Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Junjie Yao, Daria Shcherbakova, Chiye Li, Arie Krumholz, Ramon A. Lorca, Erin Reinl, Sarah K. England, Vladislav Verkhusha, Lihong V. Wang

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Article number086018
JournalJournal of Biomedical Optics
Volume19
Issue number8
DOIs
StatePublished - 2014

Fingerprint

Fluorescence microscopy
image contrast
Fluorophores
microscopy
Imaging techniques
fluorescence
Microscopic examination
Microscopes
Fluorescence
microscopes
Confocal microscopy
cells
luminous intensity
excitation
Optical microscopy
polynomials
Coloring Agents
Dyes
dyes
Polynomials

Keywords

  • contrast enhancement
  • reversibly switchable fluorophore
  • rsTagRFP
  • subdiffraction imaging

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials
  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics

Cite this

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast. / Yao, Junjie; Shcherbakova, Daria; Li, Chiye; Krumholz, Arie; Lorca, Ramon A.; Reinl, Erin; England, Sarah K.; Verkhusha, Vladislav; Wang, Lihong V.

In: Journal of Biomedical Optics, Vol. 19, No. 8, 086018, 2014.

Research output: Contribution to journalArticle

Yao, Junjie ; Shcherbakova, Daria ; Li, Chiye ; Krumholz, Arie ; Lorca, Ramon A. ; Reinl, Erin ; England, Sarah K. ; Verkhusha, Vladislav ; Wang, Lihong V. / Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast. In: Journal of Biomedical Optics. 2014 ; Vol. 19, No. 8.
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