Correlative transmission electron microscopy and high-resolution hard X-ray fluorescence microscopy of cell sections to measure trace elements concentrations at the organelle level

Abstract

Metals are essential to all forms of life and their concentration and distribution in the organisms are tightly regulated. Indeed, in their free form, metal ions are toxic. Therefore, an excess of physiologic metal ions or the uptake of non-physiologic metal ions can be highly detrimental for the organisms. It is thus fundamental to understand metals distribution and dynamics in physiologic or disrupted conditions, for instance in metal-related pathologies or upon environmental exposure to metals. Elemental imaging techniques can serve this purpose, by allowing the visualization and the quantification of metal species in a tissue or down to the interior of a cell. Among these techniques, synchrotron radiation-based X-ray fluorescence (SR-XRF) microscopy is the most sensitive to date, and great progresses were made to reach spatial resolutions as low as 20×20 nm2. Until recently, 2D XRF mapping was used on whole cells, thus summing up the signal from the whole thickness of the cell. In the last two years, we have developed a methodology to work on thin cell sections, in order to analyze the metal content at the level of the organelle. Herein, we propose a correlative method to couple SR-XRF to electron microscopy, with the aim to quantify the elemental content in an organelle of interest. As a proof-of-concept, the technique was applied to the analysis of mitochondria from hepatocytes exposed to silver nanoparticles. It was thus possible to identify mitochondria with higher concentration of Ag(I) ions compared to the surrounding cytosol. The versatility of the method makes it suitable to answer a large panel of biological questions, for instance related to metal homeostasis in biological organisms.

Imaging inorganic nanomaterial fate down to the organelle level

“…More recently, this correlative EM-XRF method on cell sections was pushed further and enabled the quantification of Ag(I) molecular species inside mitochondria, which was a challenge since mitochondria does not accumulate huge amounts of Ag. 41…”

Rhenium carbonyl complexes bearing methylated triphenylphosphonium cations as antibody-free mitochondria trackers for X-ray fluorescence imaging

“…18,19 Sealed carbon nanotubes filled with heavy metals and decorated with organelle-specific peptides were described for the XRF mapping of cell membrane, nucleus and endoplasmic reticulum. 20 Mitochondria localization, up to now, has only been identified by XRF using correlative imaging with fluorescence 21 or electron microscopy 22 or using an immunolabeling technique involving a secondary antibody functionalized with gold nanoparticles. 23,24 Hence, systems of easier access such as low molecular weight XRF imaging molecular probes with specific organelle targeting in the manner of conventional fluorophores, with an additional modality such as classical fluorescence for validation, would be highly valuable tools for colocalization studies and convenient access to the accurate localization of metal complexes by XRF.…”

Rhenium Carbonyl Complexes Conjugated with Methylated Triphenylphosphonium Cations as Sensitive Mitochondria Trackers for X-Ray Fluorescence Imaging

“…[18][19] Sealed carbon nanotubes filled with heavy metals and decorated with organelle-specific peptides were described for the XRF mapping of cell membrane, nucleus and endoplasmic reticulum. 20 Mitochondria localization, up to now, has only been identified by XRF using correlative imaging with fluorescence 21 or electron microscopy 22 or using an immunolabeling technique involving a secondary antibody functionalized with gold nanoparticles. [23][24] Hence, systems of easier access such as low molecular weight XRF imaging molecular probes with specific organelle targeting in the manner of conventional fluorophores, with an additional modality such as classical fluorescence for validation, would be highly valuable tools for colocalization studies and convenient access to the accurate localization of metal complexes by XRF.…”