Fig. 5: EMcapsulin contrast in SEM and FIB-SEM of Drosophila neurons.
From: Genetically encoded barcodes for correlative volume electron microscopy
a,b, SEM and corresponding TEM micrographs of the identical sample from a Drosophila line with pan-neuronal expression of either 1M-QtFLAG-NLS (a) or 1M-MxFLAG-NLS (b) after a standard fixation and staining protocol. Ultrathin sections were captured either on TEM grids or on silica wafers for subsequent analysis by TEM and SEM (inverted contrast), allowing for the analysis of similar cuts through the identical cell with both techniques. SEM images were obtained from a Zeiss GeminiSEM with sense-BSD, Tandem Decel with 1.5 kV. Corresponding TEM images were acquired on a Zeiss Libra120 at 120 kV, 13 µA and 100 µrad. Insets show averages (n = 30) of the respective particle from manual segmentation. White arrowheads indicate the presence of EMcapsulin particles inside the nucleus. c,d, Isotropic FIB-SEM image volumes (4 nm voxel size) of Drosophila brains expressing 1M-QtFLAG-NLS (cyan) (c) and 1M-MxFLAG-NLS (red) (d) targeted to the nucleus. EMcapsulins and nuclear membranes were manually segmented and rendered within the FIB-SEM volume bounded by the ortho-slices. The magnifications (right) show ortho-slices through three EMcapsulins. Volume acquisition was performed with an SEM beam voltage of 1.3 kV and a working distance of 5 mm, at a nominal voxel size of 4 nm using an InLens detector. The FIB Ga beam was accelerated by 30 kV voltage at a current of 700 pA. Scale bars, 500 nm (overview) and 50 nm (zoom-ins). Please also see Supplementary Video 6.
