Abstract:
Structural biology may better be fused to micro-anatomy or histochemistry, where ultrastructures inside cells are observed mainly with transmission electron microscopes (TEMs). This fusion might be very productive since the relationship between structures and functions of protein molecules and organelles could be delineated in the living stage. To have the mating successful, however, a big obstacle in the current biological TEM has to be removed. That is harsh chemical treatments in the TEM sample preparation, which naturally induces the denaturation of protein molecules and organelles in various levels. No fixation and no staining are preferred to preserve functional strfuctures but such a method has not been well accepted because of the sample invisibility.
We have solved this dilemma by introducing phase contrast methods based on phase plates. To date two phase contrast methods, Zernike phase-contrast1) and Hilbert differential contrast2), have been developed and successfully applied to biological systems3, 4). Particularly the Hilbert differential conntrast is advantageous to visualize to biological samples such as viruses, bacteria and somatic cells, which are prepared for the cryo-TEM only with a rapid freezing without any further treatment. One of the examples is shown below where ultrastructures inside cyanobacterial cells are more distinctively visible in their intact form as compared with those obtained with two conventional methods3).

Related Publications:
1. R. Danev and K. Nagayama (2001) “Transmission Electron Microsocpy with Zernike Phase Plate”, Ultramicroscopy 88, 243-252.
2. R. Danev, H. Okawara, N. Usuda, K. Kametani and K. Nagayama (2002) “A Novel Phase-contrast Transmission Electron Microscopy Producing High-contrast Topographic Images of Weak Objects”, J. Biol. Phys. 28, 627-635.
3. Y. Kaneko, R. Danev, K. Nitta and K. Nagayama (2005) “In vivo subcellular ultrastructures recognized with Hilbert-differential-contrast transmission electron microscopy”, J. Elect. Microsc. 54, 79-85.
4. Y. Kaneko, R. Danev, K. Nagayama and H. Nakamoto (2006) “Carboxysomes in Living Cyanobacterial Cell Visualized by Hilbert Differential Contrast Transmission Electron Microscopy”, J.Bacterol. 188, 805-808.