in-situ-detection-single-cell-quantification-metal-oxide

<em>In Situ</em> Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Université de Bordeaux

CNRS

Deciphering the molecular basis of toxicology mechanism induced by nanoparticles (NPs) remains an essential challenge. Ion Beam Analysis (IBA) was applied in combination with Transmission Electron Microscopy and Confocal Microscopy to analyze human keratinocytes exposed to TiO(2)-NPs. Investigating chemical elemental distributions using IBA gives rise to a fine quantification of the TiO(2)-NPs uptake within a cell and to the determination of the intracellular chemical modifications after TiO(2)-NPs internalization. In addition, fluorescent dye-modified TiO(2)-NPs have been synthesized to allow their detection, precise quantification and tracking in vitro. The internalization of these TiO(2)-NPs altered the calcium homeostasis and induced a decrease in cell proliferation associated with an early keratinocyte differentiation, without any indication of cell death. Additionally, the relation between the surface chemistry of the TiO(2)-NPs and their in vitro toxicity is clearly established and emphasizes the importance of the calcium homeostasis alteration in response to the presence of TiO(2)-NPs.

Titanium dioxide nanoparticles induced intracellular calcium homeostasis modification in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxicity.

The energy and specific energy absorbed in the main cell compartments (nucleus and cytoplasm) in typical radiobiology experiments are usually estimated by calculations as they are not accessible for a direct measurement. In most of the work, the cell geometry is modelled using the combination of simple mathematical volumes. We propose a method based on high resolution confocal imaging and ion beam analysis (IBA) in order to import realistic cell nuclei geometries in Monte-Carlo simulations and thus take into account the variety of different geometries encountered in a typical cell population. Seventy-six cell nuclei have been imaged using confocal microscopy and their chemical composition has been measured using IBA. A cellular phantom was created from these data using the ImageJ image analysis software and imported in the Geant4 Monte-Carlo simulation toolkit. Total energy and specific energy distributions in the 76 cell nuclei have been calculated for two types of irradiation protocols: a 3 MeV alpha particle microbeam used for targeted irradiation and a ²³⁹Pu alpha source used for large angle random irradiation. Qualitative images of the energy deposited along the particle tracks have been produced and show good agreement with images of DNA double strand break signalling proteins obtained experimentally. The methodology presented in this paper provides microdosimetric quantities calculated from realistic cellular volumes. It is based on open-source oriented software that is publicly available.

Monte-Carlo dosimetry on a realistic cell monolayer geometry exposed to alpha particles.

Assessing in situ nanoparticles (NPs) internalization at the level of a single cell is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of internalized NPs per cell. To in situ detect, track, and above all quantify NPs in a single cell, we propose an approach based on a multimodal correlative microscopy (MCM), via the complementarity of three imaging techniques: fluorescence microscopy (FM), scanning electron microscopy (SEM), and ion beam analysis (IBA). This MCM was performed on single targeted individual primary human foreskin keratinocytes (PHFK) cells cultured and maintained on a specifically designed sample holder, to probe either dye-modified or bare NPs. The data obtained by both FM and IBA on dye-modified NPs were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA techniques should therefore open a new field concerning specific studies on bare NPs and their toxicological impact on cells. Complementarity of SEM and IBA analyses provides surface (SEM) and in depth (IBA) information on the cell morphology as well as on the exact localization of the NPs. Finally, IBA not only provides in a single cell the in situ quantification of exogenous elements (NPs) but also that all the other endogenous elements and the subsequent variation of their homeostasis. This unique feature opens further insights in dose-dependent response analyses and adds the perspective of a better understanding of NPs behavior in biological specimens for toxicology or nanomedicine purposes.

Single cell in situ detection and quantification of metal oxide nanoparticles using multimodal correlative microscopy.

Charged-particle microbeams (CPMs) allow the targeting of sub-cellular compartments with a counted number of energetic ions. While initially developed in the late 1990s to overcome the statistical fluctuation on the number of traversals per cell inevitably associated with broad beam irradiations, CPMs have generated a growing interest and are now used in a wide range of radiation biology studies. Besides the study of the low-dose cellular response that has prevailed in the applications of these facilities for many years, several new topics have appeared recently. By combining their ability to generate highly clustered damages in a micrometric volume with immunostaining or live-cell GFP labelling, a huge potential for monitoring radiation-induced DNA damage and repair has been introduced. This type of studies has pushed end-stations towards advanced fluorescence microscopy techniques, and several microbeam lines are currently equipped with the state-of-the-art time-lapse fluorescence imaging microscopes. In addition, CPMs are nowadays also used to irradiate multicellular models in a highly controlled way. This review presents the latest developments and applications of charged-particle microbeams to radiation biology.

Advances in microbeam technologies and applications to radiation biology.

Although titanium dioxide nanoparticles (TiO2 NPs) have been extensively studied, their possible impact on health due to their specific properties supported by their size and geometry, remains to be fully characterized to support risk assessment. To further document NPs biological effects, we investigated the impact of TiO2 NPs morphology on biological outcomes. To this end, TiO2 NPs were synthesized as nanoneedles (NNs), titanate scrolled nanosheets (TNs), gel-sol-based isotropic nanoparticles (INPs) and tested for perturbation of cellular homeostasis (cellular ion content, cell proliferation, stress pathways) in three cell types and compared to the P25. We showed that TiO2 NPs were internalized at various degrees and their toxicity depended on both titanium content and NPs shape, which impacted on intracellular calcium homeostasis thereby leading to endoplasmic reticulum stress. Finally, we showed that a minimal intracellular content of TiO2 NPs was mandatory to induce toxicity enlightening once more the crucial notion of internalized dose threshold beside the well-recognized dose of exposure.

In situ quantification of diverse titanium dioxide nanoparticles unveils selective endoplasmic reticulum stress-dependent toxicity.

As well as being a significant source of environmental radiation exposure, α-particles are increasingly considered for use in targeted radiation therapy. A better understanding of α-particle induced damage at the DNA scale can be achieved by following their tracks in real-time in targeted living cells. Focused α-particle microbeams can facilitate this but, due to their low energy (up to a few MeV) and limited range, α-particles detection, delivery, and follow-up observations of radiation-induced damage remain difficult. In this study, we developed a thin Boron-doped Nano-Crystalline Diamond membrane that allows reliable single α-particles detection and single cell irradiation with negligible beam scattering. The radiation-induced responses of single 3 MeV α-particles delivered with focused microbeam are visualized in situ over thirty minutes after irradiation by the accumulation of the GFP-tagged RNF8 protein at DNA damaged sites.

Single α-particle irradiation permits real-time visualization of RNF8 accumulation at DNA damaged sites.

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 μm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria's response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

Article	Year
Titanium dioxide nanoparticles induced intracellular calcium homeostasis modification in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxici... Nanotoxicology\| PubMed ID: 21425910	2011
Monte-Carlo dosimetry on a realistic cell monolayer geometry exposed to alpha particles. Physics in medicine and biology\| PubMed ID: 22456322	2012
Single cell in situ detection and quantification of metal oxide nanoparticles using multimodal correlative microscopy. Analytical chemistry\| PubMed ID: 25006686	2014
Advances in microbeam technologies and applications to radiation biology. Radiation protection dosimetry\| PubMed ID: 25911406	2015
In situ quantification of diverse titanium dioxide nanoparticles unveils selective endoplasmic reticulum stress-dependent toxicity. Nanotoxicology\| PubMed ID: 28044465	2017
Single α-particle irradiation permits real-time visualization of RNF8 accumulation at DNA damaged sites. Scientific reports\| PubMed ID: 28139723	2017
Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential. Scientific reports\| PubMed ID: 28440317	2017

Philippe Barberet

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