Figure 2
Workflow of correlative live cell imaging and scanning electron microscopy with gold mesh grids. (A) Live cell imaging (LCI) of STM infection of host cells on a mesh grid (red arrowhead) placed in a FluoroDish. The dish was transferred to adaptable mount on the motorized stage for spinning disc confocal microscopy (SDCM). The objective was positioned directly under the grid. Prior to defining imaging areas in the software, the lid was removed to avoid loss of defined coordinates by accidently touching. (B) Fluorescence image of Lifeact-eGFP MDCK cell monolayer grown on C + F film using a 10x objective. Distinct mesh holes were defined as region of interests (ROI) for LCI (yellow square). The asymmetric landmarks (white arrowheads) in the grid centre are essential for further relocalization. (C) Magnified ROI with 40x objective, where single cells within the monolayer were identified. A small notch in left corner of mesh area serves as visible landmark (white arrowhead). The corresponding image sequence recorded over 17 min is shown in Movie 1. (D) To prevent charging of biological samples during SEM imaging, grids with dehydrated monolayer cells were carbon coated by using a sputter device specified for thin carbon layer. If the sputter chamber provides enough space, grid samples are sputtered in their cell culture dishes. By this, the risk of collecting dust particles on cellular structures or losing entire grids is reduced. (E) After completion of carbon coating, samples appear darker. (F) The mesh grid was transferred to a grid carrousel holder (red arrowhead), providing several sample positions to sequentially image multiple grids. (G) For imaging of mesh grids with C + F film facing upwards, the STEM detector (bottom right) was used to identify orientation marks in grid centre. Therefore, the STEM detector was transferred from its parking position into the vacuum chamber, and the sample in carrousel holder was positioned manually over the detector. Scale bars, (B) 100 µm, (C) 20 µm.
