Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

Togo Shimozawa, Kazuo Yamagata, Takefumi Kondo, Shigeo Hayashi, Atsunori Shitamukai, Daijiro Konno, Fumio Matsuzaki, Jun Takayama, Shuichi Onami, Hiroshi Nakayama, Yasuhito Kosugi, Tomonobu M. Watanabe, Katsumasa Fujita, Yuko Mimori-Kiyosue

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45 Citations (Scopus)

Abstract

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron- size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

Original languageEnglish
Pages (from-to)3399-3404
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number9
DOIs
Publication statusPublished - Feb 26 2013

All Science Journal Classification (ASJC) codes

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    Shimozawa, T., Yamagata, K., Kondo, T., Hayashi, S., Shitamukai, A., Konno, D., Matsuzaki, F., Takayama, J., Onami, S., Nakayama, H., Kosugi, Y., Watanabe, T. M., Fujita, K., & Mimori-Kiyosue, Y. (2013). Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging. Proceedings of the National Academy of Sciences of the United States of America, 110(9), 3399-3404. https://doi.org/10.1073/pnas.1216696110