Electron microscopy has the power to resolve even atomic details but is very limited with the field of view and sample thickness. Other imaging methods like light microscopy can image larger fields of view and easily detect fluorescently labelled areas, x-ray can even analyse bigger samples in larger volumes. Today’s efforts are focussed on combining the power of multiple imaging technologies to resolve cellular processes at high resolution.

 We were investigation with methods for correlative light and electron microscopy. First experiments were aimed at correlating fluorescent markers for light microscopy with colloidal gold particles for electron microscopy. These experiments once more confirmed the superiority of fluorescent labels over particulate markers. Sometimes the efficiency gap was so big that a clear, convincing label can be seen on the fluorescence micrograph whereas the gold label did not exceed background labelling. With this knowledge in mind we propose to use an overlay of super-resolution light microscopy data with high-resolution electron microscopy images to identify the cellular components containing the protein of interest.

 Next to correlative microscopy also volume microscopy has come of age. Today many methods are available: traditional serial sectioning, array tomography, serial block face SEM and focussed ion beam SEM.  

 Firstly, we setup focused ion beam scanning electron tomography. We succeeded in getting routinely volumes of 30 x 20 x 10 μm³ at a voxel resolution of 48.8 x 48.8 x 100 ų. Due to the current circumstances the sample preparation is restricted to classic chemical protocols. Here I will demonstrate a few projects where we use FIB-SEM tomography. It includes the study of endothelial cell layer penetration by pathogenic bacteria, ultrastructure of diatoms and the analysis of chromophore granules in the tail of Zebra fish.  

FIB tomography is suited for small volumes at high resolution. To access larger volumes serial block face SEM is the method of choice. Last but not least, array tomography offers the great advantage of easily combine localisation by fluorescence microscopy and high-resolution information gained by electron microscopy.