CLEM of GFP-C1 and mCherry-H2B in HeLa cells using pre-embedding light microscopy. (A) Maximum intensity projection of a confocal microscopy image stack showing two cells expressing GFP-C1 and mCherry-H2B and an adjacent cell which is not transfected. Electron micrographs of the boxed regions are shown in panels 1–3. For each micrograph, the closest approximate slice from the confocal image stack has been used for overlaying the fluorescent signal onto the electron micrograph. (B, C) Electron micrographs showing localisation of the fluorescent signal to specific structures within the cell; mCherry-H2B is clearly localised to the nucleus, whereas GFP-C1 expression is strongly associated with the perinuclear region. GFP-C1 is less clearly localised to the nuclear envelope and endoplasmic reticulum. (D) Electron micrographs showing a similar perinuclear region within the adjacent cell, which is not transfected. C: centriole, ER: endoplasmic reticulum, G: Golgi, M: mitochondrion, N: nucleus, NE: nuclear envelope, TGN: trans-Golgi network. Scale bars—A: inset 1/2/3: 10 μm, B–D: 5 μm (inset 1/2: 1 μm).