Matteo Cornaglia

Hyperspectral nanoscale imaging on dielectric substrates with coaxial optical antenna scan probes

An important goal in nano science is to unlock previously inaccessible physics and dynamics within nanoscale systems by combining the efficient nanoscale field confinement/optical resolution (∼10 nm) of optical antennae and the ultrafast temporal resolution (fs) inherent in optical studies with the capabilities of modern scanning probe techniques. Here we report on a significant step toward this goal using a novel nanofabricated coaxial antenna tip capable of recording useful Raman spectra in ∼50 ms to acquire 256 by 256 pixel images on dielectric substrates with a full spectrum at each pixel.

An automated microfluidic platform for C. elegans embryo arraying, phenotyping,and long-term live imaging

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population’s statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPRmt) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

Photonic Nanojet Array for Fast Detection of Single Nanoparticles in a Flow

We detect by optical microscopy Au and fluorescent nanoparticles (NPs) during their motion in water-based medium, using an array of dielectric microspheres that are patterned in a microwell array template. The microspheres act as lenses focusing the light originating from a microscope objective into so-called photonic nanojets that expose the medium within a microfluidic channel. When a NP is randomly transported through a nanojet, its backscattered light (for a bare Au NP) or its fluorescent emission is instantaneously detected by video microscopy. Au NPs down to 50 nm in size, as well as fluorescent NPs down to 20 nm in size, are observed by using a low magnification/low numerical aperture microscope objective in bright-field or fluorescence mode, respectively. Compared to the NPs present outside of the photonic nanojets, the light scattering or fluorescence intensity of the NPs in the nanojets is typically enhanced by up to a factor of ∼40. The experimental intensity is found to be proportional to the area occupied by the NP in the nanojet. The technique is also used for immunodetection of biomolecules immobilized on Au NPs in buffer and, in future, it may develop into a versatile tool to detect nanometric objects of environmental or biological importance, such as NPs, viruses, or other biological agents.

Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models

BackgroundWhile many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions – e.g. neurodegenerative diseases – often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field.ResultsWe describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms’ tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system’s ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model.ConclusionOur microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.

Magnetic Particle-Scanning for Ultrasensitive Immunodetection On-Chip

We describe the concept of magnetic particle-scanning for on-chip detection of biomolecules: a magnetic particle, carrying a low number of antigens (Ags) (down to a single molecule), is transported by hydrodynamic forces and is subjected to successive stochastic reorientations in an engineered magnetic energy landscape. The latter consists of a pattern of substrate-bound small magnetic particles that are functionalized with antibodies (Abs). Subsequationuent counting of the captured Ag-carrying particles provides the detection signal. The magnetic particle-scanning principle is investigated in a custom-built magneto-microfluidic chip and theoretically described by a random walk-based model, in which the trajectory of the contact point between an Ag-carrying particle and the small magnetic particle pattern is described by stochastic moves over the surface of the mobile particle, until this point coincides with the position of an Ag, resulting in the binding of the particle. This model explains the particular behavior of previously reported experimental dose-response curves obtained for two different ligand-receptor systems (biotin/streptavidin and TNF-α) over a wide range of concentrations. Our model shows that magnetic particle-scanning results in a very high probability of immunocomplex formation for very low Ag concentrations, leading to an extremely low limit of detection, down to the single molecule-per-particle level. When compared to other types of magnetic particle-based surface coverage assays, our strategy was found to offer a wider dynamic range (>8 orders of magnitude), as the system does not saturate for concentrations as high as 10(11) Ag molecules in a 5 μL drop. Furthermore, by emphasizing the importance of maximizing the encounter probability between the Ag and the Ab to improve sensitivity, our model also contributes to explaining the behavior of other particle-based heterogeneous immunoassays.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.

On-chip microfluidic biocommunication assay for studying male-induced demise in C. elegans hermaphrodites

Like other animals, C. elegans nematodes have the ability to socially interact and to communicate through exchange and sensing of small soluble signaling compounds that help them cope with complex environmental conditions. For the time being, worm biocommunication assays are being performed mainly on agar plates; however, microfluidic assays may provide significant advantages compared to traditional methods, such as control of signaling molecule concentrations and gradients or confinement of distinct worm populations in different microcompartments. Here, we propose a microfluidic device for studying signaling via diffusive secreted compounds between two specific C. elegans populations over prolonged durations. In particular, we designed a microfluidic assay to investigate the biological process of male-induced demise, i.e. lifespan shortening and accelerated age-related phenotype alterations, in C. elegans hermaphrodites in the presence of a physically separated male population. For this purpose, male and hermaphrodite worm populations were confined in adjacent microchambers on the chip, whereas molecules secreted by males could be exchanged between both populations by periodically activating the controlled fluidic transfer of μl-volume aliquots of male-conditioned medium. For male-conditioned hermaphrodites, we observed a reduction of 4 days in mean lifespan compared to the non-conditioned on-chip culture. We also observed an enhanced muscle decline, as expressed by a faster decrease in the thrashing frequency and the appearance of vacuolar-like structures indicative of accelerated aging. The chip was placed in an incubator at 20 °C for accurate control of the lifespan assay conditions. An on-demand bacteria feeding protocol was applied, and the worms were observed during long-term on-chip culture over the whole worm lifespan.

The Movement Tracker: A Flexible System for Automated Movement Analysis in Invertebrate Model Organisms

Phenotyping strategies in simple model organisms such as D. melanogaster and C. elegans are often broadly limited to growth, aging, and fitness. Recently, a number of physical setups and video tracking software suites have been developed to allow for accurate, quantitative, and high‐throughput analysis of movement in flies and worms. However, many of these systems require precise experimental setups and/or fixed recording formats. We report here an update to the Parallel Worm Tracker software, which we termed the Movement Tracker. The Movement Tracker allows variable experimental setups to provide cross‐platform automated processing of a variety of movement characteristics in both worms and flies and permits the use of simple physical setups that can be readily implemented in any laboratory. This software allows high‐throughput processing capabilities and high levels of flexibility in video analysis, providing quantitative movement data on C. elegans and D. melanogaster in a variety of different conditions.

Fine-tuning of magnetic and microfluidic viscous forces for specific magnetic bead-based immunocomplex formation

We investigate the working principle of a novel type of microfluidic sandwich immunoassay, as used for the detection of biomarkers. The heterogeneous assay is based on the specific interactions between an array of functionalized superparamagnetic beads and a flow of secondary superparamagnetic beads that carry the antigens and are simultaneously used as detection labels. We identify the main forces governing the immunoassay performance and develop a combined finite element method/analytical model to predict and control these forces. The clue for the improved assay specificity is in the fine-tuning of inter-bead magnetic dipolar and microfluidic viscous forces, which allows strongly reducing non-specific interactions, while enhancing the specific formation of immunocomplexes. We exploit our theoretical model to explain the enhanced sensitivity of magnetic bead-based immunoassay experiments performed in microfluidic chips.

A microfluidic array for high-content screening at whole-organism resolution

A main step for the development and the validation of medical drugs is the screening on whole organisms, which gives the systemic information that is missing when using cellular models. Among the organisms of choice, Caenorhabditis elegansis a soil worm which catches the interest of researchers who study systemic physiopathology (e.g. metabolic and neurodegenerative diseases) because: (1) its large genetic homology with humans supports translational analysis; (2) worms are much easier to handle and grow in large amounts compared to rodents, for which (3) the costs and (4) the ethical concerns are substantial.C. elegansis therefore well suited for large screens, dose-response analysis and target-discovery involving an entire organism. We have developed and tested a microfluidic array for high-content screening, enabling the selection of small populations of its first larval stage in many separated chambers divided into channels for multiplexed screens. With automated protocols for feeding, drug administration and image acquisition, our chip enables the study of the nematodes throughout their entire lifespan. By using a paralyzing agent and a mitochondrial-stress inducer as case studies, we have demonstrated large field-of-view motility analysis, and worm-segmentation/signal-detection for mode-of-action quantification with genetically-encoded fluorescence reporters.