In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Giovanna Muggiolu; Marina Simon; Nathanael Lampe; Guillaume Devès; Philippe Barberet; Claire Michelet; Marie-Hélène Delville; Hervé Seznec

doi:10.3791/55041

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Method Article

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

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DOI:

10.3791/55041

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February 3rd, 2018

Giovanna Muggiolu^*¹^,² , Marina Simon^*¹^,² , Nathanael Lampe¹^,² , Guillaume Devès¹^,² , Philippe Barberet¹^,² , Claire Michelet¹^,² , Marie-Hélène Delville³^,⁴ , Hervé Seznec¹^,²

¹Centre d'Etudes Nucléaires Bordeaux Gradignan (CENBG), Université de Bordeaux, ²Centre d'Etudes Nucléaires Bordeaux Gradignan (CENBG), CNRS, ³Institut de Chimie de la Matière Condens é e de Bordeaux (ICMCB), CNRS, ⁴Institut de Chimie de la Matière Condens é e de Bordeaux (ICMCB), Université de Bordeaux

^* These authors contributed equally

Summary

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Abstract

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 µg.g^-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

Introduction

Cellular homeostasis is determined by the uptake control, assimilation, and intracellular localization of different trace elements (ions, metals, exogenous inorganic compounds). These components are frequently in the form of traces, but nevertheless may have a considerable impact in the system physiology. Thus, the study of cell biochemistry in both normal and pathological/stressed situations is a key-step towards an overall understanding of cellular metabolic mechanisms. Therefore, the development of imaging and analytical techniques enabling the investigation of intracellular chemical abundances, structural organization and their related metabolic functions becomes ....