Neeraj Prabhakar

Cryogenic correlative light and electron microscopy (cryo-CLEM) combines specific fluorescence labelling of proteins inside cells with structural information at the angstrom-level. The introduction of super-resolution fluorescence methods in the field of cryogenic fluorescence microscopy is a necessary step to bridge the large resolution gap between the different imaging modalities. However, there are many challenges hindering the full potential of cryogenic super-resolution correlative light and electron microscopy and seamless integration with structural cell biology. One of the main limiting factors is a lack of dedicated cryogenic fluorescence microscopy systems with sufficient mechanical stability to enable the collection of high-quality super-resolution data and full compatibility with vitrified specimens for cryo-electron tomography. Here, we address this by developing a vacuum-free ultra-stable cryogenic optical microscope (VULCROM). VULCROM is a dedicated super-resolution cryo-CLEM (cryo-SR-CLEM) setup that combines the stability of a vacuum-insulated cryostat with the flexibility and modularity of an open microscopy system. We demonstrate that VULCROM enables detailed investigations of single-molecule cryo-photo-physics across timescales spanning milliseconds to hours. We furthermore demonstrate its suitability for routine cryo-SR-CLEM with a resolution in the 10 nm range in distinct vitrified biological specimen types. We resolve the nanoscale architecture of YFP-labelled PML bodies within the nucleus of mammalian cells and the distribution of ATG9-eGFP in its cellular structural context in a cryo-lift-out lamella of N. benthamiana plant tissue. Owing to its vacuum-free design, VULCROM can be readily adapted for diverse correlative workflows and other cryo-light microscopy applications.

Microfluidics has become a popular method for constructing nanosystems in recent years, but it can also be used to coat other materials with polymeric layers. The polymeric coating may serve as a diffusion barrier against hydrophilic compounds, a responsive layer for controlled release, or a functional layer introduced to a nanocomposite for achieving the desired surface chemistry. In this study, mesoporous silica nanoparticles (MSNs) with enlarged pores were synthesized to achieve high protein loading combined with high protein retention within the MSN system with the aid of a microfluidic coating. Thus, MSNs were first coated with a cationic polyelectrolyte, poly (diallyldimethylammonium chloride) (PDDMA), and to potentially further control the protein release, a second coating of a pH-sensitive polymer (spermine-modified acetylated dextran, SpAcDEX) was deposited by a designed microfluidic device. The protective PDDMA layer was first formed under aqueous conditions, whereby the bioactivity of the protein could be maintained. The second coating polymer, SpAcDEX, was preferred to provide pH-sensitive protein release in the intracellular environment. The optimized formulation was effectively taken up by the cells along with the loaded protein cargo. This proof-of-concept study thus demonstrated that the use of microfluidic technologies for the design of protein delivery systems has great potential in terms of creating multicomponent systems and preserving protein stability.

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood–brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets.

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to its high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. These resistant and aggressive sub-populations of GBs including the glioblastoma stem cells (GSCs) can circumvent treatment. GSCs act as a reservoir of cancer-initiating cells; they are a major challenge for successful therapy. We have discovered, as opposed to well-reported anti-cancer drug based therapeutical approach for GB therapy, the role of polyethylenimine (PEI) in inducing selective death of patient-derived GSCs via lysosomal membrane rupturing. Even at very low doses (1 μg/ml), PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any additional anti-cancer drug, very potently and selectively killed multiple GSC lines. Very importantly, PEI-MSNs did not affect the survival of well-established GB cells, or other type of cancer cells even at 25x higher doses. Remarkably, any sign of predominant cell death pathways such as apoptosis and autophagy was absent. Instead, as a potential explanation for their GSC selective killing function, we demonstrate that the internalized PEI-MSNs accumulated inside the lysosomes, subsequently causing a rupture of the vulnerable lysosomal membranes, exclusively in GSCs. As a further evaluation, we observed blood-brain-barrier (BBB) permeability of these PEI-MSNs in vitro and in vivo . Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting, and GSC selectivity of the PEI-MSNs described here, the results suggest that PEI-functionalized nanoparticles could have a potential role in the eradication of GSCs.

Correlative light and electron microscopy (CLEM) is revolutionizing how cell samples are studied. CLEM provides a combination of the molecular and ultrastructural information about a cell. For the execution of CLEM experiments, multimodal fiducial landmarks are applied to precisely overlay light and electron microscopy images. Currently applied fiducials such as quantum dots and organic dye-labeled nanoparticles can be irreversibly quenched by electron beam exposure during electron microscopy. Generally, the sample is therefore investigated with a light microscope first and later with an electron microscope. A versatile fiducial landmark should offer to switch back from electron microscopy to light microscopy while preserving its fluorescent properties. Here, we evaluated green fluorescent and electron dense nanodiamonds for the execution of CLEM experiments and precisely correlated light microscopy and electron microscopy images. We demonstrated that green color emitting fluorescent nanodiamonds withstand electron beam exposure, harsh chemical treatments, heavy metal straining, and, importantly, their fluorescent properties remained intact for light microscopy.

Several bioactive compounds are in use for the treatment of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Historically, willow (salix sp.) bark has been an important source of salisylic acid and other natural compounds with anti-inflammatory, antipyretic and analgesic properties. Among these, picein isolated from hot water extract of willow bark, has been found to act as a natural secondary metabolite antioxidant. The aim of this study was to investigate the unrevealed pharmacological action of picein. In silico studies were utilized to direct the investigation towards the neuroprotection abilities of picein. Our in vitro studies demonstrate the neuroprotective properties of picein by blocking the oxidative stress effects, induced by free radical generator 2-methyl-1,4-naphthoquinone (menadione, MQ), in neuroblastoma SH-SY5Y cells. Several oxidative stress-related parameters were evaluated to measure the protection for mitochondrial integrity, such as mitochondrial superoxide production, mitochondrial activity (MTT), reactive oxygen species (ROS) and live-cell imaging. A significant increase in the ROS level and mitochondrial superoxide production were measured after MQ treatment, however, a subsequent treatment with picein was able to mitigate this effect by decreasing their levels. Additionally, the mitochondrial activity was significantly decreased by MQ exposure, but a follow-up treatment with picein recovered the normal metabolic activity. In conclusion, the presented results demonstrate that picein can significantly reduce the level of MQ-induced oxidative stress on mitochondria, and thereby plays a role as a potent neuroprotectant.

Three-dimensional correlative light and electron microscopy (3D CLEM) are attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. In order to perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FNDs is biocompatible and easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the 3D CLEM method by registering multi-modal datasets.

Rapid thermal annealing of electron irradiated nanoscale type Ib diamond particles facilitates formation of various nitrogen-related fluorescent color centers, providing either red, yellow, green, or blue fluorescence for downstream multiplex imaging applications.

The ultimate goal of any scientific development is to increase well-being and human health. Novel strategies are required for the achievement of safe and effective therapeutic treatments beyond the conventional ones, and society needs new requirements for new technologies, moving towards clean and green technology development. Green nanotechnology is a branch of green technology that utilizes the concepts of green chemistry and green engineering. It reduces the use of energy and fuel by using less material and renewable inputs wherever possible. Green nanotechnology, in phytoformulations, significantly contributes to environmental sustainability through the production of nanomaterials and nanoproducts, without causing harm to human health or the environment. The rationale behind the utilization of plants in nanoparticle formulations is that they are easily available and possess a broad variability of metabolites, such as vitamins, antioxidants, and nucleotides. For instance, gold (Au) nanoparticles have attracted substantial attention for their controllable size, shape, and surface properties. A variety of copper (Cu) and copper oxide (CuO) nanoparticles have also been synthesized from plant extracts. Titanium dioxide and zinc oxide nanoparticles are also important metal oxide nanomaterials that have been synthesized from a number of plant extracts. International and domestic laws, government and private-party programs, regulations and policies are being carefully reviewed and revised to increase their utility and nurture these nanoscale materials for commercialization. Inspiring debates and government initiatives are required to promote the sustainable use of nanoscale products. In this review, we will discuss the potential of the utilization of plant extracts in the advancement of nanotechnology.