title: Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging.
abstract: Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Hanqing
authors.initials: H
authors.lastname: Xiong
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Zhenqiao
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Zhu
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Lv
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Anan
authors.initials: A
authors.lastname: Li
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Shiwei
authors.initials: S
authors.lastname: Li
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Longhui
authors.initials: L
authors.lastname: Li
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Siming
authors.initials: S
authors.lastname: Wang
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Zhongqin
authors.initials: Z
authors.lastname: Yang
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Tonghui
authors.initials: T
authors.lastname: Xu
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Qingming
authors.initials: Q
authors.lastname: Luo
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Gong
authors.affiliation: 1] Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China [2] Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Shaoqun
authors.initials: S
authors.lastname: Zeng
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002021:Buffers; D049452:Green Fluorescent Proteins; D006652:Histological Techniques / Q000379:methods*; D049408:Luminescent Agents; D008164:Luminescent Proteins; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D002021:Buffers; D049408:Luminescent Agents; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins