Particle detection and tracking in fluorescence time-lapse imaging: a contrario approach

This paper proposes a probabilistic approach for the detection and the tracking of particles in fluorescent time-lapse imaging. In the presence of a very noised and poor-quality data, particles and trajectories can be characterized by an a contrario model, that estimates the probability of observing the structures of interest in random data. This approach, first introduced in the modeling of human visual perception and then successfully applied in many image processing tasks, leads to algorithms that neither require a previous learning stage, nor a tedious parameter tuning and are very robust to noise. Comparative evaluations against a well-established baseline show that the proposed approach outperforms the state of the art.Comment: Published in Journal of Machine Vision and Application

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

Computational illumination for high-speed in vitro Fourier ptychographic microscopy

We demonstrate a new computational illumination technique that achieves large space-bandwidth-time product, for quantitative phase imaging of unstained live samples in vitro. Microscope lenses can have either large field of view (FOV) or high resolution, not both. Fourier ptychographic microscopy (FPM) is a new computational imaging technique that circumvents this limit by fusing information from multiple images taken with different illumination angles. The result is a gigapixel-scale image having both wide FOV and high resolution, i.e. large space-bandwidth product (SBP). FPM has enormous potential for revolutionizing microscopy and has already found application in digital pathology. However, it suffers from long acquisition times (on the order of minutes), limiting throughput. Faster capture times would not only improve imaging speed, but also allow studies of live samples, where motion artifacts degrade results. In contrast to fixed (e.g. pathology) slides, live samples are continuously evolving at various spatial and temporal scales. Here, we present a new source coding scheme, along with real-time hardware control, to achieve 0.8 NA resolution across a 4x FOV with sub-second capture times. We propose an improved algorithm and new initialization scheme, which allow robust phase reconstruction over long time-lapse experiments. We present the first FPM results for both growing and confluent in vitro cell cultures, capturing videos of subcellular dynamical phenomena in popular cell lines undergoing division and migration. Our method opens up FPM to applications with live samples, for observing rare events in both space and time

Robust visualization and discrimination of nanoparticles by interferometric imaging

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.R01 AI096159 - NIAID NIH HHSFirst author draf

Growth of Trichoderma atroviride and importance of the nano-motor Kinesin-1 in its development

"The vegetative growth of filamentous fungi comprises cell elongation and branching. This complex process involves Intracellular transport of molecules, vesicles and organelles within the cell interior, assisted by motor proteins, which, under the study of Nanosciences, are considered the smallest motors. Here, the growth process of Trichoderma atroviride was studied. First, a novel tool for automated tracking of growth in fungal cells (called hyphae) is introduced. This tool allows performing quantitative analysis of growth rate and morphology. An image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates was developed. Tracking records allowed to determine that T. atroviride hyphae grow with characteristic elongation rates of ~ 0.07 μm/s. Prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. From tracking data, it was found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362 μm. Second, the absence of Kinesin-1, one of the main nano-motors transporters of essential growth vesicles (reported for other filamentous fungi), and how it affects hyphal elongation, branching and morphology of T. atroviride was studied. By developing a Kinesin-1 deletion mutant strain it was shown that T. atroviride elongation rate drops up to ~ 60%, while branching highly increases. Several experiments following T. atroviride growth stages were carried out (germination, vegetative growth and conidiation) and determined that: The absence of Kinesin-1 modifies T. atroviride germination resulting in delayed conidia germination times. Also, about 50% of conidia presented bipolar germination. Vegetative growth was highly reduced with characteristic elongation rates of ~ 0.03 μm/s. Conidiation was also reduced in ΔKinesin-1 producing ~30x108 conidia vs ~8x108 WT. Altogether, these preliminary results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics and how the absence of the nano-motor protein Kinesin-1 results in severe modifications of hyphal elongation, branching, conidiation and morphology.""El crecimiento vegetativo de los hongos filamentosos se define por el alargamiento y la ramificación celular. Este crecimiento es un proceso complejo que involucra el transporte intracelular de moléculas, vesículas y organelos, mediado principalmente por nano-motores. En este proyecto, se estudió el proceso de crecimiento de Trichoderma atroviride en dos aspectos principales: Primero, se presenta una herramienta para el seguimiento automático del crecimiento de células de hongos filamentosos (llamadas hifas) para realizar un análisis cuantitativo de la tasa de crecimiento y aspectos de su morfología. Se desarrolló una rutina de procesamiento de imágenes que detecta en tiempo real la punta de una hifa y la rastrea a medida que la hifa se alarga. Los registros de seguimiento permitieron determinar que las hifas de T. atroviride crecen con tasas de elongación características de ~ 0.07 μm / s. Se encontró que antes de que ocurriera un evento de ramificación apical, la hifa parental detiene su crecimiento durante unos minutos. Finalmente, se determinó que la longitud de persistencia (una medida de la extensión del filamento antes de presentar un cambio en la dirección) asociada a las hifas de T. atroviride es de 362 μm. Segundo, se estudió que la ausencia de la Cinesina-1, uno de los principales nano-motores transportadores de vesículas de crecimiento esenciales (reportados para otros hongos filamentosos), afecta la elongación de las hifas, la ramificación y la morfología de T. atroviride. Al desarrollar en el laboratorio una cepa mutante ΔCinesina-1, se observó que la tasa de elongación de T. atroviride disminuye hasta en un 60%, mientras que la ramificación aumenta considerablemente. Se llevaron a cabo varios experimentos morfológicos siguiendo las etapas de crecimiento de T. atroviride (germinación, crecimiento vegetativo y conidiación) y se encontró que: la ausencia de la Cinesina-1 afectó la germinación de T. atroviride dando como resultado que el ~ 50% de los conidios presentan germinación bipolar. El crecimiento vegetativo se vió altamente disminuido con tasas de elongación características de ~ 0.03 μm/s. El proceso de conidiación también se vió afectado en la cepa ΔCinesina-1 produciendo ~ 30x108 conidias vs ~ 8x108 en la cepa silvestre. En conjunto, estos resultados preliminares muestran cómo la integración del análisis de imágenes y el control por computadora permiten realizar observaciones microscópicas cuantitativas de la dinámica de las hifas de los hongos y cómo la ausencia de la proteína nano-motora Cinesina-1 produce modificaciones en la elongación de hifas, la ramificación, la conidiación y la morfología.

Quantitative automated analysis of host-pathogen interactions

This work aims to broaden knowledge about neutrophil biology in their interaction with fungi species that most frequently cause invasive fungal diseases (IFD). The questions addressed include the alteration of neutrophil morphology after interaction with Candida albicans or C. glabrata, revealing factors that modulate the production and composition of neutrophil-derived extracellular vesicles (EVs) obtained in confrontation assay with conidia of Aspergillus fumigatus and analysing EVs activity against this fungus. Alongside fundamental interests, those questions have important applied aspects in the medicine of IFD. In particular, for diagnostic purposes and infection process monitoring. The results of this work include: 1 a novel segmentation and tracking algorithm which is capable of working with low-contrast cell images, producing accurate cell contours and providing data about positions of clusters, which would improve further analysis; 2 a novel workflow algorithm for analysis of neutrophil continuous morphological spectrum without consensus-based manual annotation; 3 quantitative evidence that morphodynamics of isolated neutrophils depends on the infectious agent (C. albicans or C. glabrata) used in whole blood infection assay; 4 quantitative evidence that neutrophil-derived extracellular vesicles, obtained in confrontation assays with conidia of A. fumigatus could inhibit hyphae development and damage hyphae cell wall; 5 quantitative evidence that EVs inhibition activity is strain-specific

Myosin II filament dynamics in actin networks revealed with interferometric scattering microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network

The promise and the challenges of cryo-electron tomography

Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-electron tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET