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Correlative Video-Light-Electron Microscopy

A Detailed Protocol with Useful Tricks

Nov. 09, 2009
Figure 1. Identification of the cell of interest.  (A). Growing of cells on the cover slip with gridded cover slip. (B) Growing of cells on the cover slip without coordinated grid. It is possible to use the peculiar pattern of cell position for the identification of our cell. For instance in the upper-right-upper right position there are two elongated cells (grey elongated profiles) and the cell of interest (dark profile) could be identified between them. (C, D) Growing of cells on the cover slip without coordinated grid. To label the cell of interest after fixation one should take a wooden stick and make a ring (between two ovals) without cells by their scrapping around our cell (grey asterisk). After scrapping the cell of interest (arrow in D) is easily identified
Figure 1. Identification of the cell of interest. (A). Growing of cells on the cover slip with ... more
Figure 1. Identification of the cell of interest.  (A). Growing of cells on the cover slip with ... Figure 2. Orientation of samples for sectioning. (A) The sample map after embedding. The cell of ... Figure 3. Picking up of serial sections with the donor slot grid from the water.  (A) Left. Serial ... Figure 4. In vivo dynamics and ultrastructure of individual ER-to-Golgi carriers studied using ... 

2.5. Sample Orientation and EM Sectioning from the Very First Section (see Note 6)

2.5.1. Place the sample under the transmission light microscope.
2.5.2. Find the cell of interest among the cells within the sample according to the coordinated grid (Fig. 1A) or pattern of the cell layer (Fig. 1B).
2.5.3. Put the resin block into the holder of an ultratome and examine it under a stereomicroscope.
2.5.4. Place the holder with the sample under the stereomicroscope and using a steel needle and rotating the sample in the holder, make two small cavities in such a way that they form a horizontal line (broken line in Fig. 2A and C) with our cell appearing in the center of the sample.
2.5.5. Introduce the holder into the ultratome in such a way that the segment arc of the ultratome is in the vertical position and the two cavities form a horizontal line.
2.5.6. By rotating the glass knife stage align the bottom edge of the pyramid parallel to the knife-edge. Using the segment arc, orient the plane of the sample vertically.
2.5.7. Bring the sample as close as possible towards the glass knife.
2.5.8. Adjust the gap (which is visible as a bright band if all three of the lamps of an ultratome are switched on) between the knife-edge and the surface of the sample. The gap has to be identical in width between the most upper and lower edges of the sample during the up and down movement of the resin block. This ascertains that every point of the sample surface containing the cell of interest is at the same distance from the knife-edge.
2.5.9. Slowly moving the sample up and down, continue its approach until the knife begins to cut one of the edges of the sample. The sectioning begins from either the upper or the lower part of the sample, the middle part of the sample where the cell of interest is situated will be unaffected because the length of the radius passing through the cell is shorter than the radii passing through the upper and the lower edges of the sample.

2.5.10. If the sectioning is to begin from the upper part of the sample, tilt the segment arc to approach the lower edge towards the knife. If the sectioning is to begin from the lower edge of the sample, tilt the segment arc and approach the upper edge of the sample towards the knife. A vertically oriented sample should produce equal sections from both the upper and lower edges of the samples (Fig. 2C).
2.5.11. Note down precisely all of parameters relating to the position of the sample in the ultratome, i.e. the degree of rotation of the sample in the holder, the degree of tilting of the segment arc, and the degree of rotation of the knife in its stage. Do not take the sample from the holder and do not rotate the sample inside the holder.
2.5.12. Take the sample and trim it to provide a narrow horizontal pyramid. The pyramid should be as narrow as possible, and the cell of interest should be at the centre of the pyramid. The length of the pyramid should be smaller than 0.9 mm and its height smaller than 0.1 mm. In this case you could place on the slot 18 serial sections. An experienced person can trim a pyramid directly with a razor blade.
2.5.13. Introduce the sample back into the ultratome, and lock it in exactly the same position as before (preserving all of the parameters of sample positioning; this is very important).
2.5.14. Replace the glass knife with the diamond one, and position the latter towards the pyramid. If the sample is not parallel to the knife, adjust the angle of the diamond knife by rotating the knife stage to make its edge parallel to the plane of the pyramid. Do not change any other parameters of the sample position.
2.5.15. Approach the sample towards the edge of the knife until the gap is extremely narrow. Using a 200-nm approaching step, begin the sectioning. Take serial sections according to the instructions with the ultratome. It is enough to take only 10 sections to pass 2 µm from the bottom of the cell.
2.5.16. Identify the position of the organelle of interest within the EM sample according to the Z-stacking, and select those thick sections that should correspond to this position. For instance, if the organelle of interest is situated at 500 nm from the surface of the sample (bottom of the cell), it is enough to collect only the first three 200-nm serial sections (or ten 50-nm sections). If the position is at 1 µm in height, it will be necessary to collect from the fourth to the eighth serial 200-nm sections (or twenty 50-nm sections).

2.6. Picking up the Serial Sections with the Empty Slot Grid (Fig. 3, see Note 7)

2.6.1. Clean the empty (not covered) slot grid (the donor transfer grid) and one slot grid covered with formvar-carbon film (the acceptor grid.
2.6.2. When the normal (not bent) slot grid is taken by the tweezer it is not convenient to take sections from the water, because the axes of grid cannot be oriented paralleling to the plane of water surface. One needs a holder on the grid. The holder on the slot grid could be done by bending just the small part of the grid from horizontal position. Using a tweezer bend just the small part of the edge of the empty slot grid from horizontal position. This bent rim will serve as a holder for the tweezer.
2.6.3. Take the slot grid with supporting film from a container by the self-closing tweezer and place it film-down on parallel holders covered with scotch tape (Fig. 3C, below).
2.6.4. Take the empty slot grid using the just made holder and place it over the band of serial sections not touching the water and in such a way that the slot is projected on the band of serial section.
2.6.5. Touch water with the donor grid in such a way that sections should be inside the slot and move the slot grid away. The droplet of water and sections will be inside the slot (Fig. 3A-C).
2.6.6. To avoid dirt on the sections it is better to place a small droplet of glass handled distilled water on the acceptor slot grid. Using stereomicroscope put a small droplet of water on the supporting film of the acceptor grid (Fig. 3D).
2.6.7. Put the donor grid with serial sections on acceptor grid as it is shown in Fig. 3E, F.
2.6.8. Using sharp and very narrow filter paper orient slot in parallel to each other and eliminate the excess of water from the space between slot grids (Fig. 3G).
2.6.9. Dry grids during at least 20 min.
2.6.10. Carefully eliminate the donor grid (Fig. 3H).
2.6.11. Take the dried acceptor grid and check whether sections are in correct position (Fig. 3I).

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Keywords: Beznoussenko complementary microscope technologies CVLEM Electron Microscopy EM live EM live imaging Mironov protocol EM Video-Light Microscopy

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