Abstract
Dynamic processes in cells are usually monitored by live cell fluorescence microscopy. Unfortunately, this method lacks the ultrastructural information about the structure of interest (SOI). Currently, electron microscopy (EM) is the best tool to achieve highest spatial resolution. In addition, correlative light and electron microscopy (CLEM) analysis of the same structure allows combining authentic live cell imaging with the resolution power of EM. Additionally the reference space of the SOI is revealed. Our CLEM analyses of HeLa cells allow tracing the morphology and dynamic behavior of intracellular micro-compartments in living cells and their ultrastructure and subcellular organization in a highly resolved manner.
Keywords: Sample preparation, Correlative, Electron microscopy, Eucaryotic cells, Protocol
Materials and Reagents
Equipment
Software
Procedure
Due to effort in preparation and limited shelf life the chemicals required have to be prepared at various times:
Scheme of procedure In the following the CLEM work flow from seeding cells to TEM imaging is illustrated. Figure 1. CLEM workflow. HeLa cells are seeded in a MatTek dish with a gridded coverslip. After one day, cells can be transfected or pulse-chased with fluid phase marker. The next day, cells are infected, imaged live and fixed as fast as possible on the microscope stage. The coverslip is removed from the dish and the sample is processed for TEM and flat-embedded in resin. During the removal of the coverslip the engraved coordinates are transferred to the resin surface and allows trimming around the ROI. Serial sections are transferred to EM grids and stained for contrast. The same ROI is imaged in TEM for overlaying with LM image.
Representative data
Figure 9. Examples of CLEM analyses of the intracellular lifestyle of Salmonella. HeLa cells expressing LAMP1-GFP (green) were seeded in Petri dishes with a gridded coverslip and infected with Salmonella expressing mCherry (STM, red). Live cell imaging was performed 8 h p.i. to visualize LAMP1-GFP-positive SIF (A. maximum intensity projection [MIP], C. single Z plane). Subsequently, the cells were fixed and processed for CLEM to reveal the ultrastructure of selected cells. Several low magnification images were stitched to visualize the cell morphology (B). Higher magnification images were used to align LM and TEM images (D). Note the formation of double membrane LAMP1-GFP-positive SIF. Details of a LAMP1-GFP-positive SIF (E) and an SCV linked to a SIF (F, G) are shown. G. Two additional ultrathin sections show the membrane organization of the SCV and SIF in corresponding to panel F. H. The inner and outer membrane of a SIF and SCV are outlined in orange and yellow, respectively. Light and dark red arrowheads indicate inner and outer membrane of the Salmonella cell envelope, respectively. Labels: S, Salmonella; M, mitochondria; iL, inner lumen; oL, outer lumen. A cell representative for 10 biological replicates is shown (1–3 technical replicates with each 2–4 cells). Scale bars: 10 µm (A, B), 2 µm (C, D), 500 nm (E, F, G).
Notes
Recipes
Acknowledgments
Work in our group was supported by the DFG through grant HE1964/18-1 and the Z project of SFB944 ‘Physiology and dynamics of cellular microcompartments’.
References
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