1. Image processing advances in X-ray Microscopy Carlos Óscar Sorzano National Center of Biotechnology (CSIC) Madrid 23/9/2010

2. Contents • Image formation model • Effects of the image formation model in 3D • X-ray taylored import • Image Processing GUI • Future

3. IMAGE FORMATION MODEL

4. IMAGE FORMATION MODEL

5. IMAGE FORMATION MODEL

6. IMAGE FORMATION MODEL

7. Contents • Image formation model • Effects of the image formation model in 3D • X-ray taylored import • Image Processing GUI • Future

8. TEST SPECIMEN Phantom simulation of a Candida albicans cell. Uchida et al. PNAS,106-46 (2009),p. 19375 Cell size : 5.00 x 3.75 x 3.75 mm (HxWxD) Slice in Sideview Slice in Top view Vacuole Mitochondrias Lipid droplets Nuclei Nucleoli 1 mm 1 mm

9. CHARACTERISTICS OF SIMULATED MICROSCOPES Simulations have been done for two Fresnel zone plate lenses.

10. RECONSTRUCTION FROM PERFECT PROJECTIONS Slices of the reconstruction from ideal projections, where defocus variations have not been considered, and then filtered to the X-ray microscope cut-off frequencies. Two sets of simulations have been done, with the tilt axis parallel and perpendicular to the larger cell axis. ParallelTilt Axis Cut off: 48.8 nm Cut off: 30.5 nm Perpendicular Tilt Axis

11. RECONSTRUCTION WITH SLICE IN FOCUS Reconstructed slices from simulated X-ray projections, where the observed slice has been placed in the focal plane of the microscope. Results have been computed for two orientations of tilt axis. 560 Zoneslens 900 Zoneslens ParallelTilt Axis Perpendicular Tilt Axis

12. RECONSTRUCTION WITH SLICE OUT OF FOCUS Reconstructed slices when the tilt axis does not intersect the focal plane. Results have been computed for several distances , two Fresnel lenses and two orientations of tilt axis. 560 Zoneslens 1 mm 2 mm 3 mm ParallelTilt Axis 900 Zoneslens

13. RECONSTRUCTION WITH SLICE OUT OF FOCUS Reconstructed slices when the tilt axis does not intersect the focal plane. Results have been computed for several distances , two Fresnel lenses and two orientations of tilt axis. 560 Zoneslens 1 mm 2 mm 3 mm Perpendicular Tilt Axis 900 Zoneslens

14. Contents • Image formation model • Effects of the image formation model in 3D • X-ray taylored import • Image Processing GUI • Future

15. X-ray taylored import • SPE format • Synchrotron parameters

16. Contents • Image formation model • Effects of the image formation model in 3D • X-ray taylored import • Image Processing GUI • Future

17. Image processing GUI

18. Image Processing GUI • Based on ImageJ • MRC stack load & save (non-blocking: users can interact with the plugin while remaining projections load) • User workflow is recorded (the basis for future Experiment Management) • Shows scaled tilt-series (to reduce resource usage and allow fast interaction)

19. Future Near future: • Incorporation of preprocessing tools like: • High-pass filter • Accurate downsampling • Removal of illumination fluctuations • Automatic Single tilt-series alignment • Automatic Dual tilt-axis alignment More in the future: • Manual alignment and refinement of the automatic alignment • Incorporation of the image formation model into the 3D reconstruction algorithm