RESUMO
We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow.
Assuntos
Cristalização/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas de Membrana/química , Proteínas de Membrana/ultraestruturaRESUMO
TEM image processing tools are devised for the assessment of 2D-crystallization experiments. The algorithms search for the presence and assess the quality of crystalline membranes. The retained scenario emulates the decisions of a microscopist in selecting targets and assessing the sample. Crystallinity is automatically assessed through the diffraction patterns of high magnification images acquired on pertinent regions selected at lower magnifications. Further algorithms have been developed for membrane characterization. Tests on images of different samples, acquired on different microscopes led to good results.