Stimulated Emission Depletion Microscopy Resolves Individual Nitrogen Vacancy Centers in Diamond Nanocrystals

Nitrogen-vacancy (NV) color centers in nanodiamonds are highly promising for bioimaging and sensing. However, resolving individual NV centers within nanodiamond particles and the controlled addressing and readout of their spin state has remained a major challenge. Spatially stochastic super-resolution techniques cannot provide this capability in principle, whereas coordinate-controlled super-resolution imaging methods, like stimulated emission depletion (STED) microscopy, have been predicted to fail in nanodiamonds. Here we show that, contrary to these predictions, STED can resolve single NV centers in 40–250 nm sized nanodiamonds with a resolution of ≈10 nm. Even multiple adjacent NVs located in single nanodiamonds can be imaged individually down to relative distances of ≈15 nm. Far-field optical super-resolution of NVs inside nanodiamonds is highly relevant for bioimaging applications of these fluorescent nanolabels. The targeted addressing and readout of individual NV^– spins inside nanodiamonds by STED should also be of high significance for quantum sensing and information applications

Oriented Bioconjugation of Unmodified Antibodies to Quantum Dots Capped with Copolymeric Ligands as Versatile Cellular Imaging Tools

Distinctive optical properties of inorganic quantum dot (QD) nanoparticles promise highly valuable probes for fluorescence-based detection methods, particularly for in vivo diagnostics, cell phenotyping via multiple markers or single molecule tracking. However, despite high hopes, this promise has not been fully realized yet, mainly due to difficulties at producing stable, nontoxic QD bioconjugates of negligible nonspecific binding. Here, a universal platform for antibody binding to QDs is presented that builds upon the controlled functionalization of CdSe/CdS/ZnS nanoparticles capped with a multidentate dithiol/zwitterion copolymer ligand. In a change-of-paradigm approach, thiol groups are concomitantly used as anchoring and bioconjugation units to covalently bind up to 10 protein A molecules per QD while preserving their long-term colloidal stability. Protein A conjugated to QDs then enables the oriented, stoichiometrically controlled immobilization of whole, unmodified antibodies by simple incubation. This QD–protein A immobilization platform displays remarkable antibody functionality retention after binding, usually a compromised property in antibody conjugation to surfaces. Typical QD–protein A–antibody assemblies contain about three fully functional antibodies. Validation experiments show that these nanobioconjugates overcome current limitations since they retain their colloidal stability and antibody functionality over 6 months, exhibit low nonspecific interactions with live cells and have very low toxicity: after 48 h incubation with 1 μM QD bioconjugates, HeLa cells retain more than 80% of their cellular metabolism. Finally, these QD nanobioconjugates possess a high specificity for extra- and intracellular targets in live and fixed cells. The dithiol/zwitterion QD–protein A nanoconjugates have thus a latent potential to become an off-the-shelf tool destined to unresolved biological questions

<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models

<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models

<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models

In-vivo fast non-linear microscopy reveals impairment of fast axonal transport induced by molecular motor imbalances in the brain of zebrafish larvae

Abstract Cargo transport by molecular motors along microtubules is essential for the function of eucaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and of the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈ 90 μ m×60 μ m size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole, and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having non-linear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate, and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated to disease models

<i>In Vivo</i> Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models

Non-Neurotoxic Nanodiamond Probes for Intraneuronal Temperature Mapping

Optical biomarkers have been used extensively for intracellular imaging with high spatial and temporal resolution. Extending the modality of these probes is a key driver in cell biology. In recent years, the nitrogen-vacancy (NV) center in nanodiamond has emerged as a promising candidate for bioimaging and biosensing with low cytotoxicity and stable photoluminescence. Here we study the electrophysiological effects of this quantum probe in primary cortical neurons. Multielectrode array recordings across five replicate studies showed no statistically significant difference in 25 network parameters when nanodiamonds are added at varying concentrations over various time periods, 12–36 h. The physiological validation motivates the second part of the study, which demonstrates how the quantum properties of these biomarkers can be used to report intracellular information beyond their location and movement. Using the optically detected magnetic resonance from the nitrogen-vacancy defects within the nanodiamonds we demonstrate enhanced signal-to-noise imaging and temperature mapping from thousands of nanodiamond probes simultaneously. This work establishes nanodiamonds as viable multifunctional intraneuronal sensors with nanoscale resolution, which may ultimately be used to detect magnetic and electrical activity at the membrane level in excitable cellular systems