Fig. 1.

Detailed imaging workflow. a Preparation. To acquire the (correlative) LM/EM image datasets on the iLEM, first a tiled overview image is created using a 20x lens and transmitted light, then the stage coordinates are aligned to overview image pixel coordinates and a focus map is built. b Processing. Using image analysis tools, the ribbons are recognized and the sections detected. Additionally, a normalization procedure corrects for shape changes of the sections due to cutting and other factors. Using these processing steps, the coordinates of the ROI are predicted across the ribbons, with ROIs being first defined on the overview image, by selection in one section. c Automated navigation and acquisition. Using the designated coordinates image stacks are acquired. After acquisition, the stack is registered, and the correction used can be applied to update the coordinates of the ROI. This stepwise refinement can be repeated with each switch of magnification and modality (100x LM, 4kx, 12kx, etc.)