NIH Molecular Imaging Exploratory Center

Single-Molecule Fluorophores for Cellular Imaging





 W. E. Moerner, PI Department of Chemistry, Department of Applied Physics (courtesy), Molecular Imaging Program, and Biophysics Program, Stanford University
 Lucy Shapiro Department of Developmental Biology, Stanford University School of Medicine
 Harley McAdams Department of Developmental Biology, Stanford University
 Robert J. Twieg Department of Chemistry, Kent State University, Kent, OH

Sponsor:  NHGRI, Roadmap Initiative, National Institutes of Health, P20-HG003638


In this program, an interdisciplinary team of biological, chemical, and physical scientists from Stanford University and Kent State University will design, develop and apply unique fluorophores to explore protein localization in cells, down to the single-molecule level.  This will involve (i) design, synthesis, characterization and optimization of a new class of fluorescent tags for biological imaging, (ii) development of innovative cellular targeting technologies for these new fluorophores to determine location and co-location of proteins, and (iii) demonstration of methods for detection of coordinated of protein location (or mislocation in mutant strains) and gene expression to explore the regulatory function of spatial positioning in bacteria. 

The members of the research team have recently demonstrated optical detection of fluorescent single molecules in living cells using both extrinsic fluorescent lipid probes as well as green fluorescent protein mutant fusions.  In this program, the new fluorophores to be designed, synthesized, and applied are based on a class of fluorophores (the DCDHF molecules) with favorable fluorescence properties which already allow detection at the single-copy level.  As single-molecule labels, these molecules provide more than an order of magnitude improvement in photophysical properties compared to green fluorescent protein mutants, thus the ability to detect single molecules in cells should ultimately be improved by more than an order of magnitude.  In addition to single-molecule experiments, the new reporter fluorophores generated by this project will enable advances in time-lapse fluorescence microscopy by virtue of increased signal-to-noise and photobleaching resistance, properties that will allow lower concentrations of labels to be used in cellular fluorescence investigations.

Longer Abstract (March 2007)


Thrust Area Examples :

Single-Molecule Cellular Imaging

Some Projects :

Super-resolution Imaging in Live C. Crescentus Cells Using Photoswitchable EYFP

We have demonstrated that fluorescence imaging far beyond the optical diffraction limit can be performed in living cells using optical control of single molecules of the fluorescent protein EYFP fused to the bacterial actin, MreB. Bar, 1 micron

(Ref: Nature Methods (2008))

New Photoswitchable Single-Molecule Fluorophores for Imaging in Cells

We have synthesized a covalently linked Cy3-Cy5 dimer, and have demonstrated its photoswitching properties which enable superresolution imaging of cellular structures beyond the diffraction limit.

(Ref: J. Phys. Chem. B Lett. (2008))

New Photoswitchable Single-Molecule Fluorophores for Imaging in Cells

We have invented a new class of photoswitchable single-molecule emitters, which will be useful for superresolution imaging in cells.

(Ref: JACS (2008))

Single Molecule Motions of Oligoarginine Transporter Conjugates on the Plasma Membrane of CHO Cells

This work explores the interaction of polyarginine cell-penetrating peptides with the plasma membrane of CHO cells. SM tracking and diffusion analysis are enabled by the use of one of our new DCDHF fluorophores.

(Ref: JACS (2008))

Single Molecules of the Bacterial Actin MreB Undergo Directed Treadmilling in Bacterial Cells

This provides a superresolution image of the size, shape, and location of the MreB filaments!


(Ref: PNAS 103, 10929 (2006))

Recent Progress in DCDHF Single-Molecule Emitters: Overview

Detecting Single Oligonucleotides with DCDHF Self-Quenched Intermolecular H-Dimers (SQuIDs)

For more details, see Moerner Group Web Page