(E and F) Photobleaching behavior of H2B-mCherry and H2B-mEGFP in a resin block. in adult ovaries, and dissected larvae) and FS them with 0.1% UA in dry acetone (Table S1). In contrast to other reports (Peddie et al., 2014), the addition of water was not necessary to preserve fluorescence, even though we cannot rule out that water contamination, via PD0325901 condensation, could have been introduced together with the cold high-pressure frozen planchettes in the FS cocktail. After 72 h incubation at ?90C, the temperature was increased to allow the UA to stain the biological material. We found that an optimal concentration of UA in the sample (the best compromise between EM contrast and fluorescence preservation) was achieved by increasing the heat to ?45C at a velocity of 3C/h and then incubating the samples in the UA solution for an additional 5 h at ?45C. Compared with the original on-section CLEM protocols (e.g., Kukulski et al., 2011), the heat rise rate after the FS ?90C step was slower (3C/h vs. 5C/h). This was crucial in our hands to achieve satisfactory contrast with the samples we used. For instance, in ovaries, membranes appeared with negative contrast with a rate of 5C/h (not shown). The samples were then rinsed with real acetone before PD0325901 infiltration with the resin Lowicryl HM20. This sample preparation method preserved the fluorescence of the samples, especially for red FPs, including mCherry and DsRed. We could image fluorescence signals at a depth of several hundreds of microns within the resin block when scanning with a confocal microscope over the entire block (Fig. 1, A, E, K, and O). Moreover, this sample preparation was compatible with FIB-SEM acquisition. We could achieve good imaging and milling quality for large volumes (up to 80 m 60 m 80 m; Fig. 1, B, F, PD0325901 L, and P), with sufficient contrast to visualize subcellular structures, when imaging at 8- or 10-nm voxel size. For example, we were able to visualize not only membrane-bound organelles such as mitochondria (Fig. 1, C, I, and T; cristae visible in Fig. 1, C and I), the Golgi apparatus (Fig. 1, G and M), multivesicular bodies (MVBs; Fig. 1, H and S), and the ER (Fig. 1 R) but also membrane invaginations (Fig. 1 Q), nuclear pores (Fig. 1 N), centrioles (Fig. 1 J), microtubule bundles in the midbody (Fig. 1 D), and single microtubules (Fig. 1 E). Open in a separate window Physique 1. Sample preparation provides optimal fluorescence preservation and FIB-SEM imaging quality.(ACD) 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 usually orthogonal to the confocal PD0325901 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. (ECJ) Primary mammary gland organoids expressing H2B-mCherry (reddish colored). (E) Confocal picture acquired through the resin stop. In reddish colored, the mCherry sign, overlaid towards the bright-field picture. (F) Slice from the FIB-SEM level of the complete organoid demonstrated in E, obtained at 15-nm isotropic voxel size. (GCJ) High-magnification information on single-cell volumes obtained from additional organoids at 8-nm isotropic pixel size. In G, Golgi complicated; in H, MVBs, with noticeable solitary vesicles in the lumen; in I, a mitochondrion (asterisk) and a lot of money of cytoskeleton filaments (most likely microtubules, arrowhead); in J, a centrosome with both centrioles highlighted by arrowheads. (KCN) trachea terminal cell expressing cytoplasmic DsRed. (K) Confocal cut acquired through the resin stop. In green, autofluorescence from the cells (like the tracheal pipe). In reddish colored, DsRed, indicated by trachea cells specifically. The cell is indicated from the arrowhead shown in L. (L) Slice from the FIB-SEM level of a portion from the fluorescent cell demonstrated in K, obtained at 10-nm isotropic voxel size. (M and N) Information on the same quantity, displaying the Golgi equipment and mitochondria (M) and nuclear skin pores in top look at, in the nuclear envelope (N). (OCT) ovarian FCs, with clonal manifestation of CD8-mCherry and RNAi. (O) Confocal picture acquired through the resin stop. In reddish Rabbit Polyclonal to CSGLCAT colored, the Compact disc8-mCherry.