Quantification of cellular action fibers alignment from confocal microscopic images.

The cellular rheology has recently undergone a rapid development with particular attention to the cytoskeleton mechanical properties and its main components - actin filaments, intermediate filaments, microtubules and crosslinked proteins. However it is not clear what are the cellular structural changes that directly affect the cell mechanical properties. Thus, in this work, we aimed to quantify the structural rearrangement of these fibers that may emerge in changes in the cell mechanics. We created an image analysis platform to study smooth muscle cells from different arteries: aorta, mammary, renal, carotid and coronary and processed respectively 31, 29, 31, 30 and 35 cell image obtained by confocal microscopy. The platform was developed in Matlab (MathWorks) and it uses the Sobel operator to determine the actin fiber image orientation of the cell, labeled with phalloidin. The Sobel operator is used as a filter capable of calculating the pixel brightness gradient, point to point, in the image. The operator uses vertical and horizontal convolution kernels to calculate the magnitude and the angle of the pixel intensity gradient. The image analysis followed the sequence: (1) opens a given cells image set to be processed; (2) sets a fix threshold to eliminate noise, based on Otsu's method; (3) detect the fiber edges in the image using the Sobel operator; and (4) quantify the actin fiber orientation. Our first result is the probability distribution II(Δθ) to find a given fiber angle deviation (Δθ) from the main cell fiber orientation θ0. The II(Δθ) follows an exponential decay II(Δθ) = Aexp(-αΔθ) regarding to its θ0. We defined and determined a misalignment index α of the fibers of each artery kind: coronary αCo = (1.72 ‘+ or =’ 0.36)rad POT -1; renal αRe = (1.43 + or - 0.64)rad POT -1; aorta αAo = (1.42 + or - 0.43)rad POT -1; mammary αMa = (1.12 + or - 0.50)rad POT -1; and carotid αCa = (1.01 + or - 0.39)rad POT -1. The α of coronary and carotid are statistically different (p < 0.05) among all analyzed cells. We discussed our results correlating the misalignment index data with the experimental cell mechanical properties obtained by using Optical Magnetic Twisting Cytometry with the same group of cells

Anomalous diffusion evidenced by particle tracking at high frame rates.

Diffusion is a common phenomenon in nature and generally is associated with a system trying to reach a local or a global equilibrium state, as a result of highly irregular individual particle motion. Therefore it is of fundamental importance in physics, chemistry and biology. Particle tracking in complex fluids can reveal important characteristics of its properties. In living cells, we coat the microbead with a peptide (RGD) that binds to integrin receptors at the plasma membrane, which connects to the CSK. This procedure is based on the hypothesis that the microsphere can move only if the structure where it is attached move as well. Then, the observed trajectory of microbeads is a probe of the cytoskeleton (CSK), which is governed by several factors, including thermal diffusion, pressure gradients, and molecular motors. The possibility of separating the trajectories into passive and active diffusion may give information about the viscoelasticity of the cell structure and molecular motors activity. And also we could analyze the motion via generalized Stokes-Einstein relation, avoiding the use of any active techniques. Usually a 12 to 16 Frames Per Second (FPS) system is used to track the microbeads in cell for about 5 minutes. Several factors make this FPS limitation: camera computer communication, light, computer speed for online analysis among others. Here we used a high quality camera and our own software, developed in C++ and Linux, to reach high FPS. Measurements were conducted with samples for 10£ and 20£ objectives. We performed sequentially images with different intervals, all with 2 ¹s exposure. The sequences of intervals are in milliseconds: 4 5 ms (maximum speed) 14, 25, 50 and 100 FPS. Our preliminary results highlight the difference between passive and active diffusion, since the passive diffusion is represented by a Gaussian in the distribution of displacements of the center of mass of individual beads between consecutive frames. However, the active process, or anomalous diffusion, shows as long tails in the distribution of displacements.CNPqFAPES

Physical Properties that Trigger Mechanical Changes in Live Cells

Todos os seres vivos compartilham uma característica comum na sua composição estrutural, a célula. No corpo humano, as células vasculares de músculo liso são fundamentais para o bom funcionamento dos vasos arteriais. A principal função dessas células é contrair e regular o calibre desses vasos, a pressão sanguínea e a distribuição do fluxo de sangue. Devido a isto, alterações mecânicas sofridas por estas células acarretam modificações estruturais nos vasos, podendo levar à hipertensão, vasoespasmo e arteriosclerose. O principal objetivo do nosso trabalho foi o de desenvolver uma nova plataforma de análise de imagens de células vasculares para caracterizar suas propriedades estruturais. Em nossa plataforma, analisamos parâmetros estruturais de células vasculares de músculo liso de diferentes leitos arteriais, com as fibras de actina evidenciadas com marcadores fluorescentes, obtidas por microscopia confocal. Estes parâmetros são: o Índice de Alinhamento das fibras de actina da imagem, a distribuição de comprimento dessas fibras e sua dimensão fractal. Mostramos que com esses parâmetros somos capazes de comparar células de leitos arteriais diferentes de forma quantitativa, assim como, correlacionar esses parâmetros com suas propriedades mecânicas.All living organisms share a unique characteristic in their structural composition, the cell. In the human body, vascular smooth muscle cells are fundamental for the ideal functioning of the arterial vessels. The main function of these cells is to contract and regulate these vessels caliber, as well as the blood pressure and flow distribution of blood. Due to the exposed above, mechanical alterations suffered by these cells cause structural modifications in vessels, which may lead to hypertension, vasospasm and atherosclerosis. The main objective of our research was to develop a new framework of image analysis for vessel cells in order to characterize their structural properties. In our framework we analyzed structural parameters of vascular smooth muscle cells from different arterial sites, with actin fibers labeled with fluorescent markers, obtained by confocal microscopy. These parameters are: the actin fibers alignment index of the image, the length distribution of these fibers and their fractal dimension. We presented that with these parameters we are able to quantitatively compare cells in different arterial sites as well as correlate these parameters with their mechanical properties

Development of traction force dynamics analysis tools during the contraction cycle of isolated cardiomyocytes in vitro

As propriedades mecânicas do coração têm sido estudadas extensivamente nos últimos anos, tanto em nível de órgão, tecido muscular, e recentemente em nível celular. As células cardíacas, ou cardiomiócitos, são células do músculo cardíaco, que é responsável pelo permanente fluxo sanguíneo no corpo, compondo os átrios e ventrículos, câmaras onde o sangue entra e é bombeado para o coração. A utilização de cardiomiócitos em experimentos científicos trouxe várias vantagens experimentais, assim como, a possibilidade de se estudar células isoladas de diversas áreas do coração. No presente trabalho foi desenvolvida uma metodologia de análise capaz de mensurar parâmetros relacionados a dinâmica de forças de tração de cardiomiócitos isolados durante os ciclos de contração, visando gerar um melhor entendimento na área de regeneração cardíaca. Para isso foram utilizadas duas linhagens de cardiomiócitos, WT, que são de cardiomiócitos saudáveis, e KO-CRP3, que são de cardiomiócitos que apresentam defeitos na arquitetura e funcionamento da musculatura cardíaca. A metodologia desenvolvida visa analisar parâmetros tais como, a frequência de pulsação dos cardiomiócitos, o alinhamento das forças de tração durante os ciclos de contração e a potência realizada pelo cardiomiócito durante a contração e o relaxamento. Esses parâmetros foram analisados em duas condições, com os cardiomiócitos em estados basais, e estimulados por um fármaco, o isoproterenol. Dessa forma foi possível analisar a resposta mecânica dos cardiomiócitos ao fármaco utilizado, e a diferença do comportamento dos parâmetros entre as linhagens de cardiomiócitos.The mechanical properties of the heart have been studied studied extensively in recent years, both at organ level, muscle tissue, and recently at the cellular level. Heart cells, or cardiomyocytes, are cell of the heart muscle, which is responsible for the permanent blood flow in the body, making up the atria and ventricles, chambers where blood enters and is pumped into the heart. The use of cardiomyocytes in scientific experiments has brought several experimental advantages, as well as the possibility of studying isolated cells from different areas of the heart. In the present work an analysis methodology was developed capable of measuring parameters related to the dynamics of traction forces os isolated cardiomyocytes during the contraction cycles, aiming to generate a better understanding in the area of cardiac regeneration. For this we used two cardiomyocytes line, WT, which are from healthy cardiomyocytes, an KO-CRP3, which are from cardiomyocytes that have malfunction in cardiac muscle architecture. The developed methodology aims to analyze parameters such as the pulse rate of the cardiomyocytes, the alignment of the traction forces during the contraction cycles and the power performed by the cardiomyocytes during the contraction and relaxation. These parameters were analyzed under two conditions, with cardiomyocytes at baseline, and stimulated by a drug, isoproterenol. Thus it was possible to analyze the mechanical response of cardiomyocytes to the drug used, and the difference in the behavior of teh parameters between the cardiomyocytes line