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Journal of Biomedical Optics

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast
Author(s): Junjie Yao; Daria M. Shcherbakova; Chiye Li; Arie Krumholz; Ramon A. Lorca; Erin Reinl; Sarah K. England; Vladislav V. Verkhusha; Lihong V. Wang
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Paper 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.

Paper Details

Date Published: 21 August 2014
PDF: 9 pages
J. Biomed. Opt. 19(8) 086018 doi: 10.1117/1.JBO.19.8.086018
Published in: Journal of Biomedical Optics Volume 19, Issue 8
Show Author Affiliations
Junjie Yao, Washington Univ. in St. Louis (United States)
Daria M. Shcherbakova, Albert Einstein College of Medicine of Yeshiva Univ. (United States)
Chiye Li, Washington Univ. in St. Louis (United States)
Arie Krumholz, Washington Univ. School of Medicine in St. Louis (United States)
Ramon A. Lorca, Washington Univ. School of Medicine in St. Louis (United States)
Erin Reinl, Washington Univ. School of Medicine in St. Louis (United States)
Sarah K. England, Washington Univ. School of Medicine in St. Louis (United States)
Vladislav V. Verkhusha, Albert Einstein College of Medicine of Yeshiva Univ. (United States)
Lihong V. Wang, Washington Univ. in St. Louis (United States)


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