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- Manual and automatic image analysis segmentation methods for blood flow studies in microchannelsPublication . Carvalho, Violeta Meneses; Gonçalves, Inês M.; Souza, Andrews Victor Almeida; Souza, Maria Sabrina; Bento, David; Ribeiro, J.E.; Lima, Rui A.; Pinho, DianaIn blood flow studies, image analysis plays an extremely important role to examine raw data obtained by high-speed video microscopy systems. This work shows different ways to process the images which contain various blood phenomena happening in microfluidic devices and in microcirculation. For this purpose, the current methods used for tracking red blood cells (RBCs) flowing through a glass capillary and techniques to measure the cell-free layer thickness in different kinds of microchannels will be presented. Most of the past blood flow experimental data have been collected and analysed by means of manual methods, that can be extremely reliable, but they are highly time-consuming, user-intensive, repetitive, and the results can be subjective to user-induced errors. For this reason, it is crucial to develop image analysis methods able to obtain the data automatically. Concerning automatic image analysis methods for individual RBCs tracking and to measure the well know microfluidic phenomena cell-free layer thickness, two developed methods are present and discuss in order to demonstrate their feasibility for accurate data acquisition in such studies Additionally, a comparison analysis between manual and automatic methods was performed.
- Determinação e caracterização das trajectórias dos glóbulos vermelhos: um método semi-automáticoPublication . Pinho, Diana; Pereira, Ana I.; Gayubo Rojo, Fernando; Lima, Rui A.Neste trabalho procedeu-se ao desenvolvimento de uma aplicação, RBC-Dt 1.0, com base em conceitos de processamento de imagem/vídeo, capaz de efectuar o seguimento semi-automático dos glóbulos vermelhos ao longo de uma sequência de imagens captadas no centro de um microcanal de vidro com 100μm de diâmetro. A aplicação foi elaborada em programação Matlab, e deverá ser capaz de extrair características, tais como centróides, ou seja a posição (x, y) num dado instante t. Os dados numéricos extraídos são guardados em ficheiros, e tratados por técnicas de optimização, pretendendo-se caracterizar o comportamento das trajectórias dos glóbulos vermelhos a escoarem no centro do microcanal em estudo. Foi ainda calculado o coeficiente de dispersão radial, Dyy, das mesmas trajectórias. Foram realizadas comparações entre os dados obtidos pela aplicação, RBC–Dt 1.0, e os dados obtidos em estudos anteriores usando um método manual, MtrackJ, plugin do Image J. Conclui-se que os dados obtidos por RBC-Dt 1.0 são similares aos obtidos pelo método manual. In this work we have developed an application,RBC-Dt 1.0, based on concepts of image/video processing, in order to track in a semi-automatic way the red blood cells over a sequence of images captured in the center of a glass microchannel with 100μm in diameter. The application was developed in Matlab programming, and it should be able to extract features such as centroids, i.e. the position (x, y) in a given interval of time t. The extracted numerical data is stored in files, and processed by techniques of optimization, which is aimed to characterize the trajectories behavior of red blood cells flowing at the center of the microchannel. The radial dispersion coefficient, Dyy, was also calculated for the same trajectories. We compared the data obtained by the current study, with the past results obtained in similar studies using a manual tracking method, i.e., MtrackJ, a plugin from Image J. The results from the present study suggest that both data are in good agreement.
- A passive microfluidic device based on crossflow filtration for cell separation measurements: a spectrophotometric characterizationPublication . Faustino, Vera; Catarino, Susana; Pinho, Diana; Lima, Rui A.; Minas, GraçaMicrofluidic devices have been widely used as a valuable research tool for diagnostic applications. Particularly, they have been related to the successful detection of different diseases and conditions by assessing the mechanical properties of red blood cells (RBCs). Detecting deformability changes in the cells and being able to separate those cells may be a key factor in assuring the success of detection of some blood diseases with diagnostic devices. To detect and separate the chemically modified RBCs (mimicking disease-infected RBCs) from healthy RBCs, the present work proposes a microfluidic device comprising a sequence of pillars with different gaps and nine different outlets used to evaluate the efficiency of the device by measuring the optical absorption of the collected samples. This latter measurement technique was tested to distinguish between healthy RBCs and RBCs chemically modified with glutaraldehyde. The present study indicates that it was possible to detect a slight differences between the samples using an optical absorption spectrophotometric setup. Hence, the proposed microfluidic device has the potential to perform in one single step a partial passive separation of RBCs based on their deformability.
- A microfluidic device for partial cell separation and deformability assessmentPublication . Pinho, Diana; Yaginuma, Tomoko; Lima, Rui A.Blood flow in microcirculation shows several interesting phenomena that can be used to develop microfluidic devices for blood separation and analysis in continuous flow. In this study we present a novel continuous microfluidic device for partial extraction of red blood cells (RBCs) and subsequent measurement of RBC deformability. For this purpose, we use polydimethylsiloxane (PDMS) microchannels having different constrictions (25%, 50% and 75%) to investigate their effect on the cell-free layer (CFL) thickness and separation efficiency. By using a combination of image analysis techniques we are able to automatically measure the CFL width before and after an artificial constriction. The results suggest that the CFL width increases with enhancement of the constriction and contributes to partial cell separation. The subsequent measurements of RBCs deformation index reveal that the degree of deformation depends on the constriction geometries and hematocrit after the cell separation module. The proposed microfluidic device can be easily transformed into a simple, inexpensive and convenient clinical tool able to perform both RBC separation and deformability analysis in one single device. This would eliminate the need for external sample handling and thus reducing associated labor costs and potential human errors.
- Flow of red blood cells suspensions through hyperbolic microcontractionsPublication . Faustino, Vera; Pinho, Diana; Yaginuma, Tomoko; Calhelha, Ricardo C.; Kim, Geyong; Arana, Sergio; Ferreira, Isabel C.F.R.; Oliveira, Mónica S.N.; Lima, Rui A.The present study uses a hyperbolic microchannel with a low aspect ratio (AR) to investigate how the red blood cells (RBCs) deform under conditions of both extensional and shear induced flows. The deformability is presented by the degree of the deformation index (DI) of the flowing RBCs throughout the microchannel at its centerline. A suitable image analysis technique is used for semi-automatic measurements of average DIs, velocity and strain rate of the RBCs travelling in the regions of interest. The results reveal a strong deformation of RBCs under both extensional and shear stress dominated flow conditions.
- Shrinkage and colour in the production of micro-sized PDMS particles for microfluidic applicationsPublication . Anes, Cláudia Filipa; Pinho, Diana; Muñoz-Sánchez, Beatriz N.; Vega, E.J.; Lima, Rui A.Polydimethylsiloxane (PDMS), due to its remarkable properties, is a suitable polymer for the production of microparticles with industrial and medical applications. The micro-sized PDMS liquid droplets suffer a pronounced shrinkage while curing to turn into solid particles. In this article, we report the calibration of the shrinkage phenomenon in the production of PDMS microparticles. Our results show that this shrinkage does not depend on the amount of curing agent in the PDMS precursor or on the addition of micro/nanoparticles to the mixture, but on the surface effects due to the relatively large droplet surface-to-volume ratio. Moreover, we have also investigated the addition of colour to the particles to improve their visualization/detection. The addition of colour by using pigments enhances the visualization of the contours of the PDMS microparticles, and reveals the capability of this technique to microencapsulate micro/nanoparticles in PDMS spheres with diameters below 10 μm. We demonstrate that the technique used in this work is able to work with a wide range of viscosities obtaining an acceptable degree of monodispersity.
- In vitro blood flow and cell-free layer in hyperbolic microchannels: visualizations and measurmentsPublication . Rodrigues, Raquel Oliveira; Lopes, Raquel; Pinho, Diana; Pereira, Ana I.; Garcia, Valdemar; Gassmann, Stefan; Sousa, Patrícia C.; Lima, Rui A.Red blood cells (RBCs) in microchannels has tendency to undergo axial migration due to the parabolic velocity profile, which results in a high shear stress around wall that forces the RBC to move towards the centre induced by the tank treading motion of the RBC membrane. As a result there is a formation of a cell free layer (CFL) with extremely low concentration of cells. Based on this phenomenon, several works have proposed microfluidic designs to separate the suspending physiological fluid from whole in vitro blood. This study aims to characterize the CFL in hyperbolic-shaped microchannels to separate RBCs from plasma. For this purpose, we have investigated the effect of hyperbolic contractions on the CFL by using not only different Hencky strains but also varying the series of contractions. The results show that the hyperbolic contractions with a Hencky strain of 3 and higher, substantially increase the CFL downstream of the contraction region in contrast with the microchannels with a Hencky strain of 2, where the effect is insignificant. Although, the highest CFL thickness occur at microchannels with a Hencky strain of 3.6 and 4.2 the experiments have also shown that cells blockage are more likely to occur at this kind of microchannels. Hence, the most appropriate hyperbolic-shaped microchannels to separate RBCs from plasma is the one with a Hencky strain of 3.
- Automatic tracking of labeled red blood cells in microchannelsPublication . Pinho, Diana; Lima, Rui A.; Pereira, Ana I.; Gayubo, FernandoThe current study proposes an automatic method for the segmentation and tracking of red blood cells flowing through a 100- m glass capillary. The original images were obtained by means of a confocal system and then processed in MATLAB using the Image Processing Toolbox. The measurements obtained with the proposed automatic method were compared with the results determined by a manual tracking method. The comparison was performed by using both linear regressions and Bland–Altman analysis. The results have shown a good agreement between the two methods. Therefore, the proposed automatic method is a powerful way to provide rapid and accurate measurements for in vitro blood experiments in microchannels.
- Red blood cells radial dispersion in blood flowing through microchannels: the role of temperaturePublication . Pinho, Diana; Rodrigues, Raquel Oliveira; Faustino, Vera; Yaginuma, Tomoko; Exposto, José; Lima, Rui A.The behavior of suspensions of individual blood cells, such as red blood cells (RBCs), flowing through microvessels and microfluidic systems depend strongly on the hematocrit (Hct), microvessel topology and cell properties. Although it is well known that blood rheological properties are temperature dependent, to the best of our knowledge no work has studied the role of the temperature on the RBCs dispersion. A powerful way to investigate this latter effect is through a high-speed video microscopy system, which provides detailed flow measurements of each individual RBC. Hence, the effect of temperature on the RBCs dispersion flowing through a 100 μm glass capillary was examined by means of a confocal micro-PTV system. Hundreds of labeled RBCs were tracked at moderate Hct (12%) and at four different temperatures, i.e., 25 °C, 32 °C, 37 °C and 42 °C. The results yielded an enhancement of the RBCs diffusion as the temperature increases. Hence, our findings show that RBCs radial dispersion is temperature dependent and as a result the temperature should not be ignored in future blood flow studies. We believe that this finding is important for a better understanding of blood mass transport mechanisms under both physiological and pathological conditions.
- Fabricação de microcanais utilizando o método de xurografia: visualização da camada livre de célulasPublication . Pinto, Elmano; Pinho, Diana; Bento, David; Correia, Teresa Montenegro; Garcia, Valdemar; Dias, Ricardo P.; Miranda, João MárioNo presente estudo, pretendeu-se desenvolver/melhorar uma metodologia de microfabricação de baixo custo, conhecida por xurografia e efectuar a visualização e quantificação da camada livre de células para diferentes escoamentos sanguíneos em microcanais.