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.      

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.      

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).      

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.     

Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart

Efficient separation of blood and cardiac wall in the beating embryonic heart is essential and critical for experiment‐based computational modelling and analysis of early‐stage cardiac biomechanics. Although speckle variance optical coherence tomography (SV‐OCT) relying on calculation of intensity variance over consecutively acquired frames is a powerful approach for segmentation of fluid flow from static tissue, application of this method in the beating embryonic heart remains challenging because moving structures generate SV signal indistinguishable from the blood. Here, we demonstrate a modified four‐dimensional SV‐OCT approach that effectively separates the blood flow from the dynamic heart wall in the beating mouse embryonic heart. The method takes advantage of the periodic motion of the cardiac wall and is based on calculation of the SV signal over the frames corresponding to the same phase of the heartbeat cycle. Through comparison with Doppler OCT imaging, we validate this speckle‐based approach and show advantages in its insensitiveness to the flow direction and velocity as well as reduced influence from the heart wall movement. This approach has a potential in variety of applications relying on visualization and segmentation of blood flow in periodically moving structures, such as mechanical simulation studies and finite element modelling. Picture : Four‐dimensional speckle variance OCT imaging shows the blood flow inside the beating heart of an E8.5 mouse embryo.     

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.     

Speckle contrast diffuse correlation tomography of cerebral blood flow in perinatal disease model of neonatal piglets

We adapted and tested an innovative noncontact speckle contrast diffuse correlation tomography (scDCT) system for 3D imaging of cerebral blood flow (CBF) variations in perinatal disease models utilizing neonatal piglets, which closely resemble human neonates. CBF variations were concurrently measured by the scDCT and an established diffuse correlation spectroscopy (DCS) during global ischemia, intraventricular hemorrhage, and asphyxia; significant correlations were observed. Moreover, CBF variations associated reasonably with vital pathophysiological changes. In contrast to DCS measurements of mixed signals from local scalp, skull and brain, scDCT generates 3D images of CBF distributions at prescribed depths within the head, thus enabling specific determination of regional cerebral ischemia. With further optimization and validation in animals and human neonates, scDCT has the potential to be a noninvasive imaging tool for both basic neuroscience research in laboratories and clinical applications in neonatal intensive care units.     

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.     

Confocal laser scanning microscopy of fluorescently stained wood cells: a new method for three-dimensional imaging of xylem elements

Summary Xylem cells were fluorescently stained with periodic acid — Schiff reaction or with Schiffs reagent alone and studied by confocal laser scanning microscopy. Single images with extremely low depth of focus, series of optical sections, computed stereo scopic images and shadow casting images as well as x-z images are obtained. In contrast to scanning electron microscopy, not only are the surfaces imaged, but elements concealed by other structures can be visualized by this system. Quantitative data on cell depth are provided and differences in lignification are detected.     

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.     

Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non‐invasive determination of these characteristics. We present an image‐processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source‐code for MATLAB and ImageJ is freely available under a permissive open‐source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc.     

Spectral correction for handheld optoacoustic imaging by means of near‐infrared optical tomography in reflection mode

In vivo imaging of tissue/vasculature oxygen saturation levels is of prime interest in many clinical applications. To this end, the feasibility of combining two distinct and complementary imaging modalities is investigated: optoacoustics (OA) and near‐infrared optical tomography (NIROT), both operating noninvasively in reflection mode. Experiments were conducted on two optically heterogeneous phantoms mimicking tissue before and after the occurrence of a perturbation. OA imaging was used to resolve submillimetric vessel‐like optical absorbers at depths up to 25 mm, but with a spectral distortion in the OA signals. NIROT measurements were utilized to image perturbations in the background and to estimate the light fluence inside the phantoms at the wavelength pair (760 nm, 830 nm). This enabled the spectral correction of the vessel‐like absorbers' OA signals: the error in the ratio of the absorption coefficient at 830 nm to that at 760 nm was reduced from 60%‐150% to 10%‐20%. The results suggest that oxygen saturation (SO ₂) levels in arteries can be determined with <10% error and furthermore, that relative changes in vessels' SO ₂ can be monitored with even better accuracy. The outcome relies on a proper identification of the OA signals emanating from the studied vessels.      

Probe‐based confocal laser endomicroscopy (pCLE) – a new imaging technique for in situ localization of spermatozoa

In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe‐based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non‐invasive, real‐time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real‐time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively.

Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe‐based laser endomicroscopy (pCLE, right) using Pro Flex^TM UltraMini O after staining with acriflavine.     

Visualizing vinca alkaloids in the petal of Catharanthus roseus using functionalized titanium oxide nanowire substrate for surface-assisted laser desorption/ionization imaging mass spectrometry

Imaging mass spectrometry (IMS) is a powerful technique that enables analysis of various molecular species at a high spatial resolution with low detection limits. In contrast to the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approach, surface-assisted laser desorption/ionization (SALDI) can be more effective in the detection of small molecules due to the absence of interfering background signals in low m/z ranges. We developed a functionalized TiO₂ nanowire as a solid substrate for IMS of low-molecular-weight species in plant tissues. We prepared TiO₂ nanowires using an inexpensive modified hydrothermal process and subsequently functionalized them chemically with various silane analogs to overcome the problem of superhydrophilicity of the substrate. Chemical modification changed the selectivity of imprinting of samples deposited on the substrate surface and thus improved the detection limits. The substrate was applied to image distribution of the metabolites in very fragile specimens such as the petal of Catharanthus roseus. We observed that the metabolites are distributed heterogeneously in the petal, which is consistent with previous results reported for the C. roseus plant leaf and stem. The intermediates corresponding to the biosynthesis pathway of some vinca alkaloids were clearly shown in the petal. We also performed profiling of petals from five different cultivars of C. roseus plant. We verified the semi-quantitative capabilities of the imprinting/imaging approach by comparing results using the LC-MS analysis of the plant extracts. This suggested that the functionalized TiO₂ nanowire substrate-based SALDI is a powerful technique complementary to MALDI-MS.     

Evaluating the accuracy of geometrical distortion correction of magnetic resonance images for use in intracranial brain tumor radiotherapy

AimDetermine the 1) effectiveness of correction for gradient-non-linearity and susceptibility effects on both QUASAR GRID^3D and CIRS phantoms; and 2) the magnitude and location of regions of residual distortion before and after correction.BackgroundUsing magnetic resonance imaging (MRI) as a primary dataset for radiotherapy planning requires correction for geometrical distortion and non-uniform intensity.Materials and MethodsPhantom Study: MRI, computed tomography (CT) and cone beam CT images of QUASAR GRID^3D and CIRS head phantoms were acquired. Patient Study: Ten patients were MRI-scanned for stereotactic radiosurgery treatment. Correction algorithm: Two magnitude and one phase difference image were acquired to create a field map. A MATLAB program was used to calculate geometrical distortion in the frequency encoding direction, and 3D interpolation was applied to resize it to match 3D T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) images. MPRAGE images were warped according to the interpolated field map in the frequency encoding direction. The corrected and uncorrected MRI images were fused, deformable registered, and a difference distortion map generated.ResultsMaximum deviation improvements: GRID^3D, 0.27 mm y-direction, 0.07 mm z-direction, 0.23 mm x-direction. CIRS, 0.34 mm, 0.1 mm and 0.09 mm at 20-, 40- and 60-mm diameters from the isocenter. Patient data show corrections from 0.2 to 1.2 mm, based on location. The most-distorted areas are around air cavities, e.g. sinuses.ConclusionsThe phantom data show the validity of our fast distortion correction algorithm. Patient-specific data are acquired in <2 min and analyzed and available for planning in less than a minute.