Multimodal correlative microscopy for in situ detection and quantification of chemical elements in biological specimens. Applications to nanotoxicology

Abstract

International audienceCorrelative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological and structural information that exceeds what is possible with any single technique to answer a biological question. We propose an approach based on a multimodal correlative microscopy, via two imaging and analytical techniques: fluorescence microscopy (FM) and ion beam analysis (IBA) to investigate in vitro nanoparticles (NPs) interactions. Indeed, the explosive growth in Nanotechnology has led to their utilization in a wide range of applications from therapeutics to multimodal imaging labeling. However, the risks for adverse health effects have not been clearly established. Detecting and tracking nanomaterials in biological systems are thus challenging and essential to understand the possible NPs-induced adverse effects. Indeed, assessing in situ NPs internalization at the single cell level 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 NPs internalized per cell. The data obtained by both FM and IBA were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA provides the in situ quantification not only of exogenous elements in a single cell but also of all the other endogenous elements and the subsequent variation in their cellular homeostasis. This unique property gives access to dose-dependent response analyses and therefore new perspectives for a better insight on the effect of metal oxide NPs on cellular homeostasis

The High Resolutive Detection of TiO2 Nanoparticles in Human Corneocytes via TEM/NanoSIMS Correlation

“…A large list of combinations includes the following: mass spectrometry imaging and magnetic resonance imaging [23]; multiphoton tomography with fluorescence lifetime imaging microscopy [24]; TEM and SEM/EDX (scanning electron microscopy/electron dispersive X-ray spectroscopy) [14]; stimulated emission depletion and SIMS [25]; TEM and confocal microscopy [26]; electron microscopy and SIMS [27][28][29][30][31][32][33][34][35]; or even more techniques, such as autoradiography, SIMS and TEM [36], TEM, fluorescence microscopy, and SIMS [37,38]; or EDX, electron energy loss spectroscopy, cathodoluminescence, and SIMS [39]. Such strategies have been used to study TiO 2 NPs using PIXE/RBS/STIM (particle-induced X-ray emission/Rutherford backscattering spectrometry/scanning transmission ion microscopy) [15,17,40,41] or PIXE/RBS/STIM with TEM [10,12], SEM [42], or autoradiography [13], or using secondary electron and mass spectral imaging in a gas field ion microscope [43][44][45].…”

Quantifying nanodiamonds biodistribution in whole cells with correlative iono-nanoscopy

“…In contrast to electrons, hard X-rays and mega-electron-volt (MeV) ions, such as helium ions, offer a significant advantage of being able to penetrate a whole cell with little broadening of the beam spot 8 , 9 . Fluorescence microscopic techniques have been previously combined with X-ray microscopy or ion beam analysis techniques to perform high-resolution correlative imaging 10 , 11 . However, these techniques are based on using dual beams to generate images independently and rely on post-image alignment, which might degrade the correlation accuracy.…”

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

“…None of them can provide a precise evaluation of the subcellular NPs content nor the NPs distribution without the use of fractionation methods. A systematic assessment of the dose-response is thus impossible with these methods, as opposed to methods based on atomic spectroscopy such as nuclear microprobe analysis 12,13 , synchrotron X-ray fluorescence microscopy 14 , and Secondary Ion Mass Spectrometry (SIMS). 15,16 These methods are particularly interesting as they complement observations made using fluorescence microscopy, especially when NPs cannot be labeled with fluorescent molecules and are thus studied in their native state.…”