Correlative microscopy for elemental analysis by NanoSIMS and Electron microscopy: optimization of sample preparation methods

Electron microscopy (EM) and nano secondary ion mass spectrometry (NanoSIMS) aim to acquiring nanometric information, which also imply ultraresolution and therefore these techniques require the best preservation of samples. Analytical techniques such as X‐ray spectroscopy and NanoSIMS are able to identify, localize and quantify chemical elements both at the whole cell and at the intracellular level. These techniques can be coupled with biological structural analysis. The goal in sample preparation is to maintain chemical elements at their original localization site as well as at their physiological active site. Therefore, sample preparation has to prevent also delocalization of the biological molecules (e.g. proteins, lipids). In EM and NanoSIMS, samples are subjected to drastic conditions such as high vacuum and beam energy. Thus, due to the characteristics of these devices, analyses are incompatible with native biological systems. Moreover, these techniques require thin sections of samples (TEM/X‐EDS (70‐100nm) and NanoSIMS (200‐300nm)). The sample should not be destroyed too quickly under the beam or by vacuum sublimation and must be stable chemically. Diffusible elements are quickly lost during dehydration and embedding step used for the routine preparation of biological specimens and so these methods can only be used if it is already known that the elements of interest are tightly bound. Sample preparation for EM and NanoSIMS must immobilize the biological elements, must eliminate water and must allow sectioning of the sample. As generally accepted in the literature, cryofixation by high pressure freezing followed by cryosubstitution are the best methods to limit redistribution of metal ions. In general, the preparation of biological samples for TEM and NanoSIMS is rather similar. Therefore, both techniques can be easily applied together and in a complementary way for bioimaging. In this work, different biological sample preparation techniques will be presented. The goal is to compare several cryomethods like cryosubstitution with different resins or freeze‐drying. An advanced sample preparation protocol was developed basing on high pressure freezing cryofixation follow by cryosubstitution or freeze‐drying, in order to limit metal redistribution, and preparation of adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Best results were obtained by correlative imaging of a single cell by TEM and NanoSIMS combining the advantages of both techniques. As an illustration, the ultrastructure of a C. reinhardtii cell (Fig. 2) could be directly related to the spatial distribution of macro and trace elements present at basal levels in the cell (Figs. 1 and 3). Thus, metals could be localized in different cell organelles such as the pyrenoid and granules. For another example, epidermal cells on adherent culture (keratinocytes), localization of elements (e.g. Ni) demonstrates the importance of sample preparation. Correlative TEM and NanoSIMS shows potential for many future applications of subcellular imaging of trace elements in medicine and biology.