Combined use of X-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

C Gramaccioni,S Bohic,A Procopio,G Farruggia,L Massimi,M Fratini,S Iotti,P Valenti,E Malucelli,F Berlutti,A Notargiacomo,L Rosa,S Lagomarsino,A Cutone,A Pacureanu,Y Yang,P Cloetens

doi:10.1088/1742-6596/849/1/012008

Abstract

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging.

Highlights

Iron is an essential element for cell growth and proliferation being involved in fundamental processes, such as DNA replication and energy production
In inflammation and infection induced by lipopolysaccharide (LPS) or bacterial pathogens, the synthesis of ferroportin, the sole protein able to export iron from macrophages to blood, is inhibited hindering iron export, increasing intracellular iron, bacterial multiplication and inflammatory processes
The present work has two main aims: i) to demonstrate the possibility to quantitatively map the intracellular iron mass fraction and concentration at nanoscale spatial resolution in macrophages using combination of several techniques, i.e. X-ray Fluorescence Microscopy (XRFM), Atomic Force Microscopy (AFM) and Phase Contrast Imaging (PCI); ii) to compare the iron maps of the cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf

Summary

Introduction

Iron is an essential element for cell growth and proliferation being involved in fundamental processes, such as DNA replication and energy production. The present work has two main aims: i) to demonstrate the possibility to quantitatively map the intracellular iron mass fraction and concentration at nanoscale spatial resolution in macrophages using combination of several techniques, i.e. X-ray Fluorescence Microscopy (XRFM), Atomic Force Microscopy (AFM) and Phase Contrast Imaging (PCI); ii) to compare the iron maps of the cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf (bLf).

Results

Conclusion