Image processing on reconfigurable hardware for continuous monitoring of fluorescent biomarkers in cell cultures

Fluorescence microscopy is a widespread tool in biological research. It is the primary modality for bioimaging and empowers the study and analysis of multitudes of biological processes. It can be applied to fixed biosamples, that is samples with frozen biological features by mean of chemical linkers, or live biosamples providing useful insights on the spatio-temporal behavior of fluorescently stained biomarkers. Current fluorescent microscopy techniques use digital image sensors which are used to leverage quantitative studies instead qualitative outcomes. However, state-of-the-art techniques are not suitable for integration in small, contained and (semi-)autonomous systems. They remain costly, bulky and rather quantitatively inefficient methods for monitoring fluorescent biomarkers, which is not on par with the design constraints found in modern Lab-on-a-Chip or Point-of-Use systems requiring the use of miniaturized and integrated fluroscence microscopy. In this thesis, I summarize my research and engineering efforts in bringing an embedded image processing system capable of monitoring fluorescent biomarkers in cell cultures in a continuous and real-time manner. Three main areas related to the problem at hand were explored in the course of this work: simulation, segmentation algorithms and embedded image processing. n the area of simulation, a novel approach for generating synthetic fluorescent 2D images of cell cultures is presented. This approach is dichotomized in a first part focusing on the modeling and generation of synthetic populations of cells (i.e. cell cultures) at the level of single fluorescent biomarkers and in a second part simulating the imaging process occurring in a traditional digital fluorescent microscope to produce realistic images of the synthetic cell cultures. The objective of the proposed approach aims at providing synthetic data at will in order to test and validate image processing systems and algorithms. Various image segmentation algorithms are considered and compared for the purpose of segmenting fluorescent spots in microscopic images. The study presented in this thesis includes a novel image thresholding technique for spot extraction along with three well-known spot segmentation techniques. The comparison is undertaken on two aspects. The segmentation masks provided by the methods are used to extract further metrics related to the fluorescent signals in order to (i) evaluate how well the segmentation masks can provide data for classifying real fluorescent biological samples from negative control samples and (ii) quantitatively compare the segmentations masks based on simulated data from the previously stated simulation tool. Finally, the design of an embedded image processing system based on FPGA technologies is showcased. A semi-autonomous smart camera is conceived for the continuous monitoring of fluorescent biomarkers based on one of the segmentation methods incorporated in the previously stated comparison. Keeping the focus on the need for integration in fluorescence microscopy, the image processing core at the heart of the smart camera results from the use of a novel image processing suite; a suite of IP cores developed under the constraints dictated by the bioimaging needs of fluorescence microscopy for use in FPGA and SoC technologies. As a proof of concept, the smart camera is applied to the monitoring of the kinetics of the uptake of fluorescent silica nano-particles in cell cultures

Simulated biological cells for receptor counting in fluorescence imaging

Digital image processing and epi-fluorescence microscopy provide one of the main and basic tools for living biological cells analysis and studying. Developing, testing and comparing those image processing methods properly require the use of a controlled environment. A verified and trustworthy database of images and meta-information is needed to control the validity of the processing results. Manually generating that golden database is a long process involving specialists being able to apprehend and extract useful data out of fluorescent images. More and more, we need to automate this process. Having enough cases in the database to challenge the processing methods and gain trust in them cannot be realistically be achieved manually. This paper presents a framework implementing a novel approach to generate synthetic fluorescent images of fluorescently-stained cell populations by simulating the imaging process of fluorescent molecules. Ultimately, the proposed simulator allows us to generate images and golden data to populate the database, thus providing tools for the development, evaluation and testing of processing algorithms meant to be used in automated systems

Empowering Low-Cost CMOS Cameras by Image Processing to Reach Comparable Results with Costly CCDs

Despite the huge research effort to improve the performance of the complementary metal oxide semiconductor (CMOS) image sensors, charge-coupled devices (CCDs) still dominate the cell biology-related conventional fluorescence microscopic imaging market where low or ultra-low noise imaging is required. A detailed comparison of the sensor specifications and performance is usually not provided by the manufacturers which leads the end users not to go out of the habitude and choose a CCD camera instead of a CMOS one. However, depending on the application, CMOS cameras, when empowered by image processing algorithms, can become cost-efficient solutions for conventional fluorescence microscopy. In this paper, we introduce an application-based comparative study between the default CCD camera of an inverted microscope (Nikon Ti-S Eclipse) and a custom-designed CMOS camera and apply efficient image processing algorithms to improve the performance of CMOS cameras. Quantum micro-bead samples (emitting fluorescence light at different intensity levels), breast cancer diagnostic tissue cell samples, and Caco-2 cell samples are imaged by both CMOS and CCD cameras. The results are provided to show the reliability of CMOS camera processed images and finally to be of assistance when scientists select their cameras for desired applications

Image thresholding techniques for localization of sub-resolution fluorescent biomarkers

In this article, we explore adaptive global and local segmentation techniques for a lab-on-chip nutrition monitoring system (NutriChip). The experimental setup consists of Caco-2 intestinal cells that can be artificially stimulated to trigger an immune response. The eventual response is optically monitored using immunofluoresence techniques targeting toll-like receptor 2 (TLR2). Two problems of interest need to be addressed by means of image processing. First, a new cell sample must be properly classified as stimulated or not. Second, the location of the stained TLR2 must be recovered in case the sample has been stimulated. The algorithmic approach to solving these problems is based on the ability of a segmentation technique to properly segment fluorescent spots. The sample classification is based on the amount and intensity of the segmented pixels, while the various segmenting blobs provide an approximate localization of TLR2. A novel local thresholding algorithm and three well-known spot segmentation techniques are compared in this study. Quantitative assessment of these techniques based on real and synthesized data demonstrates the improved segmentation capabilities of the proposed algorithm

How I Investigate...The Coronary Arteries in 2007: Contributions of Ct Coronary Angiography

peer reviewedCardiac imaging has always been a challenge because of the continuous movement of the heart. Cardiac computed tomography (CT) has undergone an accelerated progression over the past decade, due to the combination of the high-speed rotation of the X-ray tube, the ECG-gating technique and the infra-millimeter spatial resolution. Multidetector CT allows visualisation of the coronary artery lumen and the detection of coronary stenosis after intravenous injection of contrast medium. Studies have demonstrated a high negative predictive value of CT coronary angiography (CTCA). CTCA may be reasonably used for the assessment of symptomatic patients, especially in the setting of equivocal treadmill or functional testing. Also, CTCA allows assessment of coronary bypass graft patency and recognition of aberrant coronary arteries. Limitations in the use of this technique exist: atrial fibrillation and other cardiac arrhythmias remain a contraindication; severe calcifications are the most frequent reason for impaired assessment of coronary arteries. High radiation doses prohibit the use of this test as a screening tool for asymptomatic patients

Can CT pulmonary angiography allow assessment of severity and prognosis in patients presenting with pulmonary embolism? What the radiologist needs to know

peer reviewedComputed tomographic (CT) pulmonary angiography has been established as a first-line diagnostic technique in patients suspected of having pulmonary embolism. Risk stratification is important in patients with pulmonary embolism because optimal management, monitoring, and therapeutic strategies depend on the prognosis. Acute right-sided heart failure is known to be responsible for circulatory collapse and death in patients with severe pulmonary embolism. Acute right-sided heart failure can be assessed at CT pulmonary angiography by measuring the dimensions of right-sided heart cavities or upstream venous structures, such as the superior vena cava or azygos vein. The magnitude of pulmonary embolism can be calculated at CT pulmonary angiography by applying angiographic scores adapted for CT (Miller and Walsh scores) or dedicated CT scores (Qanadli and Mastora scores). The advent of CT pulmonary angiography performed with electrocardiographic gating permits new advances in assessment of acute right-sided heart failure, such as measurement of the ventricular ejection fraction. Although such findings may be useful for assessment of treatment effectiveness, their effect on prognosis in patients with severe pulmonary embolism is debated in the literature. (C) RSNA, 2006