Booth et al., 2011 Booth D.G.

Hood F.E.

Prior I.A.

Royle S.J. A TACC3/ch-TOG/clathrin complex stabilises kinetochore fibres by inter-microtubule bridging. (A) Cells seeded onto gridded dishes (MatTek) are transfected to express fluorescent fusion proteins. Cells are imaged live or fixed for 1 hr with glutaraldehyde before being treated with dyes (for example, DAPI, to visualize DNA) or probed with antibodies as appropriate ().

(B) Both overview (20×) and high-magnification (100×) light microscopy (LM) images are acquired for the cells of interest. Coverslip coordinates were recorded using phase contrast, aiding location of target cells during later stages of the method.

(C) Samples were stained with osmium, tannic acid, uranyl acetate, and lead aspartate to generate contrast for electron microscopy, then dehydrated with a graded ethanol series before infiltration with resin. Samples were covered in 100% resin and cured at 60°C for 48 hr.

Booth et al., 2013 Booth D.G.

Cheeseman L.P.

Prior I.A.

Royle S.J. Studying kinetochore-fiber ultrastructure using correlative light-electron microscopy. (D) The sample was separated from the dish (), excess resin excised, and the remaining 1 × 1 mm block glued to a pin (left and central panels). Using the coordinates imprinted on the block face, the area of resin containing the cell of interest was fine-trimmed into a 100 × 100 μm block using an ultra-microtome (right panel) and coated with silver paint and gold palladium. A single cell can be observed (far right image).

(E) The sample was mounted into a Gatan 3View microtome, and the block face was repeatedly imaged during the removal of consecutive sections. This provides lossless acquisition in which the entire cell can be imaged and reconstructed. CLEM registration, merging both LM and EM data, was used to identify cells and structures of interest.

(F) Cells and structures of interest were annotated and segmented using Amira software (FEI), resulting in a nanometer resolution, three-dimensional model suitable for further geometric analysis. For preliminary tests, a DT40 chicken lymphoma cell was analyzed and modeled. From start to finish, this method requires ≥5.5 days, subject to the time dedicated to image annotation and data analysis. The typical resolution of the generated models is 12–24 × 12–24 × 60 nm in x, y, and z, respectively (60 nm was the thickness of the sections cut in the 3View).

Scale bars, 20 μm (B); 200 μm (D); 1 μm (F).