‘Nature loves to hide’. These elegant words, written by Heraclitus of Ephesus about 2500 years ago, can be interpreted in many ways. As researchers, we may recognise the great game of hide-and-seek that Nature loves to play. The development of new analytical tools has always been a key parameter to answer the many questions that arise in biology. Multiple and complementary analytical techniques are needed to unravel the mechanisms underlying complex biological processes and their pathological dysfunctions. This is particularly true for the study of metals in biology. The information about the sole localisation of the metal in tissues or cells is generally not enough to understand the molecular mechanisms of action. Ideally, metal localisation should be compared to the structural organisation of the cell or tissue, and/or to the localisation of biomolecules (proteins, nucleic acids, lipids…).

The main techniques for imaging metals in biological samples are based either on X-ray emission spectrometry, e.g., particle induced X-ray emission (PIXE) and synchrotron X-ray fluorescence (SXRF), on mass spectrometry (MS), e.g., laser ablation inductively coupled plasma MS (LA-ICP-MS) and secondary ion MS (SIMS), or on fluorescence microscopy of metal specific fluorescent probes (Table 1, Table 2). The characteristics of these imaging techniques in terms of sensitivity, spatial resolution and quantitative analysis as well as their capabilities to address biological questions have been exhaustively reviewed [1, 2, 3, 4, 5]. On the other hand, the imaging of biological molecules can be achieved by a variety of instruments available to characterise the biological tissues (Table 1, Table 2), from optical and fluorescence microscopy (e.g., histological staining, immuno-staining), electron microscopy, infrared microscopy, and mass spectrometry for organic compounds analysis.

Another important requirement to understand the effects of metals on biological processes is that different scales of observation are needed, from the tissue level down to the subcellular level. Depending on the nature of the studied samples, tissue sections or single cells, the sample preparation may differ drastically (Table 1, Table 2). In this article, we will review the last two years research dedicated to the imaging of chemical elements and their correlation with biological molecules or structures. We will first review the state-of-the-art for correlative imaging of biological tissues, as a continuation and update of the review article from Perry et al. [6] in this journal, and secondly we will present the correlative imaging at the subcellular level. As we will see, although some chemical element imaging techniques can be used over several length scales, there is actually a separation between the analysis of biological tissues from organ sections, which is generally performed at micrometer resolution (Table 1), and the analysis of isolated cells, which can be carried out at the nanometer scale (Table 2). This difference is mainly due to sample preparation methods, with the ability to preserve and identify chemical elements in subcellular compartments more easily from isolated cells in culture rather than from organ sections.