Fig. 4. Correlative fluorescence and electron spectroscopic imaging microscopy of heat shocked cells. (A) Fluorescence images of six serial physical sections were obtained by ultramicrotomy (thickness approximately 60 nm) of cells labeled by immunofluorescence against PML protein (yellow). The sections are numbered 1-6. The PML body remnant indicated by the arrow in the low magnification image of section 1 (left) is also represented in the zoomed and rotated image of the same section (right) (the zoomed image was rotated approximately 230°). The light blue structures are nucleoli. A single microstructure (a) is detected primarily in section 2 only. (B) Nitrogen map obtained by electron spectroscopic imaging of section 2 (in A). The upper PML body remnant indicated by the arrow is the same remnant indicated in A. Two nucleolar domains can be seen at the left and right of the field (upper panel). A second PML body remnant, below the one indicated by the arrow, is in the center of the box region. This region is shown at higher magnification in the lower panel, where the remnant is indicated by an arrowhead. (C) High magnification phosphorus (P) and nitrogen maps (N) of the PML body indicated by the arrowhead in the lower section of B. Comparison of the directions of the arrowheads in B and C indicate the relative rotation of the images in these panels. A microstructure labeled `a' in the merged P and N maps is the same microstructure referred to in the fluorescence image of section 2 in A. Microstructures were identified by triangulation by center-to-center measurements of PML-containing structures in the fluorescence images, relative to other fiduciary objects, such as nucleoli, with comparative measurements made with low, medium and high magnification electron spectroscopic images. Outlines of large open channels devoid of chromatin are shown in the phosphorus map, and a more concentrated region of chromatin fibers is indicated by am asterisk. A microstructure in the open region would be expected to be more mobile than one trapped in the region of higher chromatin fiber concentration. Scale bar (shown in B) represents 1100 (upper) and 700 (lower) nm in B, and 400 nm in C.