The understanding at an atomic level of a broad range of cellular functions is a vital and strategic goal in biology. In many instances, these functions are performed by large macromolecular complexes, which have to be understood in a cellular context for a range of resolutions. We report on two interrelated lines of research aimed at achieving such understanding.
1.Three-dimensional electron microscopy (3D-EM) with defocus variation correction. The microscope
depth of field is a resolution limiting factor in 3D-EM, specially for larger objects. We
propose new techniques that can effectively eliminate the depth of field limitation. They are applicable for all data collection geometries used in 3D-EM. In addition to their interest for 3D-EM, the methods are also needed for reconstruction from soft X-ray images, since in that case the changes in focus over a specimen of many microns (a whole cell) are critically important.
2.Reconstruction from soft X-ray projections. Soft X-ray tomography has become a reality in the
last years. The range of resolution of this technique fits between confocal and electron microscopies and will play a key role in the elucidation of the organization between the molecular and the organelle levels. Due to the quite complex nature of this imaging process, development of algorithms capable of 3D reconstruction from soft X-ray projections is a challenging task that we propose to solve by novel methods under development by us.
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