The increasing complexity of protein and organelle investigations in cell biology sometimes demands a combination of different imaging approaches. The CLEM method in particular couples technologies such as light and electron microscopy. The key aim is to display images obtained with a light microscope at electron microscopy level, i.e. to correlate dynamic events in living cells with ultrastructural and 3D information. Using LMD, a system of reference coordinates can be produced on the surface of a culture substrate to enable quick and precise location of labeled details during sample preparation. This process is impressively illustrated in Figure 1.
Fig. 1: Reference grid imprinted onto the cell culture substrate. (A) Scheme of the reference grid for the laser microdissection microscope. The patterned aclar substrate was imaged under an epifluorescence microscope using excitation filters BP 545/30 (B), BP 480/40 (C) and BP 360/40 (D). The grid fluorescence is much fainter than the GFP signal of tagged proteins. (E) The reference grid is visible in brightfield and by scanning electron microscopy (F). (G–I) Due to the melting of the aclar, positive and negative patterns are imprinted as shown by scanning EM (G). As a result, after polymerization, and removal of the culture substrate (H), the pattern appears as negative and positive marks leaving visible holes (I) on the first EM sections. (J) Pictures of the pre-patterned substrate mounted onto gold plated live cell carriers .
Bacteria analysis is performed using NanoSIMS (Nano Secondary Ion Mass Spectrometry) coupled with LMD and