Last update:
November 13, 2012

© University of Michigan



Toward high-resolution, high-sensitivity biomedical imaging
based on single-molecule fluorescence and nanophotonics

     By beating the diffraction limit that restricts traditional light microscopy, single-molecule fluorescence imaging is a flexible, noninvasive way to sensitively probe position and dynamics.

Our multidisciplinary efforts are focused on:
  • Applying super-resolution and ultrasensitive imaging to live bacteria cells
  • Improving single-molecule emission and resolution with particle plasmonics
  • Developing novel methods for imaging complex structures
Bacteria cell biology: Tackling problems of biomedical importance

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale. This allows us to unlock the mysteries of how biomolecules work together in cells. We have developed novel methods for single-molecule investigations and apply them to three prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each presents unique challenges, and we discuss the adaptations developed for each system, including a comparison of membrane and soluble proteins, extensions to two-color and 3D imaging, and live anaerobic cell studies.

Plasmon-enhanced fluorescence for single-molecule imaging

Upon resonance excitation of a noble metal nanoparticle (NP) much smaller than the incident wavelength, collective oscillations of the free electrons establish a strongly localized local surface plasmon mode, producing an enhanced field in the particle vicinity. Plasmonic metal NPs plasmon modes are central to many important optical effects, from surface-enhanced Raman spectroscopy (SERS) to photovoltaic concentrators, and in the Biteen Lab, we are particularly concerned with the effect of plasmon-enhanced fluorescence.

We have demonstrated enhanced emission from quantum dots, organic dyes, and fluorescent proteins, have used the enhancement effect to map the local field, and have begun to extend this work to membrane proteins in live cells. We are interested in exploring the fundamental optical properties of the nanoparticles as well as using this enhancement to improve the resolution of single-molecule fluorescence imaging.

Visualization of nano-features in complex structures
The Biteen Group is developing new techniques for imaging single fluorescent molecules with nanometer-scale resolution with applications ranging from biomolecules in live bacteria cells to the nanofluidic devices. Figure A – Single-Molecule Micelle-Assisted Blink (MAB) Microscopy allows super-resolution imaging in constrained geometries inaccessible by conventional super-resolution microscopy. The method, based on micelles and thiol-related photoswitching, is used to measure reversibly size-adjustable PDMS nanochannels in situ. A constant delivery of thiol is required to restore emission in reversibly non-fluorescent dye molecules, and transport of such reagents in the nanofluidic channels is permitted by SDS micelles. We use MAB microscopy to probe with better than 40-nm accuracy the nano-environment and reveal biologically relevant information about the sizes of the nanochannels. Figure B - The diffusion of individual Nile red molecules in three different crystalline microporous coordination polymers (MCPs) is visualized with single-molecule fluorescence microscopy. By localizing molecules with high spatial resolution, the trajectories of the diffusing dyes are reconstructed with nanometer-scale precision. A detailed analysis of these tracks reveals different dynamics and guest−host interactions in each crystal as well as distinct motion types within the same system, suggesting the presence of structural heterogeneities in local environments.
Our experiments rely on careful sample preparation, quantitative analysis,
and of course, lasers: