Plenary Session 2 – Imaging
8/31 4:00 - 6:00, Memorial Auditorium
How electron cryotomography will provide the ultrastructural information needed for spatially explicit systems analyses
California Institute of Technology. Howard Hughes Medical Institute.
Cells are not simply bags of enzymes, and in many cases the spatial arrangement of macromolecules is critical to their function. Electron cryotomography is a rapidly emerging technique that can produce 3-D images of intact cells in their native states to “molecular” resolution, thereby revealing some of these critical spatial arrangements. The power of this technique and its importance to systems biological studies will be illustrated through images and computational analyses of the ultrastructure of Halothiobacillus neapolitanus, a small bacterium that accelerates carbon fixation by packing RuBisCO molecules within proteinaceous microcompartments called carboxysomes.
Light Sheet Based Fluorescence Microscopes (LSFM, SPIM, DSLM) Reduce Phototoxic Effects by Several Orders of Magnitude
Ernst H. K. Stelzer
European Molecular Biology Laboratory (EMBL), Heidelberg.
Most biochemical compounds absorb light and suffer photo-toxic degradation inducing malfunction or death of a specimen. This is particularly dramatic in conventional/confocal fluorescence micro-scopy. Even though a single plane is observed, the entire specimen is illuminated. Recording stacks of images along the optical axis thus illuminates entire specimens once for each plane. Hence cells are illuminated 10-20 and fish embryos even 100-300 times more often than observed. This can be avoided by re-arranging the optics. We use light sheets, which are fed into the specimen from the side to overlap with the focal plane of a wide-field fluorescence microscope. Optical sectioning and no photo-toxic damage outside a small volume around the focal plane are intrinsic properties. Light sheet-based fluorescence microscopes (LSFM) use modern cameras with a signal to noise ratio that is at least thirty times better than in a confocal microscope. LSFM can be combined with essentially every contrast and specimen manipulation tool. Recently, LSFM were used to record zebrafish (Da-nio rerio) development in vivo and in toto from the 32-cell stage until late neurulation with sub-cellular resolution and 60-90sec sampling.
University of Washington.