Microscopy

Light-microscopy (LM) of fluorescentically labeled samples has cellular and sub-cellular resolution, allowing proteins to be localized within cells.

The information obtained by LM can be extended by Serial Blockface Imaging by electron microscopy (EM). For this the sample is embedded in resin and imaged in an FEI Quanta-200 scanning electron microscope (SEM; collaboration with the FMI) that has an in-microscope ultramicrotome. Slices are serially removed from the sample block using the microtome, and the new block face is imaged using the SEM. Together these images reveal the three-dimensional (3-D) structure of the resin-embedded sample at a resolution of 30 nm.

To increase the resolution, once areas of interest have been located, the room-temperature sample can be removed and mounted in a conventional ultramicrotome, where thicker (e.g. 100 to 400 nm) sections are cut from the block. These sections are then analyzed by 3-D electron tomography in a transmission electron microscope, e.g., the FEI Titan Krios.

The highest resolution can be achieved by cryo-electron tomography, single particle cryo-electron microscopy, or electron crystallography, for which the transmission electron microscopes of different acceleration voltages and beam qualities are available at C-CINA.