DE Luca G M R, Desclos E, Breedijk R M P, Dolz-Edo L, Smits G J, Nahidiazar L, Bielefeld P, Picavet L, Fitzsimons C P, Hoebe R, Manders E M M
Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
J Microsc. 2017 May;266(2):166-177. doi: 10.1111/jmi.12526. Epub 2017 Mar 3.
The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The signal-to-noise ratio of Re-scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re-scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re-scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re-scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re-scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca measurements) and FRAP imaging.
新型高灵敏度和高分辨率技术——重新扫描共聚焦显微镜（RCM），是在标准共聚焦显微镜的基础上扩展了一个重新扫描检测单元。重新扫描单元包括一对重新扫描镜，它们以扫描方式将发射光投射到相机上。与标准共聚焦显微镜相比，重新扫描共聚焦显微镜的信噪比提高了4倍，其横向分辨率为170纳米（相比之下，对于488纳米激发、1.49数值孔径的衍射极限分辨率为240纳米）。除了提高灵敏度和分辨率外，重新扫描共聚焦显微镜的光学设置在镜子、激光器和滤光片的控制方面配置灵活。由于这种灵活性，重新扫描共聚焦显微镜可以配置用于特定的生物学应用。在本文中，我们探索了重新扫描共聚焦显微镜针对多色、荧光共振能量转移（FRET）、比率测量（例如pH和细胞内钙测量）以及光漂白荧光恢复（FRAP）成像等特定生物医学应用的多种可能配置。
