Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.

Structure of crystallized particles in sputter-deposited amorphous germanium films

Pristine thin films of amorphous Ge prepared by sputtering are unstable and form coarse crystalline particles of 100 nm in size upon crystallization by electron irradiation. These crystalline particles exhibit unusual diffraction patterns that cannot be understood from the diamond cubic structure. The structure has previously been assumed to be a metastable hexagonal form. In the present work, the structure of the coarse crystalline particles has been analysed in detail by transmission electron microscopy, considering the possibility that those diffraction patterns might occur with the diamond cubic structure if the particle consists of thin twin layers. By high-resolution lattice imaging the particles have been shown to be of the diamond cubic structure containing a high density of twins and stacking faults parallel to {111}. With such defects, diffraction patterns can be complex because of the following effects: superposition of two or more diffraction patterns of the same structure but of different orientations, double diffraction through twin crystals, and streaks parallel to the thin crystal which give rise to extra diffraction spots. It is found that diffraction patterns taken from various orientations can be explained in terms of these effects.

Environmental TEM, A CTF Approach for Atmospheric Lattice Imaging

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.