Tuning of the Zernike phase-plate for visualization of detailed ultrastructure in complex biological specimens.
In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state.
Fukuda
Yoshiyuki
Y
Division of Nano-Structure Physiology, Okazaki Institute for Integrative Bioscience, Myodaiji, Okazaki, Japan.
Fukazawa
Yugo
Y
Danev
Radostin
R
Shigemoto
Ryuichi
R
Nagayama
Kuniaki
K
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002478:Cells, Cultured; D020285:Cryoelectron Microscopy / Q000379:methods*; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008858:Microscopy, Phase-Contrast / Q000379:methods*