The Coral Life workflow provides a streamlined live-cell CLEM solution for getting insight about structural changes of cellular components over time.
Besides the technical handling described in the workflow manual, this article provides additional knowledge for a successful experimental run.
For this example, the main interest was the final separation of two mitotic cells, called abscission. After cytokinesis, the two dividing cells are only connected by an intercellular bridge which needs to be resolved. Because of its size, this mechanism can’t be fully understood by live-cell imaging. Therefore, the ultrastructural analysis is needed to understand the underlying mechanism of final cell separation.
The 6 mm sapphire disks need to be cleaned before the actual experiments. Cleaning is performed according to the following steps:
- In a fume hood place the sapphire disks in concentrated hydrochloric acid (HCl 30%) for 2 h.
- Wash the sapphire disks 3x for 5 min with doubly distilled (dd) H20.
- Let the sapphire disks dry on filter paper afterwards.
After the cleaning process, a finder grid pattern needs to be evaporated onto the clean sapphire disks. This step is critical! The pattern ensures easy retrieval of the sample position and regions of interests later in the resin block. Coating the sapphire with the pattern is performed as follows:
- Place a clean sapphire disk into each respective position of the coating mount and add a 6 mm finder mask on top (Fig. 1). The finder grid masks have a recess at the periphery which indicates the mask orientation. All masks shall have the same orientation to consistently indicate the correct orientation of the sapphires during the subsequent steps. It is recommended to place the finder grid in the orientation which produces readable letters.
- For evaporation of the mask, you can either use gold or carbon. Here, a carbon coating was applied with an EM ACE 600 carbon thread using pulse mode (double thread, 100 mm distance, 210 W, 150 ms pulse length). A carbon layer of 10 nm thickness was deposited onto
- Make sure to turn the rotation off. This will result in better clarity of the grid pattern.
- Depending on the coater and the application technique it may be necessary to apply a thicker carbon layer to obtain good visibility.
- If you use carbon, do not forget to stabilize the carbon layer at 180° C over night.
- The sapphires are now ready to be used for setting up the SampLink chambers as described in the workflow manual (Ref. Section 5.1). Make sure that the carbon layer faces the inside of the SampLink chamber. The cells need to be grown on top of the carbon layer.
Before seeding the cells into the assembled SampLink chambers, the chambers need to be cleaned again and sterilized. Perform the following steps in a cell culture hood:
- Fill the chamber with 1 ml 70% Ethanol and incubate for 5 min.
- Wash 3x with dd H2O.
- Let it dry overnight.
- UV sterilize it for at least 1 h.
The next day:
- Wash it 3x with PBS.
- Add 1 ml PBS or culture media and place the chamber in the incubator overnight.
- After incubation rinse it with dd H2O.
The chambers are now ready for use. Seed the cells either directly on the sapphire or on an additional polymer or polypeptide coating. Here, the sapphires were coated with Poly-L-Lysin (broid P1274, mol wt 70,000-150,000, Sigma-Aldrich) on top of the carbon grid pattern. Coating was performed with 0.1 mg/ml diluted in ddH2O. 50 μl were added onto each sapphire for 5 min, rinsed afterwards (3x ddH2O), and dried for 2 h in the hood.
For these experiments, a HeLa Kyoto HKF1, H2B-mCherry, alphaTubulin, mEGFP cell line cultivated and seeded in DMEM medium (10 %