From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy
Figure 3
Correlative light and electron microscopy of the needle-like structures.
COS-1 cells transfected with GFP-tagged MTM1 and plated onto collagen coated pre-patterned aclar grids were treated with hypo-osmotic shock, imaged by time-lapse confocal microscopy then fixed by high pressure freezing. (A–C) The reference coordinates are used to record the position of the selected cell with fluorescence (A), bright field microscopy (B) or both (C). (D) Representative images of the time-lapse video (Supplementary video 4, online), the image at 235 s was the last image before high pressure freezing. (E–F) Examples of a needle structure in immuno-EM labeling using anti-GFP antibody. Arrows in (D–F) point to the same structure. Needles are found associated with the plasma membrane. The cell position, its global shape, the position of the nucleus (N) and of a large vacuole (star) were used to confirm the identity of the cell and to perform the correlation. (G) Micrographs of consecutive sections from the same region as in F showing the distribution of the gold labeling (anti-GFP) at the plasma membrane.