CE09 - Nanomatériaux et nanotechnologies pour les produits du futur

SIlicon Carbide NAnoProbes and optical Signal Enhancement for intracellular transport investigation in 3D cultures of neurons – SINAPSE

Silicon Carbide nanoprobes and optical signal enhancement for intracellular transport investigation in 3D cultures of neurons

In this project, we propose to analyze the optical response of SiC nanocrystals excited in the infrared (second window of transparency of biological tissues). Such nanoprobes can be used to analyze the intracellular movement of vesicles in 3D neural networks. The project plans to develop an agile microscope to detect these nanoprobes in deep tissues, but also to take advantage of metallic nanoantennas to enhance the optical signals from the probes.

General objective

An important issue of this study is to explore physical possibilities to access optical information in thick biological tissue. The objective is to be able to measure the parameters of intraneuronal transport in complex and mature networks, in order to compare different conditions.

There are mainly three physical development axes in the SINAPSE project. The first is the development of a microscope for fast 3D scanning in the super-localization regime, using a digital holography device: an array of micro-mirrors acts as a spatial light modulator and allows to position a laser spot at different points in the sample space. The microscope will be able to localize the particle with a precision of a few tens of nanometers at a rate of more than one kHz. Another axis is dedicated to the fabrication and characterization of nanocrystals that can be used as nanoprobes in cells. SiC is studied. An irradiation allows to include crystalline defects for fluorescence emission in the IR. Other particles are studied, such as BaTiO3, in which it is possible to include rare earth ions for fluorescence emission. Both types of particles have efficient nonlinear properties for second harmonic emission, which makes them good candidates for two-photon microscopy applications in deep biological tissues. The last line of work concerns the modeling, fabrication and characterization of hybrid metal-dielectric nanoprobes for the exaltation of optical signals in order to gain in signal-to-noise ratio and/or to decrease the excitation power to avoid photo-induced damage.

The microscopy setup is now mounted and being validated for translational tracking of NPs. Measurement of the orientation of the NPs using the second harmonic emission anisotropy will be implemented in the next 6 months.

Work on SiC nanoparticles has been hampered by the fact that the nanoparticles are aggregated. Several attempts have been made to separatee them, but we have not yet found a reliable solution. From an experimental point of view, we have for the moment refocused the study on the second harmonic generation (GSH) in order to continue the work on the fabrication of hybrid nano-probes and to progress on the microscopy device. We are using KTiOPO4 (KTP) or BaTiO3 (BTO) NPs. The nanoBTOs have been doped with rare earths (Erbium/Ytterbium) which emit in the near-IR range, and represent an alternative to SiCs for the purpose of bi-modal emission. However, the infrared emission (at 1.5 µm for Er doping) remains too low.

The SHG characterization setup of the NPs at SPEC allowed us to estimate the nonlinear coefficients of individual KTP, BTO and doped BTO NPs. BTO doping reduces GSH efficiency (2-fold reduction in SHG intensity for 0.5% Er doping).

The fabrication process of the hybrid core-shell systems (BTO NPs in the core and gold shell) has been validated by LPEM. BTO-Au and KTP-Au samples should be analyzed soon. Preliminary measurements have verified that the gold shell was not a source of additional SHG signal.

Numerical simulations on the core-shell system have been completed, allowing to target the right geometries for the best results in terms of brightness on the one hand and temperature rise limitation on the other hand.

We will get back on SiC nanocrystals studies in the coming year, this time using a poly-4H SiC wafer substrate irradiated with protons (to create lacunar defects) and then ground into nanoparticles, unlike previous experiments conducted on cubic poly-type nanoparticles.

The hybrid metal-dielectric nanoparticles will be fabricated and characterized.

We are starting to prepare the microscopy device to receive biological samples (purchase of a thermal chamber and controlled CO2 atmosphere in particular). The microscope will be tested first on nanoparticle transport in microfluidic wells, then validated in 2D neuron cultures, before observing mouse brain slices or brain organoids made from pluripotent human cells.

- Intraneuronal transport abnormalities revealed by optically active photostable nanoparticle tracking, IBS conference on advanced optical imaging, Seoul, Corée du Sud, oral invite (F. Marquier, LuMIn, juin 2019)
- Towards 3D single-particle tracking for intraneuronal transport characterization, Live cell single molecule tracking symposium, Ulm, Allemagne, poster (F. Semmer LuMIn, novembre 2019)
- Suivi de nanoparticules en mouvement dans des réseaux de neurones, workshop académique-industriel les nanos pour le vivant, LPS, Orsay, poster (F. Marquier LuMIn, avril 2019)
- Nanoparticle orientation tracking in neuronal networks using second harmonic signal detection, Journée du Labex Nanosaclay, C2N, Orsay, poster (F. Semmer LuMIn, septembre 2019)
- Second-harmonic generation (SHG) of single dielectric nanoparticles for bio-imaging, 25ième Congrès général de la SFP, Nantes, présentation orale (W. DJAMPA TAPI, CEA SPEC Juil (2019))
- Probing intraneuronal transport in vivo with optically active photostable nanocrystals, séminair e invité au LAMBE, EVRY (F. Treussart et F. Marquier, LuMIn, janvier 2020)

SINAPSE is an interdisciplinary project, taking advantage of a large range of expertise at the interfaces of physics, chemistry and neurobiology. The main goal of the project is the development of a novel class of bright crystalline nanoparticles (NPs) with both linear and non-linear optical responses in the near-infrared (NIR) spectral range for bio-imaging applications. More specifically, these nano-labels will be optimized to enable the measurement of intracellular transport parameters in the axon and dendrites of neurons in a 3D neuronal network. Precise quantification of the intraneuronal transport can indeed serve as a readout of genetic risk factor functional impact of neuropsychiatric or neurodegenerative disease, as we already demonstrated in 2D cultures. To this aim, the optically active NPs are spontaneously internalized by neurons, ending up in endosomal vesicles, that are further transported by molecular motors along cytoskeleton tracks. Translational and rotational motion of the NPs will be inferred from two complementary optical signals from the same single particle: NIR fluorescence and second-harmonic generation (SHG), both excited by the same infrared pump laser. The NIR spectral range (excitation, and fluorescence) fits perfectly with the transparency window of biological systems, which is of crucial importance to image deep in brain tissues.

We propose to investigate for the first time the bi-modal optical response that originates from the NIR excitation of silicon carbide nanocrystals (nanoSiC). Compared to the present state-of-the-art of imaging biolabels, nanoSiC has the advantage to allow the two-mode emission: SHG originating from the host matrix and infrared fluorescence originating from crystalline point defects, generated by proton irradiation and thermal annealing. Another key task of SINAPSE project will be to enhance both signals taking advantage of metallic (plasmonic) nanoantennas associated to the nanoSiC, in order to achieve unprecedented large signal-to-noise ratio. The two complementary light-emission processes at the single-particle level will be ultimately used to analyze fine features of the vesicular motion dynamics into 3D neuronal networks, at high spatio-temporal resolution, and large depth (up to ˜100 µm).

SINAPSE gathers 4 complementary skilled partners (Laboratoire Aimé Cotton, CEA-SPEC, Laboratoire Charles Fabry, laboratoire de Physique et d’études des matériaux) who have obtained promising preliminary results. In practice, the project will proceed through three main workpackages corresponding to the three logical axis of the project:
- Production and characterization of the optically-active bi-modal nanoparticles
- Enhancement of the optical properties using plasmonic nanoantennas
- Nanoparticle translational and orientational dynamics measurements into neurons
Our consortium comprises a unique range of expertise in nanochemistry, non-linear optics, nanomaterial science, bioimaging with nanoparticles, and molecular genetics applied to neuropsychiatric/degenerative diseases, required to tackle the ambitious goal of the project. Importantly, the project addresses the challenge of a better understanding of neuropsychiatric/degenerative diseases mechanisms. It could thus have a significant impact on health, in the long run, considering the high prevalence of these diseases worldwide and the limited knowledge regarding the key processes leading to the appearance of the first symptoms. Understanding these processes is essential to develop drugs capable of stopping the disease development at its pre-symptomatic stage.

Project coordination

Francois Marquier (Laboratoire Aimé Cotton)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LPEM Laboratoire de Physique et d'Etude des Matériaux
LAC Laboratoire Aimé Cotton
SPEC Service de physique de l'état condensé
LCF Laboratoire Charles Fabry

Help of the ANR 446,488 euros
Beginning and duration of the scientific project: March 2019 - 42 Months

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