Fig. 5.

Overview of the images acquired at one location with different magnifications and their combination/correlation. a 20x transmitted light image. The section is visible, and the red frame is indicating a region of interest containing one Golgi impregnated cell. b 100x LM transmitted light image of a Golgi impregnated neuron of the red area in a. c 4kx EM image of the region indicated by the red frame in b. d 12kx EM image of the red square in c. e 30kx image. Zoom to the region of the red square in d. f, g, h Overlay of b with c, d, and e, respectively (using ec-CLEM [11]). The LM information is displayed as a false-color image with red indicating the Golgi impregnation deposit in a and b to overlay and correlate it with the EM information. The figure illustrates how the stepwise refinement, by using the image registration information as feedback, at each magnification allows imaging the same location at different magnifications. This allows acquiring a pyramid of images at different magnifications that scales between more context and more detail in opposing directions. The images of the different magnifications and modalities can then be combined and correlated. Pixel sizes: 20x (2.96 [pixel/μm]) and 100x (15.38 [pixel/μm]) for the LM, 4kx (43.03 [pixel/μm]), 12kx (129.08 [pixel/μm]), and 30kx (322.69 [pixel/μm] for the EM. Values refer to the x,y-plane. Scale bars: a 20x = 200 μm; b 100x = 10 μm; c 4kx = 5 μm; d 12kx = 2 μm; e 30kx = 1 μm