Article pubs.acs.org/ac
Single Cell In Situ Detection and Quantification of Metal Oxide Nanoparticles Using Multimodal Correlative Microscopy Quentin Le Trequesser,†,‡,§,∥ Guillaume Devès,†,‡ Gladys Saez,†,‡ Laurent Daudin,†,‡ Philippe Barberet,†,‡ Claire Michelet,†,‡ Marie-Hélène Delville,*,§,∥ and Hervé Seznec*,†,‡ †
Université de Bordeaux, Centre d’Etudes Nucléaires Bordeaux Gradignan (CENBG), Chemin du Solarium, 33175 Gradignan, France ‡ CNRS, UMR5797, Centre d’Etudes Nucléaires Bordeaux Gradignan (CENBG), Chemin du Solarium, 33175 Gradignan, France § CNRS, UPR9048, Institut de Chimie de la Matière Condensée de Bordeaux (ICMCB), 87 Avenue du Dr. A. Schweitzer, Pessac, F-33608, France ∥ Université de Bordeaux, Institut de Chimie de la Matière Condensée de Bordeaux (ICMCB), 87 Avenue du Dr. A. Schweitzer, Pessac, F-33608, France ABSTRACT: 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.
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characteristics of NPs and, more especially, to assess the biological effects of these highly reactive materials.13,14 The behavior of NPs inside living cells or organisms and the specific metabolic responses that they induce are, to date, not fully understood. Furthermore, as pointed out in a recent review about NPs, nothing is clearer than the certainty that NPs enter the environment, but when, how, and where this happens is still unclear.15 Finally, analytical methods able to detect, track, as well as quantify these NPs are scarce and add to the trouble of understanding their impact and interactions with the environment and within living species.16−20 In this context, improving the NPs metrology, especially in biological specimens, appears crucial to assess their potential hazard to health. There is a real need for the development of analytical methods, which can detect and in situ quantify NPs, whatever their nature, size, and
he explosive growth in nanotechnology led to an increasing number of new nanomaterials (NMs) in order to create unique devices with novel targeted physical and chemical functional properties.1 Nanoparticles with sizes ranging from 1 to 100 nm in at least one dimension are currently widely manufactured and used in industry, because of their particular physical and chemical properties (high surface area, increased reactivity, and uncommon electric properties in the case of metallic or semiconductor NPs).2,3 Their size approaches that of proteins, DNA, and other biological species. They are used in bioapplications such as therapeutics,4 antimicrobial agents,5 and transfection vectors6 and for multimodal imaging labeling.7−10 However, the risks for adverse health effects due to prolonged or repetitive exposures at various concentration levels in biological species and in the environment have not yet been clearly established and results are scarce.11 Governmental agencies12 and several public authorities worldwide are concerned and have already notified that the current methods for chemical safety assessments have to be modified and reevaluated so as to address the particular © 2014 American Chemical Society
Received: November 19, 2013 Accepted: July 9, 2014 Published: July 9, 2014 7311
dx.doi.org/10.1021/ac501318c | Anal. Chem. 2014, 86, 7311−7319
Analytical Chemistry
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resin-embedding samples and sectionning required high resolution ultrastructure preservation
lyophilized sample required 10−100 μg/g
electron transmission TEM
1 nm
surface
