From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy
Figure 1
Integrated methods for CLEM applications.
(A) Outline of the correlative light-electron microscopy (CLEM) methods on living cells. The micro-patterned culture substrates are useful to perform different types of CLEM, combining confocal microscopy with immuno-labeling on serial sections (transmission-EM), with EM tomography and with scanning-EM. (B) The culture substrates are prepared from aclar films [24] (1) on which a reference grid has been micro-patterned with a laser microdissection microscope. The patterns are cut as 1.4 mm discs with a punch (2) and mounted onto gold plated live cell carriers (3). (C) Cells are seeded on the montage (1) and cultured under normal conditions. For LM recording, the montage is installed on the rapid loader of the EMPACT-2 and placed on the adapted stage of an inverted microscope, allowing continuous perfusion or replacement of the medium (2). After high pressure freezing, freeze substitution and embedding, the carrier is removed from the block (3), leaving prints of the reference coordinates at the block face. Trimming is performed around the region containing the cell of interest that is cut serial and collected on EM grids. (D) COS-1 cells expressing GFP-MTM1 migrated for 7.5 h on the pre-patterned aclar grid coated with collagen, were fixed by high pressure freezing and processed as described above. Coordinates from the grid are still on the first sections, facilitating the retrieval of the previously visualized cell. Laminin and poly-L-lysin coating were successfully tested (not shown). From left to right: bright field, fluorescence and electron microscopy. Examples of different time points and the video are shown online (Fig. S2 and Video S1, online).