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Mol Brain. 2018 Mar 15;11(1):17. doi: 10.1186/s13041-018-0361-z.

Superresolution fluorescence microscopy for 3D reconstruction of thick samples.

Park S1,2,3, Kang W1,2,3, Kwon YD3,4, Shim J5, Kim S5, Kaang BK5, Hohng S6,7,8.

Author information

1
Department of Physics and Astronomy, Seoul National University, Seoul, Republic of Korea.
2
Institute of Applied Physics, Seoul National University, Seoul, Republic of Korea.
3
National Center of Creative Research Initiatives, Seoul National University, Seoul, Republic of Korea.
4
Research Institute of Basic Sciences, Seoul National University, Seoul, Republic of Korea.
5
School of Biological Sciences, Seoul National University, Seoul, South Korea.
6
Department of Physics and Astronomy, Seoul National University, Seoul, Republic of Korea. shohng@snu.ac.kr.
7
Institute of Applied Physics, Seoul National University, Seoul, Republic of Korea. shohng@snu.ac.kr.
8
National Center of Creative Research Initiatives, Seoul National University, Seoul, Republic of Korea. shohng@snu.ac.kr.

Abstract

Three-dimensional (3D) reconstruction of thick samples using superresolution fluorescence microscopy remains challenging due to high level of background noise and fast photobleaching of fluorescence probes. We develop superresolution fluorescence microscopy that can reconstruct 3D structures of thick samples with both high localization accuracy and no photobleaching problem. The background noise is reduced by optically sectioning the sample using line-scan confocal microscopy, and the photobleaching problem is overcome by using the DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). As demonstrations, we take 3D superresolution images of microtubules of a whole cell, and two-color 3D images of microtubules and mitochondria. We also present superresolution images of chemical synapse of a mouse brain section at different z-positions ranging from 0 μm to 100 μm.

KEYWORDS:

DNA-PAINT; Line-scan confocal microscopy; Single-molecule localization microscopy; Superresolution microscopy; Three-dimensional reconstruction

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