Figure 1.
Sample preparation provides optimal fluorescence preservation and FIB-SEM imaging quality.(A–D) HeLa cells expressing H2B-mEGFP (green) or H2B-mCherry (red). (A) Confocal image of the resin block. (B) FIB-SEM slice of the dividing cells shown in A, acquired at 10-nm isotropic voxel size. Note that the imaging plane at the FIB-SEM is orthogonal to the confocal one. (C and D) High-resolution details of FIB-SEM acquisitions. In C, a group of mitochondria with visible cristae; in D, a midbody with cytoskeleton bundles. (E–J) Primary mammary gland organoids expressing H2B-mCherry (red). (E) Confocal image acquired from the resin block. In red, the mCherry signal, overlaid to the bright-field image. (F) Slice of the FIB-SEM volume of the entire organoid shown in E, acquired at 15-nm isotropic voxel size. (G–J) High-magnification details of single-cell volumes acquired from other organoids at 8-nm isotropic pixel size. In G, Golgi complex; in H, MVBs, with visible single vesicles in the lumen; in I, a mitochondrion (asterisk) and a bundle of cytoskeleton filaments (probably microtubules, arrowhead); in J, a centrosome with the two centrioles highlighted by arrowheads. (K–N)Drosophila trachea terminal cell expressing cytoplasmic DsRed. (K) Confocal slice acquired from the resin block. In green, autofluorescence of the tissue (including the tracheal tube). In red, DsRed, specifically expressed by trachea cells. The arrowhead indicates the cell shown in L. (L) Slice of the FIB-SEM volume of a portion of the fluorescent cell shown in K, acquired at 10-nm isotropic voxel size. (M and N) Details of the same volume, showing the Golgi apparatus and mitochondria (M) and nuclear pores in top view, at the nuclear envelope (N). (O–T)Drosophila ovarian FCs, with clonal expression of Dhc RNAi and CD8-mCherry. (O) Confocal image acquired from the resin block. In red, the CD8-mCherry signal, overlaid to the bright-field image. Oocyte and FCs are indicated. (P) Slice of the FIB-SEM volume of the same cell shown in O, acquired at 10-nm isotropic voxel size. (Q–T) Details of the same volume: in Q, invaginations of the oocyte plasma membrane; in R, area rich in ER cisternae in an FC; in S, MVBs; and in T, mitochondria.

Sample preparation provides optimal fluorescence preservation and FIB-SEM imaging quality. (A–D) HeLa cells expressing H2B-mEGFP (green) or H2B-mCherry (red). (A) Confocal image of the resin block. (B) FIB-SEM slice of the dividing cells shown in A, acquired at 10-nm isotropic voxel size. Note that the imaging plane at the FIB-SEM is orthogonal to the confocal one. (C and D) High-resolution details of FIB-SEM acquisitions. In C, a group of mitochondria with visible cristae; in D, a midbody with cytoskeleton bundles. (E–J) Primary mammary gland organoids expressing H2B-mCherry (red). (E) Confocal image acquired from the resin block. In red, the mCherry signal, overlaid to the bright-field image. (F) Slice of the FIB-SEM volume of the entire organoid shown in E, acquired at 15-nm isotropic voxel size. (G–J) High-magnification details of single-cell volumes acquired from other organoids at 8-nm isotropic pixel size. In G, Golgi complex; in H, MVBs, with visible single vesicles in the lumen; in I, a mitochondrion (asterisk) and a bundle of cytoskeleton filaments (probably microtubules, arrowhead); in J, a centrosome with the two centrioles highlighted by arrowheads. (K–N)Drosophila trachea terminal cell expressing cytoplasmic DsRed. (K) Confocal slice acquired from the resin block. In green, autofluorescence of the tissue (including the tracheal tube). In red, DsRed, specifically expressed by trachea cells. The arrowhead indicates the cell shown in L. (L) Slice of the FIB-SEM volume of a portion of the fluorescent cell shown in K, acquired at 10-nm isotropic voxel size. (M and N) Details of the same volume, showing the Golgi apparatus and mitochondria (M) and nuclear pores in top view, at the nuclear envelope (N). (O–T)Drosophila ovarian FCs, with clonal expression of Dhc RNAi and CD8-mCherry. (O) Confocal image acquired from the resin block. In red, the CD8-mCherry signal, overlaid to the bright-field image. Oocyte and FCs are indicated. (P) Slice of the FIB-SEM volume of the same cell shown in O, acquired at 10-nm isotropic voxel size. (Q–T) Details of the same volume: in Q, invaginations of the oocyte plasma membrane; in R, area rich in ER cisternae in an FC; in S, MVBs; and in T, mitochondria.

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