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Microvascular blood flow is crucial for tissue and organ function and is often severely affected by diseases. Therefore, investigating the microvasculature under different pathological circumstances is essential to understand the role of the microcirculation in health and sickness. Microvascular blood flow is generally investigated with Intravital Video Microscopy (IVM), and the captured images are stored on a computer for later off-line analysis. The analysis of these images is a manual and challenging process, evaluating experiments very time consuming and susceptible to human error. Since more advanced digital cameras are used in IVM, the experimental data volume will also increase significantly. This study presents a new two-step image processing algorithm that uses a trained Convolutional Neural Network (CNN) to functionally analyze IVM microscopic images without the need for manual analysis. While the first step uses a modified vessel segmentation algorithm to extract the location of vessel-like structures, the second step uses a 3D-CNN to assess whether the vessel-like structures have blood flowing in it or not. We demonstrate that our two-step algorithm can efficiently analyze IVM image data with high accuracy (83%). To our knowledge, this is the first application of machine learning for the functional analysis of microvascular blood flow in vivo.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Over the last few years, many researchers have directed their efforts and interests toward in vivo studies of the cellular and molecular mechanisms in the microcirculation of many tissues under different inflammatory conditions. These studies’ main goal is to develop more effective therapeutic strategies for the treatment of inflammatory and autoimmune diseases. Leukocyte recruitment analysis is a crucial step to understand the interactions between leukocytes and endothelial cells in the microcirculation of living animals. Performed preferably by the intravital video microscopy (IVM) technique, this procedure usually requires an expert to perform visual analysis, which is prone to the inter- and intra-observer variability, besides being a tedious and time-consuming task. This problem claims, therefore, an automated method to detect and track these cells. To this end, this work aims to study and develop computational techniques for the detection and tracking of leukocytes in IVM images. We proposed an automatic computational pipeline where, after a preprocessing stage, we combined the results of frame-basis detection (2D – spatial processing) with those from three-dimensional analysis (3D=2D+t – spatiotemporal processing) of volumetric images formed by stacking all the video frames. While the 2D processing focuses on leukocytes detection without worrying about their tracking, 2D+t processing was intended to assist in the dynamic analysis of cell movement (tracking). We tested three different detection approaches for the spatial processing, named as MTM-PCA, MTM-DCNN, and DCNN. Our results were obtained by qualitative and quantitative evaluations performed over six different IVM videos, where the detected cells were compared with the manual annotations of an expert. They showed the combination of these both processing stages minimized most of the problems involved in IVM cell detection and tracking, such as cell occlusion and the proper discrimination of cell trajectories. Nos últimos anos, um grande número de pesquisadores tem direcionado seus esforços e interesses para estudos in vivo dos mecanismos celulares e moleculares na microcirculação de vários tecidos e em várias condições inflamatórias. O principal objetivo desses estudos é desenvolver estratégias terapêuticas mais eficazes para o tratamento de doenças inflamatórias e autoimunes. A análise do recrutamento leucocitário é um passo importante para entender as interações entre os leucócitos e as células endoteliais na microcirculação de animais vivos. Realizado preferencialmente através da técnica de microscopia intravital (MI), esse procedimento geralmente requer a análise visual de um especialista, que é propensa à intra- e inter-variabilidade do observador, além de ser uma atividade tediosa e demorada. Tal problema reivindica, portanto, um método automatizado para a detecção e rastreamento dessas células. Para tanto, este trabalho visa o estudo e o desenvolvimento de técnicas computacionais para a detecção e rastreamento de leucócitos em imagens de MI. Para isso, propusemos um arcabouço de desenvolvimento computacional automático que, após uma etapa de pré-processamento, combina os resultados da detecção quadro-a-quadro do vídeo (processamento espacial – 2D) com os resultados de uma análise tridimensional (processamento espaço-temporal – 3D=2D+t) feita em imagens volumétricas formadas pelo empilhamento de todos os quadros do vídeo. Neste caso, enquanto o processamento 2D visa a detecção dos leucócitos sem se preocupar com a tarefa de rastreamento, o processamento 2D+t tem o objetivo de auxiliar na análise da dinâmica celular (rastreamento). Nós testamos três abordagens diferentes para o processamento espacial, denominadas MTM-PCA, MTM-DCNN e DCNN. Nossos resultados foram obtidos por meio de avaliações qualitativas e quantitativas realizadas em seis diferentes vídeos de MI, em que as células detectadas foram comparadas com as marcações manuais de um especialista. Esses resultados mostraram que a combinação das duas etapas de processamento foi capaz de minimizar a maioria dos problemas envolvidos na detecção e rastreamento celular em imagens de MI, como a oclusão e a discriminação adequada das trajetórias das células. CAPES: Código de Financiamento 001 CAPES: 88881.187616/2018-01

Testicular torsion is a surgical emergency in urologic practice. Many models have demonstrated the relationship between the degree and duration of the torsion and the subsequent damages of the testis, but there are as yet no suitable methods for visualization of the testicular microcirculation in this condition. We aimed to use orthogonal polarization spectral (OPS) imaging to observe the microcirculatory changes in the testis after ischemia-reperfusion (I/R) challenge, and to compare this technique with that of fluorescence intravital video microscopy (IVM). Twelve rats subjected to 60-minute ischemia following 720° testicular torsion were divided into two groups for IVM or OPS imaging examinations of the microcirculatory network of the testis at 60, 120, 180, and 240 minutes after reestablishment of perfusion. Significant microcirculatory failure was demonstrated after I/R in both groups. The absolute values of the microcirculatory parameters recorded with the OPS and IVM imaging methods did not differ statistically. The microcirculatory disturbances are present during the later phases of testicular torsion. OPS imaging technique represents an accurate noninvasive method to detect physiologic and pathophysiologic changes in the microcirculation of the testis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Over the last few years, a large number of researchers have directed their efforts and interests for the in vivo study of the cellular and molecular mechanisms of leukocyte-endothelial interactions in the microcirculation of many tissues under different inflammatory conditions. The main goal of these studies is to develop more effective therapeutic strategies for the treatment of inflammatory and autoimmune diseases. Nowadays, analysis of the leukocyte-endothelial interactions in small animals is performed by visual assessment from an intravital microscopy image sequences. Besides being time consuming, this procedure may cause visual fatigue of the observer and, therefore, generate false statistics. In this context, this work aims to study and develop computational techniques for the automatic detection and tracking of leukocytes in intravital video microscopy. For that, results from frame to frame processing (2D – spatial analysis) will be combined with those from the three-dimensional analysis (3D=2D+t – spatio-temporal analysis) of the volume formed by stacking the video frames. The main technique addressed for both processings is based on the analysis of the eigenvalues of the local Hessian matrix. While the 2D image processing aims the leukocyte detection without worrying about their tracking, 2D+t processing is intended to assist on the dynamic analysis of cell movement (tracking), being able to predict cell movements in cases of occlusion, for example. In this work we used intravital video microscopy obtained from a study of Multiple Sclerosis in mice. Noise reduction and registration techniques comprise the preprocessing step. In addition, techniques for the analysis and definition of cellular pathways comprise the post processing step. Results of 2D and 2D+t processing steps, compared with conventional visual analysis, have shown the effectiveness of the proposed approach. Nos últimos anos, um grande número de pesquisadores tem voltado seus esforços e interesses para o estudo in vivo dos mecanismos celulares e moleculares da interação leucócitoendotélio na microcirculação de vários tecidos e em várias condições inflamatórias. O principal objetivo desses estudos é desenvolver estratégias terapêuticas mais eficazes para o tratamento de doenças inflamatórias e autoimunes. Atualmente, a análise de interações leucócito-endotélio em pequenos animais é realizada de maneira visual a partir de uma sequência de imagens de microscopia intravital. Além de ser demorado, esse procedimento pode levar à fadiga visual do observador e, portanto, gerar falsas estatísticas. Nesse contexto, este trabalho de pesquisa tem como objetivo estudar e desenvolver técnicas computacionais para a detecção e rastreamento automáticos de leucócitos em vídeos de microscopia intravital. Para isso, resultados do processamento quadro a quadro do vídeo (2D – análise espacial) serão combinados com os resultados da análise tridimensional (3D=2D+t – análise espaço-temporal) do volume formado pelo empilhamento dos quadros do vídeo. A principal técnica abordada para ambos os processamentos é baseada na análise local dos autovalores da matriz Hessiana. Enquanto o processamento de imagens 2D tem como objetivo a detecção de leucócitos sem se preocupar com seu rastreamento, o processamento 2D+t pretende auxiliar na análise dinâmica de movimentação das células (rastreamento), sendo capaz de prever movimentos celulares em casos de oclusão, por exemplo. Neste trabalho foram utilizados vídeos de microscopia intravital obtidos a partir de um estudo da Esclerose Múltipla realizado com camundongos. Técnicas de redução de ruído e estabilização do movimento das sequências de imagens compõem a etapa de pré-processamento, assim como técnicas para a definição e análise dos caminhos celulares compõem a etapa de pós-processamento. Resultados das etapas de processamento 2D e 2D+t, comparados com a convencional análise visual, demonstraram a eficácia da abordagem proposta. FAPESP: 2013/26171-6

Acute limb compartment syndrome (ACS) causes a unique form of limb ischaemia, which induces intense inflammatory response resulting in microcirculatory dysfunction, neutrophil activation and cell injury. Increased intracompartmental pressure is the hallmark of ACS. Decompression by fasciotomy is the gold standard treatment. While fasciotomy saves the limb from ischaemic threat, paradoxically, it causes further damage to the muscle by reperfusion injury. In addition, it does not address the inflammatory element purported to increase the tissue injury in ACS. Recent evidence suggests that hydrogen sulfide (H2S) can mitigate the damage associated with ischaemia-reperfusion injury. The purpose of this thesis was to examine the value of H2S treatment in a rat model of ACS, using GYY4137 as H2S-releasing molecule. We have demonstrated significant cytoprotective role of H2S on the skeletal muscle following ACS. These results suggest a potential therapeutic value of H2S as an adjunctive to fasciotomy, for patients suffering ACS.

Detection of rolling and adhered leukocytes in intravital microscopy image sequences is an important task in studies of leukocyte-endothelial interactions in the microcirculation of living small animals under different inflammatory conditions. This procedure is usually performed by visual assessment of the image sequences. However, despite being tedious and time consuming, this procedure is prone to the inter- and intra-observer variability. In this work, we developed an automated computer system for the detection of leukocytes in intravital video microscopy. First, the video frames were processed by the bilateral filter to reduce noise while preserving sharp edges. Then, a demons-based image registration technique was applied to minimize animal motion. Finally, the detection of leukocytes was performed by local analysis of Hessian matrix Eigen values. Quantitative and qualitative evaluation of the proposed method were conducted by using 220 video frames obtained from an experimental study performed on the brain microvasculature of mice. The proposed method was compared with the template matching technique using precision, recall and F1-Score measures. For the Hessian-based method, the results of precision, recall and F1-Score were, respectively, equal to 0.81, 0.86, and 0.83. For direct comparison, the results obtained for the template matching technique were 0.86, 0.73 and 0.79.

Objective: The present study describes the development of a novel in vivo microvascular preparation for the investigation of capillary blood flow in the extensor carpi radialis longus (ECRL) muscle...

Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration.

Objective We compare RMN to PCA under several simulated physiological conditions to determine how the use of different vascular geometry affects oxygen transport solutions. Methods Three discrete networks were reconstructed from intravital video microscopy of rat skeletal muscle (84 × 168 × 342 μm, 70 × 157 × 268 μm, and 65 × 240 × 571 μm), and hemodynamic measurements were made in individual capillaries. PCAs were created based on statistical measurements from RMNs. Blood flow and O2 transport models were applied, and the resulting solutions for RMN and PCA models were compared under four conditions (rest, exercise, ischemia, and hypoxia). Results Predicted tissue PO2 was consistently lower in all RMN simulations compared to the paired PCA. PO2 for 3D reconstructions at rest were 28.2 ± 4.8, 28.1 ± 3.5, and 33.0 ± 4.5 mmHg for networks I, II, and III compared to the PCA mean values of 31.2 ± 4.5, 30.6 ± 3.4, and 33.8 ± 4.6 mmHg. Simulated exercise yielded mean tissue PO2 in the RMN of 10.1 ± 5.4, 12.6 ± 5.7, and 19.7 ± 5.7 mmHg compared to 15.3 ± 7.3, 18.8 ± 5.3, and 21.7 ± 6.0 in PCA. Conclusions These findings suggest that volume matched PCA yield different results compared to reconstructed microvascular geometries when applied to O2 transport modeling; the predominant characteristic of this difference being an over estimate of mean tissue PO2. Despite this limitation, PCA models remain important for theoretical studies as they produce PO2 distributions with similar shape and parameter dependence as RMN.

Motility is an important property of immune system cells. It provides cells with the ability to perform their function not only at the right time but also at the right place. In this paper, we introduce the problem of modeling and estimating an effective force field directing cell movement by the analysis of intravital video microscopy. A computational approach is proposed for solving this problem without dealing with a parametrized spatial model of the field in order to avoid potential errors due to inaccurate spatial model assumptions. We consider the dynamics of cells similar to the dynamics of distributed agents typically used in the field of swarm robotics. The method utilizes a fixed-interval Kalman filter based smoother. Its application results in a map giving the intensity and direction of the effective force field. The results show that real-time video images are a source of data, enabling us to visualize intriguing spatiotemporal phenomena inside immune system organs. The proposed approach can fill the existing gap between contemporary technology and quantitative data analyses present in the field of biosystems.

Experimental studies on angiogenesis are clarifying many aspects of this important process and are leading to new approaches to use this information clinically. Histology of fixed tissues is a commonly used “gold standard” for assessing development of tumor vasculature during disease progression or changes in vasculature in response to genetic manipulation or therapy. However, histology provides only a static snapshot‐in‐time of vascular status, and can provide only limited information about vessel function or dynamics. Here we describe microscopy techniques and image processing approaches for using intravital video microscopy (IVVM) for the study of normal and tumor vascular morphology and function. IVVM provides powerful, high‐resolution approaches for observing the vasculature in multiple organs or experimental animals. In addition to providing informative images, IVVM combined with video postprocessing and image analysis approaches can be used to extract valuable quantitative information from video images. This information includes morphological parameters such as vascular diameter, density, branching, and three‐dimensional vascular geometry, as well as functional and physiological information such as the identification of vessels that are perfused with red blood cells (RBCs) or plasma, rate of RBC flow, and oxygen status of RBCs. An added strength of IVVM is the ability to provide longitudinal information, looking at changes in vascular morphology and function over time in individual animals. In this chapter, we describe methods and analytical approaches for using IVVM to study vascular morphology and dynamics.

Simultaneous Fourier domain optical coherence tomography and dark-field intravital video microscopy were used for in-vivo imaging of alveolar dynamics in the ventilated mouse. Quantification of the images revealed an alveolar expansion with increased end-inspiratory-pressure.

A new feature-based tracking algorithm was developed for measuring diameter of vessels in intravital video microscopy image sequences. Testing was performed with ten synthetic image sequences with known diameters and ten intravital microscopy sequences. The automatic measurements were compared with the manual measurements using linear regression analysis. The regression analysis indicated that our method is in agreement with manual measurements by human observers. The reproducibility of the method was evaluated based on Bland-Altman analysis. Bland-Altman statistic for the automated method was narrower than for the manual measurements, indicating that the automated method has a better reproducibility than human raters

Vascular-disrupting agents (VDA) kill tumor cells by selectively disrupting blood circulation in tumors. In vivo analysis of this intensely studied class of anticancer agents is invaluable for preclinical assessment of pharmacodynamic end points and effective therapeutic windows. In this review, we consider the role of intravital video microscopy in measuring tumor vascular response to VDAs, the potential of which lies in the opportunity to quantitate specific variables and to obtain real-time information on how VDAs affect tumor microcirculation. (Cancer Res 2006; 66(24): 11517-9)

The Optical Triplicator produces three copies of a portion of a microscopic image and places them side-by-side on the face of a video image tube, so that all three images can be viewed simultaneously in each video frame. The Optical Triplicator was used in an intravital microscopy assembly to obtain simultaneous images of a microvessel at three visible wavelengths selected to enable the accurate determination of oxygen saturation in microvessels of the hamster retractor muscle. An image processing system was used to obtain light intensity and optical density from video recordings made using the triplicator. Lumenal oxygen saturation profiles were determined using the measured intensity values and a published three wavelength photometric method.

It has been slightly over 300 years since Marcello Malpighi and Antoni van Leeuwenhoek used microscopic techniques to observe microvascular pathways and follow the path of red cells in microvessels.1 Although the microscope utilized by these pioneers was simple by today’s standards, consisting of only a single lens, the innovative approach and unique measurements have provided the basis for contemporary studies utilizing sophisticated techniques of intravital video microscopy. In the more recent past, measurements of microvascular pressure were completed about 60 years ago by Landis.2 Compared to contemporary methods and techniques, the pressure measuring device used by Landis was simple, but it provided the first direct measurements of ntravascular capillary hydrostatic pressures. These measurements are difficult even using today’s technology. It is my intent in this chapter to summarize microvascular methodological procedures used for acquisition of measurements of diameters, red cell flow velocities, and microvascular pressures in resistance arteries. pecialized be changed by gently snaring the abdominal aorta to increase pressure or by gently snaring the inferior vena cava to decrease pressure. Microvascular pressure must follow the change in aortic pressure, although the magnitude of the pressure change is expected to be less.

Growth of malignant tumors relies on development of new vessels from the existing vessels, a process called angiogenesis. Little is known about the pattern of chronic vascular growth in the human germ cell testicular cancer. To shed light on this topic, we injected human CRL-2073 testicular cancer cells or saline (sham treated mice) into the testes of SCID-mice and studied the growth of the tumor's vasculature using intravital video microscopy (IVM) on days 7, 14, 21, 28 and 37 after injection. On each day, 4 mice were studied. To better identify microvessels, fluorescein isothiocyanate-dextran (FITC) was injected i.v. prior to IVM. After IVM, one testis was cast with Mercox and studied in a scanning electron microscope (SEM), the other was used for histology. IVM images were transferred to the computer and analyzed using the Lucia morphometry software.

This study was undertaken to elucidate the role of thrombosis in blood vessels less than 0.1 mm in diameter in skeletal muscle after an ischemic episode. Capillaries were examined after normothermic ischemia using intravital video microscopy of the cremaster muscle of an anesthetized rat. Histologic sections of the cremaster muscle and silicone rubber intravascular casts were also analyzed. Our model demonstrates that, after 3 hours of warm ischemia, up to two-thirds of capillaries are clotted, as is most of the venous system. Our findings also indicate that capillaries may reopen during 30 minutes of reperfusion. These findings suggest that ischemia may cause thrombosis in the microvasculature of skeletal muscle but the thrombosis appears to be partially reversible.