autofluorescence detection enables parathyroid identification, while laser speckle contrast imaging allows assessment of parathyroid vascularity. Here, we present an imaging system combining the two techniques to perform both functions, simultaneously and label-free. An algorithm to automate the segmentation of a parathyroid gland in the fluorescence image to determine its average speckle contrast is also presented, reducing a barrier to clinical translation. Results from imaging ex vivo tissue samples show that the algorithm is equivalent to manual segmentation. Intraoperative images from representative procedures are presented showing successful implementation of the device to identify and assess vascularity of healthy and diseased parathyroid glands.     

Laser speckle spatiotemporal variance analysis for noninvasive widefield measurements of blood pulsation and pulse rate on a camera‐phone

Photoplethysmography is a well‐established technique for the noninvasive measurement of blood pulsation. However, photoplethysmographic devices typically need to be in contact with the surface of the tissue and provide data from a single contact point. Extensions of conventional photoplethysmography to measurements over a wide field‐of‐view exist, but require advanced signal processing due to the low signal‐to‐noise‐ratio of the photoplethysmograms. Here, we present a noncontact method based on temporal sampling of time‐integrated speckle using a camera‐phone for noninvasive, widefield measurements of physiological parameters across the human fingertip including blood pulsation and resting heart‐rate frequency. The results show that precise estimation of these parameters with high spatial resolution is enabled by measuring the local temporal variation of speckle patterns of backscattered light from subcutaneous skin, thereby opening up the possibility for accurate high resolution blood pulsation imaging on a camera‐phone.

Camera‐phone laser speckle imager along with measured relative blood perfusion maps of a fingertip showing skin perfusion response to a pulse pressure applied to the upper arm. The figure is for illustration only; the imager was stabilized on a stand throughout the experiments.     

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.     

Enhancing vascular visualization in laser speckle contrast imaging of blood flow using multi‐focus image fusion

Laser speckle contrast imaging (LSCI) is a full‐field optical imaging method for monitoring blood flow and vascular morphology with high spatiotemporal resolution. However, due to the limited depth of field of optical system, it is difficult to capture a clear blood flow image with all blood vessels focused, especially for the non‐planar biological tissues. In this study, a multi‐focus image fusion method based on contourlet transform is introduced to reduce the misfocus effects in LSCI. The experimental results suggest that this method can provide an all‐in‐focus blood flow image, which is convenient to observe the blood vessels.      

Interplay of temperature,thermal‐stresses and strains in laser‐assisted modification of collagenous tissues: Speckle‐contrast and OCT‐based studies

Moderate heating of collagenous tissues such as cartilage and cornea by infrared laser irradiation can produce biologically nondestructive structural rearrangements and relaxation of internal stresses resulting in the tissue reshaping. The reshaping results and eventual changes in optical and biological properties of the tissue strongly depend on the laser‐irradiation regime. Here, a speckle‐contrast technique based on monochromatic illumination of the tissue in combination with strain mapping by means of optical coherence elastography (OCE) is applied to reveal the interplay between the temperature and thermal stress fields producing tissue modifications. The speckle‐based technique ensured en face visualization of cross correlation and contrast of speckle images, with evolving proportions between contributions of temperature increase and thermal‐stresses determined by temperature gradients. The speckle‐technique findings are corroborated by quantitative OCE‐based depth‐resolved imaging of irradiation‐induced strain‐evolution. The revealed relationships can be used for real‐time control of the reshaping procedures (e.g., for laser shaping of cartilaginous implants in otolaryngology and maxillofacial surgery) and optimization of the laser‐irradiation regimes to ensure the desired reshaping using lower and biologically safer temperatures. The figure of waterfall OCE‐image demonstrates how the strain‐rate maximum arising in the heating‐beam center gradually splits and drifts towards the zones of maximal thermal stresses located at the temperature‐profile slopes.     

In vivo noninvasive visualization of retinal perfusion dysfunction in murine cerebral malaria by camera‐phone laser speckle imaging

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection associated with impaired cerebral blood flow. Visualization of the eye vasculature, which is embryologically derived from that of the brain, is used clinically to diagnose the syndrome. Here, we introduce camera‐phone laser speckle imaging as a new tool for in vivo, noncontact two‐dimensional mapping of blood flow dynamics in the experimental cerebral malaria (ECM) murine model of Plasmodium berghei ANKA. In a longitudinal study, we show that the camera‐phone imager can detect an overall decrease in the retinal blood‐flow‐speed (BFS) as ECM develops in P. berghei ANKA infected mice, with no similar change observed in uninfected control mice or mice infected with a non‐ECM inducing strain (P. berghei NK65). Furthermore, by analyzing relative alterations in the BFS of individual retinal vessels during the progression of ECM, we illustrate the strength of our imager in identifying different BFS‐change heterogeneities in the retinas of ECM and uninfected mice. The technique creates new possibilities for objective investigations into the diagnosis and pathogenesis of CM noninvasively through the eye. The camera‐phone laser speckle imager along with measured spatial blood perfusion maps of the retina of a mouse infected with P. berghei ANKA—a fatal ECM model—on different days during the progression of the infection (top, day 3 after infection; middle, day 5 after infection; and bottom, day 7 after infection).      

Remote photonic sensing of cerebral hemodynamic changes via temporal spatial analysis of acoustic vibrations

A novel photonic method for remote monitoring of task‐related hemodynamic changes in human brain activation is presented. Physiological processes associated with neural activity, such as nano‐vibrations due to blood flow and tissue oxygenation in the brain, are detected by remote sensing of nano‐acoustic vibrations using temporal spatial analysis of defocused self‐interference random patterns. Temporal nanometric changes of the speckle pattern due to visual task‐induced hemodynamic responses were tracked by this method. Reversing visual checkerboard stimulation alternated with rest epochs, and responsive signals were identified in occipital lobe using near‐infrared spectroscopy. Temporal vibrations associated with these hemodynamic response functions were observed using three different approaches: (a) single spot illumination at active and control areas simultaneously, (b) subspots cross‐correlation‐based analysis, and (c) multiwavelength measurement using a magnitude‐squared wavelet coherence function. Findings show remote sensing of task‐specific neural activity in the human brain.     

Biospeckle‐characterization of hairy root cultures using laser speckle photometry

Monitoring is indispensable for the optimization and simulation of biotechnological processes. Hairy roots (hr, plant tissue cultures) are producers of valuable relevant secondary metabolites. The genetically stable cultures are characterized by a rapid filamentous growth, making monitoring difficult with standard methods. This article focuses on the application of laser speckle photometry (LSP) as an innovative, non‐invasive method to characterize Beta vulgaris (hr). LSP is based on the analysis of time‐resolved interference patterns. Speckle interference patterns of a biological object, known as biospeckles, are characterized by a dynamic behavior that is induced by physical and biological phenomena related to the object. Speckle contrast, a means of measuring the dynamic behavior of biospeckles, was used to assess the biospeckle activity. The biospeckle activity corresponds to processes modifying the object and correlates with the biomass growth. Furthermore, the stage of the cultures’ physiological development was assessed by speckle contrast due to the differentiation between active and low active behavior. This method is a new means of monitoring and evaluating the biomass growth of filamentous cultures in real time. As a potential tool to characterize hairy roots, LSP is non‐invasive, time‐saving, can be used online and stands out for its simple, low‐cost setup.     

Optically derived metabolic and hemodynamic parameters predict hippocampal neurogenesis in the BTBR mouse model of autism

In this study, we made use of dual‐wavelength laser speckle imaging (DW‐LSI) to assess cerebral blood flow (CBF) in the BTBR‐genetic mouse model of autism spectrum disorder, as well as control (C57Bl/6J) mice. Since the deficits in social behavior demonstrated by BTBR mice are attributed to changes in neural tissue structure and function, we postulated that these changes can be detected optically using DW‐LSI. BTBR mice demonstrated reductions in both CBF and cerebral oxygen metabolism (CMRO₂), as suggested by studies using conventional neuroimaging technologies to reflect impaired neuronal activation and cognitive function. To validate the monitoring of CBF by DW‐LSI, measurements with laser Doppler flowmetry (LDF) were also performed which confirmed the lowered CBF in the autistic‐like group. Furthermore, we found in vivo cortical CBF measurements to predict the rate of hippocampal neurogenesis, measured ex vivo by the number of neurons expressing doublecortin or the cellular proliferation marker Ki‐67 in the dentate gyrus, with a strong positive correlation between CBF and neurogenesis markers (Pearson, r = 0.78; 0.9, respectively). These novel findings identifying cortical CBF as a predictive parameter of hippocampal neurogenesis highlight the power and flexibility of the DW‐LSI and LDF setups for studying neurogenesis trends under normal and pathological conditions.      

Decreased cerebral blood flow and hemodynamic parameters during acute hyperglycemia in mice model observed by dual‐wavelength speckle imaging

In this study, we use dual‐wavelength optical imaging‐based laser speckle technique to assess cerebral blood flow and metabolic parameters in a mouse model of acute hyperglycemia (high blood glucose). The effect of acute glucose levels on physiological processes has been extensively described in multiple organ systems such as retina, kidney, and others. We postulated that hyperglycemia also alters brain function, which in turn can be monitored optically using dual‐wavelength laser speckle imaging (DW‐LSI) platform. DW‐LSI is a wide‐field, noncontact optical imaging modality that integrates the principles of laser flowmetry and oximetry to obtain macroscopic information such as hemoglobin concentration and blood flow. A total of eight mice (C57/BL6) were used, randomized into two groups of normoglycemia (control, n = 3) and hyperglycemia (n = 5). Hyperglycemia was induced by intraperitoneal injection of a commonly used anesthetic drug combining ketamine and xylazine (KX combo). We found that this KX combo increases blood glucose (BG) levels from 150 to 350 mg/dL, approximately, when measured 18 minutes post‐administration. BG continues to increase throughout the test period, with BG reaching an average of 463 ± 20.34 mg/dL within 60 minutes. BG levels were measured every 10 minutes from tail blood using commercially available glucometer. Experimental results demonstrated reductions in cerebral blood flow (CBF) by 55%, tissue oxygen saturation (SO₂) by 15%, and cerebral metabolic rate of oxygen (CMRO₂) by 75% following acute hyperglycemia. The observed decrease in these parameters was consistent with results reported in the literature, measured by a variety of experimental techniques. Measurements with laser Doppler flowmetry (LDF) were also performed which confirmed a reduction in CBF following acute hyperglycemia. In summary, our findings indicate that acute hyperglycemia modified brain hemodynamic response and induced significant changes in blood flow and metabolism. As far as we are aware, the implementation of the DW‐LSI to monitor brain hemodynamic and metabolic response to acute hyperglycemia in intact mouse brain has not been previously reported.      

Contact‐free endoscopic photoacoustic sensing using speckle analysis

Photoacoustic endoscopy (PAE) is an emerging imaging modality, which offers a high imaging penetration and a high optical contrast in soft tissue. Most of the developed endoscopic photoacoustic sensing systems use miniaturized contact ultrasound transducers or complex optical approaches. In this work, a new fiber‐based detection technique using speckle analysis for contact‐free signal detection is presented. Phantom and ex vivo experiments are performed in transmission and reflection mode for proof of concept. In summary, the potential of the technique for endoscopic photoacoustic signal detection is demonstrated. The new technique might help in future to broaden the applications of PAE in imaging or guiding minimally invasive laser procedures.      

Multiparameter wide‐field integrated optical imaging system‐based spatially modulated illumination and laser speckles in model of tissue injuries

In this report, an integrated optical platform based on spatial illumination together with laser speckle contrast technique was utilized to measure multiple parameters in live tissue including absorption, scattering, saturation, composition, metabolism, and blood flow. Measurements in three models of tissue injury including drug toxicity, artery occlusion, and acute hyperglycemia were used to test the efficacy of this system. With this hybrid apparatus, a series of structured light patterns at low and high spatial frequencies are projected onto the tissue surface and diffuse reflected light is captured by a CCD camera. A six position filter wheel, equipped with four bandpass filters centered at wavelengths of 650, 690, 800 and 880 nm is placed in front of the camera. Then, light patterns are blocked and a laser source at 650 nm illuminates the tissue while the diffusely reflected light is captured by the camera through the two remaining open holes in the wheel. In this manner, near‐infrared (NIR) and laser speckle images are captured and stored together in the computer for off‐line processing to reconstruct the tissue's properties. Spatial patterns are used to differentiate the effects of tissue scattering from those of absorption, allowing accurate quantification of tissue hemodynamics and morphology, while a coherent light source is used to study blood flow changes, a feature which cannot be measured with the NIR structured light. This combined configuration utilizes the strengths of each system in a complementary way, thus collecting a larger range of sample properties. In addition, once the flow and hemodynamics are measured, tissue oxygen metabolism can be calculated, a property which cannot be measured independently. Therefore, this merged platform can be considered a multiparameter wide‐field imaging and spectroscopy modality. Overall, experiments demonstrate the capability of this spatially coregistered imaging setup to provide complementary, useful information of various tissue metrics in a simple and noncontact manner, making it attractive for use in a variety of biomedical applications.     

Improved detectability of microcirculatory dynamics by laser speckle flowmetry

Mechanisms of renal autoregulation generate oscillations in arterial blood flow at several characteristic frequencies. Full‐field laser speckle flowmetry provides a real‐time imaging of superficial blood microcirculation. The possibility to detect changes in oscillatory dynamics is an important issue in biomedical applications. In this paper we show how laser power density affects quality of the recorded signal and improves detectability of temporal changes in microvascular perfusion.

     

Optical incoherent technique for noninvasive assessment of blood flow in tissues: Theoretical model and experimental study

Laser noninvasive methods for assessment of a tissue blood flow (BF), for example, the Laser Doppler Flowmetry (LDF), are well-known today. However, in such methods, low-frequency fluctuations (LFFs) in the registered optical signal caused by blood volume changes inside a tissue have not been studied in details until now. The aim of this study is to investigate the LFFs formation and to justify the LFFs-based diagnostic technique for cutaneous BF assessment. LFFs are theoretically described and experimentally shown in the input LDF signal inside the frequency range 0 to 10 Hz. They are substantiated as the basis of the new diagnostic method, in which BF is defined as the magnitude of blood volume changes in a tissue per unit time. The hand-made prototype of the promising diagnostic tool with light emitted diodes is used to validate the technique in experiments in vivo on 16 healthy volunteers in comparison with the LDF method. Experimental results show a good similarity of the recorded BF for both coherent and incoherent method. The proposed technique makes it possible the creation of inexpensive diagnostic equipment for assessment of cutaneous BF without using lasers and coherent light, completely and functionally comparable to LDF devices.     

Optical Thromboelastography to evaluate whole blood coagulation

Measurement of blood viscoelasticity during clotting provides a direct metric of haemostatic conditions. Therefore, technologies that quantify blood viscoelasticity at the point‐of‐care are invaluable for diagnosing coagulopathies. We present a new approach, Optical Thromboelastography (OTEG) that measures the viscoelastic properties of coagulating blood by evaluating temporal laser speckle fluctuations, reflected from a few blood drops. During coagulation, platelet‐fibrin clot formation restricts the mean square displacements (MSD) of scatterers and decelerates speckle fluctuations. Cross‐correlation analysis of speckle frames provides the speckle intensity temporal autocorrelation, g₂(t), from which MSD is deduced and the viscoelastic modulus of blood is estimated. Our results demonstrate a close correspondence between blood viscoelasticity evaluated by OTEG and mechanical rheometry. Spatio‐temporal speckle analyses yield 2‐dimensional maps of clot viscoelasticity, enabling the identification of micro‐clot formation at distinct rates in normal and coagulopathic specimens. These findings confirm the unique capability of OTEG for the rapid evaluation of patients' coagulation status and highlight the potential for point‐of‐care use. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Wave optical simulation of retinal images in laser safety evaluations

Lasers with wavelengths in the visible and near infrared region, pose a potential hazard to vision as the radiation can be focused on the retina. The laser safety standard IEC 60825–1:2014 provides limits and evaluation methods to perform a classification for such systems. An important parameter is the retinal spot size which is described by the angular subtense of the apparent source. In laser safety evaluations, the radiation is often described as a Gaussian beam and the image on the retina is calculated using the wave optical propagation through a thin lens. For coherent radiation, this method can be insufficient as the diffraction effects of the pupil aperture influence the retinal image. In this publication, we analyze these effects and propose a general analytical calculation method for the angular subtense. The proposed formula is validated for collimated and divergent Gaussian beams.     

Jones matrix‐based speckle‐decorrelation angiography using polarization‐sensitive optical coherence tomography

We show that polarization‐sensitive optical coherence tomography angiography (PS‐OCTA) based on full Jones matrix assessment of speckle decorrelation offers improved contrast and depth of vessel imaging over conventional OCTA. We determine how best to combine the individual Jones matrix elements and compare the resulting image quality to that of a conventional OCT scanner by co‐locating and imaging the same skin locations with closely matched scanning setups. Vessel projection images from finger and forearm skin demonstrate the benefits of Jones matrix‐based PS‐OCTA. Our study provides a promising starting point and a useful reference for future pre‐clinical and clinical applications of Jones matrix‐based PS‐OCTA.