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).
Figure 2.
Dynamic needle-like structure formation.
(A) COS-1 cells were transfected with GFP or YFP-tagged wild-type myotubularin (MTM1) or close homologous proteins MTMR2 and MTMR4, either untreated or switched to a hypo-osmotic medium for 10 min, and imaged by confocal microscopy. Treatment does not change the subcellular localization of MTMR4 (right panel) and several other YFP-tagged proteins tested (not shown), while it promotes recruitment of MTMR2 (middle panel) to big vacuoles induced by the treatment, and the formation of needle-like structures by GFP-MTM1 (left panel). (B) The formation of needles is reversible for at least 3 times (two medium switches shown here). (C) The needles do not colocalize with actin (labeled with phalloidin-Texas Red).
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.
Figure 4.
Correlative light and scanning electron microscopy.
COS-1 cells transfected with GFP-tagged MTM1 were treated with a hypo-osmotic shock for 10 min, imaged with confocal microscopy and processed for scanning-EM. (A) Confocal fluorescence, (B) bright field and (C) scanning-EM images of a GFP-positive cell on top of the reference grid. (D) Enlargement of (C). (E) The superposition of confocal (z-stack of 3.52 µm) and scanning-EM images shows that the needles do not fit with filopodia but correspond to dorsal ruffles.
Figure 5.
COS-1 cells transfected with GFP-tagged BIN1 were imaged by confocal microscopy, fixed by high pressure freezing and processed for 60 nm thick serial sections. Sections were then labeled with anti-GFP antibody and gold particles. (A) Representative image (z projection of 3 confocal sections of 0.28 µm in depth) of the fluorescent BIN1 tubules radiating from/to the perinuclear region. (B) One over 30 serial thin sections showing the transmission-EM image of the same cell as in (A). (C) At higher magnification, the immunostaining is visible over various perinuclear structures highlighted in yellow. Other organelles such as mitochondria, Golgi complex, endosomal vesicles, that were unstained, are modeled in white. (D) When rendering the whole stack of serial sections, the immunogold labeling can be projected onto the representative transmission-EM picture, showing a good correlation with the dense perinuclear GFP signal recorded on living cells. (E and F) The model displays the distribution of the anti GFP immunogold staining in the volume of the perinuclear region, (G–J) The full z stack recording was processed with Imaris to reconstruct a 3D model (green) of the GFP fluorescence. The region analyzed by EM has been color-coded in yellow, showing a good correlation with the gold labeling displayed in D-F. (K–M) Correlation analysis of other z planes showed localization of GFP-BIN1 at plasma membrane invaginations. The confocal image shown in K corresponds to the Z projection of the five stacks shown in the Figure S6 (online). The EM micrograph of the corresponding thin sections as well as additional BIN1-positive membrane tubules and structures are shown in Figure S6 (online).
Figure 6.
CLEM and tomography reconstruction.
COS-1 cells transfected with GFP-BIN1 were imaged by confocal microscopy, fixed by high pressure freezing and processed to 200 nm thick sections. A tomogram was reconstructed from 139 tilted images (from −69 to 69°) and manually segmented to highlight the fluorescent tubules observed previously by confocal microscopy. (A) Representative confocal z-stack projection of a transfected cell, showing GFP-BIN1 tubules. Insert: magnified view of a tubule adjacent to the nucleus. (B) Corresponding transmission-EM picture. (C and D) Higher magnifications of the area selected in (B) showing a bundle of tubules (arrowhead) passing next to the cell nucleus, in the same region highlighted in (A). (E) Top view of the 3D model depicting membrane tubules in yellow, mitochondria, nuclear envelope, endosomal vesicles and lysosomes in grey, microtubules in red and actin filament in black. A 3D video of the tomogram is available (Video S8, online). L: lysosome; M: mitochondria; N: nucleus.