title: Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast.
abstract: Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells.
authors.affiliation: Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States.
authors.forename: Junjie
authors.initials: J
authors.lastname: Yao
authors.affiliation: Albert Einstein College of Medicine, Gruss-Lipper Biophotonics Center, Department of Anatomy and Structural Biology, Bronx, New York 10461, United States.
authors.forename: Daria M
authors.initials: DM
authors.lastname: Shcherbakova
authors.affiliation: Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States.
authors.forename: Chiye
authors.initials: C
authors.lastname: Li
authors.affiliation: Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States.
authors.forename: Arie
authors.initials: A
authors.lastname: Krumholz
authors.affiliation: Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, Missouri 63130, United States.
authors.forename: Ramon A
authors.initials: RA
authors.lastname: Lorca
authors.affiliation: Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, Missouri 63130, United States.
authors.forename: Erin
authors.initials: E
authors.lastname: Reinl
authors.affiliation: Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, Missouri 63130, United States.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: England
authors.affiliation: Albert Einstein College of Medicine, Gruss-Lipper Biophotonics Center, Department of Anatomy and Structural Biology, Bronx, New York 10461, United States.
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.affiliation: Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States.
authors.forename: Lihong V
authors.initials: LV
authors.lastname: Wang
mesh_terms: D000818:Animals; D007089:Image Enhancement / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D041681:NIH 3T3 Cells