Detecting abnormal cell behaviors from dry mass time series.

The prediction of pathological changes on single cell behaviour is a challenging task for deep learning models. Indeed, in self-supervised learning methods, no prior labels are used for the training and all of the information for event predictions are extracted from the data themselves. We present here a novel self-supervised learning model for the detection of anomalies in a given cell population, StArDusTS. Cells are monitored over time, and analysed to extract time-series of dry mass values. We assessed its performances on different cell lines, showing a precision of 96% in the automatic detection of anomalies. Additionally, anomaly detection was also associated with cell measurement errors inherent to the acquisition or analysis pipelines, leading to an improvement of the upstream methods for feature extraction. Our results pave the way to novel architectures for the continuous monitoring of cell cultures in applied research or bioproduction applications, and for the prediction of pathological cellular changes.

Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses.

We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

3D time-lapse imaging of a mouse embryo using intensity diffraction tomography embedded inside a deep learning framework.

We present a compact 3D diffractive microscope that can be inserted directly in a cell incubator for long-term observation of developing organisms. Our setup is particularly simple and robust, since it does not include any moving parts and is compatible with commercial cell culture containers. It has been designed to image large specimens (>100×100×100µm3) with subcellular resolution. The sample's optical properties [refractive index (RI) and absorption] are reconstructed in 3D from intensity-only images recorded with different illumination angles produced by an LED array. The reconstruction is performed using the beam propagation method embedded inside a deep-learning network where the layers encode the optical properties of the object. This deep neural network is trained for a given multiangle intensity acquisition. After training, the weights of the neural network deliver the 3D distribution of the optical properties of the sample. The effect of spherical aberrations due to the sample holder/air interfaces are taken into account in the forward model. Using this approach, we performed time-lapse 3D imaging of preimplantation mouse embryos over six days. Images of embryos from a single cell (low-scattering regime) to the blastocyst stage (highly scattering regime) were successfully reconstructed. Due to its subcellular resolution, our system can provide quantitative information on the embryos' development and viability. Hence, this technology opens what we believe to be novel opportunities for 3D label-free live-cell imaging of whole embryos or organoids over long observation times.

A Bottom-Up Approach to Red-Emitting Molecular-Based Nanoparticles with Natural Stealth Properties and their Use for Single-Particle Tracking Deep in Brain Tissue.

Fluorescent nanoparticles dedicated to bioimaging applications should possess specific properties that have to be maintained in the aqueous, reactive, and crowded biological environment. These include chemical and photostability, small size (on the scale of subcellular structures), biocompatibility, high brightness, and good solubility. The latter is a major challenge for inorganic nanoparticles, which require surface coating to be made water soluble. Molecular-based fluorescent organic nanoparticles (FONs) may prove a promising, spontaneously water-soluble alternative, whose bottom-up design allows for the fine-tuning of individual properties. Here, the critical challenge of controlling the interaction of nanoparticles with cellular membranes is addressed. This is a report on bright, size-tunable, red-emitting, naturally stealthy FONs that do not require the use of antifouling agents to impede interactions with cellular membranes. As a proof of concept, single FONs diffusing up to 150 µm deep in brain tissue are imaged and tracked.

Near-infrared nanoscopy with carbon-based nanoparticles for the exploration of the brain extracellular space.

Understanding the physiology and pathology of the brain requires detailed knowledge of its complex structures as well as dynamic internal processes at very different scales from the macro down to the molecular dimensions. A major yet poorly described brain compartment is the brain extracellular space (ECS). Signalling molecules rapidly diffuse through the brain ECS which is complex and dynamic structure at numerous lengths and time scales. In recent years, characterization of the ECS using nanomaterials has made remarkable progress, including local analysis of nanoscopic dimensions and diffusivity as well as local chemical sensing. In particular, carbon nanomaterials combined with advanced optical technologies, biochemical and biophysical analysis, offer novel promises for understanding the ECS morphology as well as neuron connectivity and neurochemistry. In this review, we present the state-of-the-art in this quest, which mainly focuses on a type of carbon nanomaterial, single walled carbon nanotubes, as fluorescent nanoprobes to unveil the ECS features in the nanometre domain.

Synucleinopathy alters nanoscale organization and diffusion in the brain extracellular space through hyaluronan remodeling.

In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathology remains unknown. Here we study the nanoscale organization of pathological ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resolution imaging analysis, we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, experimental hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathology, unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.

Nanoscale exploration of the extracellular space in the live brain by combining single carbon nanotube tracking and super-resolution imaging analysis.

The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.

The Effect of Nanoparticles on the Cluster Size Distributions of Activated EGFR Measured with Photobleaching Image Correlation Spectroscopy.

The epidermal growth factor receptor (EGFR) is an important cell surface receptor in normal physiology and disease. Recent work has shown that EGF-gold nanoparticle conjugates can influence cell behaviour, but the underlying mechanism at the receptor quaternary structural level remains poorly understood.In the present work, the cluster density and cluster size of activated (phosphorylated) EGFR clusters in HeLa cells were determined with photobleaching image correlation spectroscopy. EGFR activation was probed via immunofluorescence-detected phosphorylation of tyrosines (pY-mAb) located in the kinase domain of EGFR (Y845) and at the EGFR cytoplasmic tail (Y1173). Cell activation was probed via nuclear extracellular-regulated kinase (ERK) phosphorylation. The cluster size of activated EGFR was 1.3-2.4 pY-mAb/cluster in unstimulated HeLa cells. EGF or nanorod treatment led to an increase in EGFR oligomers containing multiple phosphotyrosines (>2 phosphotyrosines per EGFR oligomer, average cluster size range = 3-5 pY-mAb/cluster) which paralleled increases in nuclear p-ERK. In contrast, EGF-nanorods decreased the contribution from higher-order phospho-clusters and decreased nuclear p-ERK relative to the nanorod control. These studies provide direct evidence that targeted nanotechnology can manipulate receptor organization and lead to changes in receptor activation and subsequent signalling processes.

Gold Nanoparticles for Modulating Neuronal Behavior.

Understanding the detailed functioning and pathophysiology of the brain and the nervous system continues to challenge the scientific community, particularly in terms of scaling up techniques for monitoring and interfacing with complex 3D networks. Nanotechnology has the potential to support this scaling up, where the eventual goal would be to address individual nerve cells within functional units of both the central and peripheral nervous system. Gold nanoparticles provide a variety of physical and chemical properties that have attracted attention as a light-activated nanoscale neuronal interface. This review provides a critical overview of the photothermal and photomechanical properties of chemically functionalized gold nanoparticles that have been exploited to trigger a range of biological responses in neuronal tissues, including modulation of electrical activity and nerve regeneration. The prospects and challenges for further development are also discussed.

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Inhibiting EGFR clustering and cell proliferation with gold nanoparticles.

Gold nanoparticles are functionalized with epidermal growth factor (EGF) molecules and incubated with HeLa cells. These new complexes mechanically interfere with the activation of EGF receptors in a length-dependent manner. Protein-functionalized gold nanoparticles hold great potential for unveiling the fundamental characteristics of cell receptors and for future pharmacological studies on receptor targeting.

Nanoparticle-enhanced infrared neural stimulation.

OBJECTIVE: Recent research has demonstrated that nerves can be stimulated by transient heating associated with the absorption of infrared light by water in the tissue. There is a great deal of interest in using this technique in neural prostheses, due to the potential for increased localization of the stimulus and minimization of contact with the tissue. However, thermal modelling suggests that the full benefits of increased localization may be reduced by cumulative heating effects when multiple stimulus sites and/or high repetition rates are used. APPROACH: Here we review recent in vitro and in vivo results suggesting that the transient heating associated with plasmon absorption in gold nanorods can also be used to stimulate nerves. MAIN RESULTS: Patch clamp experiments on cultured spiral ganglion neurons exhibited action potentials when exposed to 780 nm light at the plasmon absorption peak, while the amplitude of compound action potentials in the rat sciatic nerve were increased by laser irradiation of gold nanorods in the vicinity of the plasma membrane. Similarly, calcium imaging studies of NG108-15 neuronal cells incubated with Au nanorods revealed an increased level of intracellular calcium activity synchronized with laser exposure. SIGNIFICANCE: Given that the plasmon absorption peak of gold nanorods can be matched with the transparency window of biological tissues, these results demonstrate that nanorod absorbers hold great promise to enhance the process of infrared neural stimulation for future applications in neural prostheses and fundamental studies in neuroscience.

Laser exposure of gold nanorods can induce intracellular calcium transients.

Uncoated and poly(styrene sulphonate) (PSS)-coated gold nanorods were taken up by NG108-15 neuronal cells. Exposure to 780 nm laser light at the plasmon resonance wavelength of the gold nanorods was found to induce intracellular Ca(2+) transients. The higher Ca(2+) peaks were observed at lower laser doses, with the highest levels obtained at a radiant exposure of 0.33 J/cm(2) . In contrast, the cells without nanoparticles showed a consistently small response, independent of the laser dose. These initial results open up new opportunities for peripheral nerve regeneration treatments and for more efficient optical stimulation techniques.

Laser exposure of gold nanorods can increase neuronal cell outgrowth.

The usage of gold nanoparticles (Au NPs) in biological applications has risen significantly over the last 10 years. With the wide variety of chemical and biological functionalization available and their distinctive optical properties, Au NPs are currently used in a range of biological applications including sensing, labeling, drug delivery, and imaging applications. Among the available particles, gold nanorods (Au NRs) are particularly useful because their optical absorption can be tuned across the visible to near infrared region. Here, we present a novel application of Au NRs associated with low power laser exposure of NG108-15 neuronal cells. When cells were irradiated with a 780 nm laser, the average number of neurons with neurites increased. A similar stimulatory effect was observed for cells that were cultured with poly-(4-styrenesulfonic acid)-coated and silica-coated Au NRs. Furthermore, when the NG108-15 cells were cultured with both bare and coated Au NRs and then irradiated with 1.2-7.5 W/cm(2) at 780 nm, they showed a neurite length increase of up to 25 µm versus control. To the best of our knowledge, this effect has never been reported before. While the pathways of the stimulation is not yet clear, the data presented here demonstrates that it is linked to the absorption of light by the Au NRs. These initial results open up new opportunities for peripheral nerve regeneration treatments and for novel approaches to addressing central nervous system axons following spinal cord injury.