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Register a new userNanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging
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Added by
Louis-Serge Bouchard
Publication date
2015
fields
Physics
and research's language is
English
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There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a subcellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable zooming-in to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery.
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This paper describes an x-ray microtomographic technique for imaging the three-dimensional structure of the human cerebral cortex. Neurons in the brain constitute a neural circuit as a three-dimensional network. The brain tissue is composed of light elements that give little contrast in a hard x-ray transmission image. The contrast was enhanced by staining neural cells with metal compounds. The obtained structure revealed the microarchitecture of the gray and white matter regions of the frontal cortex, which is responsible for the higher brain functions.
Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly due to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 um wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging.
Next generation wound care technology capable of diagnosing wound parameters, promoting healthy cell growth and reducing pathogenic infections noninvasively will provide patients with an improved standard of care and an accelerated wound repair. Temperature is one of the indicating biomarkers specific to chronic wounds. This work reports a hybrid, multifunctional optical platform: nanodiamond-silk membranes as bioinspired dressings capable of temperature sensing and wound healing. The hybrid was fabricated through electrospinning and formed sub-micron fibrous membranes with high porosity. The silk fibres are capable of compensating for the lack of extracellular matrix at the wound site, supporting the wound healing. The negatively charged nitrogen vacancy (NV-) color centres in nanodiamonds (NDs) exhibit optically detected magnetic resonance (ODMR) properties and act as fluorescent nanoscale thermometers, capable of sensing temperature variations associated to the presence of infection or inflammation in a wound, without physically removing the dressing. Our results show that the presence of NDs in the hybrid ND-silk membranes improve the thermal stability of silk fibres. The NV- color centres in NDs embedded in silk fibres exhibit well-retained fluorescent and ODMR. Using the NV- centres as fluorescent nanoscale thermometers, we achieved temperature sensing at a range of temperatures, including the biologically relevant temperature window, on cell-cultured ND-silk membranes. Enhancement in the temperature sensitivity of the NV- centres was observed for the hybrids. The membranes were further tested in vivo in a murine wound healing model and demonstrated biocompatibility and equivalent wound closure rates as the control wounds. Additionally, the hybrid ND-silk membranes showed selective antifouling and biocidal propensity toward Gram-negative Pseudomonas aeruginosa and Escherichia coli.
Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.
Optical bio-markers 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 NV centre in nanodiamond has emerged as a promising candidate for bio-imaging and bio-sensing with low cytotoxicity and stable photoluminescence. Here we study the electrophysiological effects of this quantum probe in primary cortical neurons. Multi-electrode array (MEA) 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 hr. 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 multi-functional intraneuronal sensors with nanoscale resolution, that may ultimately be used to detect magnetic and electrical activity at the membrane level in excitable cellular systems.
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