Research Papers: Imaging

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

[+] Author Affiliations
Junjie Yao

Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States

Daria M. Shcherbakova

Albert Einstein College of Medicine, Gruss-Lipper Biophotonics Center, Department of Anatomy and Structural Biology, Bronx, New York 10461, United States

Chiye Li

Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States

Arie Krumholz

Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States

Ramon A. Lorca

Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, Missouri 63130, United States

Erin Reinl

Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, Missouri 63130, United States

Sarah K. England

Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, Missouri 63130, United States

Vladislav V. Verkhusha

Albert Einstein College of Medicine, Gruss-Lipper Biophotonics Center, Department of Anatomy and Structural Biology, Bronx, New York 10461, United States

Lihong V. Wang

Washington University, Department of Biomedical Engineering, St. Louis, Missouri 63130, United States

J. Biomed. Opt. 19(8), 086018 (Aug 21, 2014). doi:10.1117/1.JBO.19.8.086018
History: Received June 9, 2014; Revised July 31, 2014; Accepted July 31, 2014
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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.

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© 2014 Society of Photo-Optical Instrumentation Engineers

Citation

Junjie Yao ; Daria M. Shcherbakova ; Chiye Li ; Arie Krumholz ; Ramon A. Lorca, et al.
"Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast", J. Biomed. Opt. 19(8), 086018 (Aug 21, 2014). ; http://dx.doi.org/10.1117/1.JBO.19.8.086018


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