Figure 6.

Correlative light and electron microscopy of isolated chromosomes. High-resolution correlative light microscopy, scanning electron microscopy and focused ion beam/SEM of specifically labeled spelt metaphase chromosomes, fixed onto carbon-coated slides. Chromosomes, immuno-labeled for α-tubulin, were selected with phase contrast combined with fluorescence (a1), re-located in SEM (a2) with secondary electrons (SE) and analyzed with both, SE- and backscattered electron (BSE)-signal for detection of FluoroNanogold^™ labels enhanced with silver (yellow) located at bundles of tubulin attached to the centromere (a3; merging of SE-image with the colored BSE image). Holocentric chromosomes of Luzula elegans were visualized with phase contrast (b1), and fluorescence of DAPI (b2), re-located in SEM (b3). The gold-labeled antibodies against a centromere-specific phosphorylated histone (anti-Histone-H2A) were localized within the centromeric groove (b4; merged SE-image and BSE image). For verification of label distribution, the specimen was carbon coated before FIB/SEM milling. Gold-labeled antibodies were detected on both sides of the chromosomes, predominantly located at the surface (b5; framed area). Barley metaphase chromosomes were fixed onto slides (c1; c2=framed area of c1) stained for DNA distribution with platinum blue and embedded in the water-soluble resin Moviol (c3; SE-image) to prevent shrinkage during dehydration in ethanol/acetone. FIB/SEM tomography (c4=BSE) shows the global Pt (DNA) distribution in three dimension (c5). C=centromere. A migrating human platelet collecting fibrin-trapped E. coli (d1) (orange=tdTomato; green=fibrin-Alexa-488-10 nm-gold). SEM micrograph of the platelet of (d1) after thin embedding and platinum deposition (d2). FIB-SEM section shows E. coli accumulating at the surface of the platelets (d3). A 3D-rendered FIB-SEM stack of the same platelet (d4) demonstrates the accumulation of the E. coli (orange) via fibrin (immuno-gold labeled=yellow).