Single-molecule fluorescence characterization in native environment

Thomas P. Burghardt, Katalin Ajtai

Research output: Contribution to journalReview article

8 Scopus citations

Abstract

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.

Original languageEnglish (US)
Pages (from-to)159-167
Number of pages9
JournalBiophysical Reviews
Volume2
Issue number4
DOIs
StatePublished - Nov 9 2010

Keywords

  • Molecular crowding
  • Muscle fiber
  • Myosin
  • Myosin light chain
  • Photoactivatable probe
  • Single-molecule detection
  • Super-resolution
  • Total internal reflection fluorescence

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Molecular Biology

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