Improved localization precision via restricting confined biomolecule stochastic motion in single-molecule localization microscopy
Ni Jielei,
Cao Bo,
Niu Gang,
Chen Danni,
Liang Guotao,
Xia Tingying,
Li Heng,
Xu Chen,
Wang Jingyu,
Zhang Wanlong,
Zhang Yilin,
Yuan Xiaocong,
Ni Yanxiang
Affiliations
Ni Jielei
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Cao Bo
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Niu Gang
Phil Rivers Technology, Beijing100871, China
Chen Danni
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Liang Guotao
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Xia Tingying
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Li Heng
Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, Shenzhen518055, China
Xu Chen
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Wang Jingyu
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Zhang Wanlong
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Zhang Yilin
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Yuan Xiaocong
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Ni Yanxiang
Nanophotonics Research Center, Shenzhen Key Laboratory of Micro-Scale Optical Information Technology and Institute of Microscale Optoelectronics, College of Physics and Optoelectronic Engineering, College of Electronics and Information Engineering, Shenzhen University, Shenzhen518060, China
Single-molecule localization microscopy (SMLM) plays an irreplaceable role in biological studies, in which nanometer-sized biomolecules are hardly to be resolved due to diffraction limit unless being stochastically activated and accurately located by SMLM. For biological samples preimmobilized for SMLM, most biomolecules are cross-linked and constrained at their immobilizing sites but still expected to undergo confined stochastic motion in regard to their nanometer sizes. However, few lines of direct evidence have been reported about the detectability and influence of confined biomolecule stochastic motion on localization precision in SMLM. Here, we access the potential stochastic motion for each immobilized single biomolecule by calculating the displacements between any two of its localizations at different frames during sequential imaging of Alexa Fluor-647-conjugated oligonucleotides. For most molecules, localization displacements are remarkably larger at random frame intervals than at shortest intervals even after sample drift correction, increase with interval times and then saturate, showing that biomolecule stochastic motion is detected and confined around the immobilizing sizes in SMLM. Moreover, localization precision is inversely proportional to confined biomolecule stochastic motion, whereas it can be deteriorated or improved by enlarging the biomolecules or adding a post-crosslinking step, respectively. Consistently, post-crosslinking of cell samples sparsely stained for tubulin proteins results in a better localization precision. Overall, this study reveals that confined stochastic motion of immobilized biomolecules worsens localization precision in SMLM, and improved localization precision can be achieved via restricting such a motion.
