Imaging of single-chromophore molecules in aqueous solution near a fused-silica interface

Lloyd M. Davis, Wesley C. Parker, David A. Ball, John G.K. Williams, Greg R. Bashford, Pamela Sheaff, Robert Eckles, Don T. Lamb, Lyle R. Middendorf

Research output: Contribution to journalArticlepeer-review


Single molecules of unconjugated Bodipy-Texas Red (BTR), BTR-dimer, and BTR conjugated to cysteine, in aqueous solutions are imaged using total-internal-reflection excitation and through-sample collection of fluorescence onto an intensified CCD camera, or a back-illuminated flame transfer CCD. The sample excitation is provided by the beam from a continuous-wave krypton ion laser, or a synchronously-pumped dye laser, operating at 568 nm. In order to essentially freeze molecular motion due to diffusion and thereby enhance image contrast, the laser beam is first passed through a mechanical shutter, which yields a 3-millisecond laser exposure for each camera frame. The laser beam strikes the fused-silica/sample interface at an angle exceeding the critical angle by about 1 degree. The resultant evanescent wave penetrates into the sample a depth of approximately 0.3 microns. Fluorescence from the thin plane of illumination is then imaged onto the camera by a water immersion apochromat (NA 1.2, WD 0.2 mm). A Raman notch filter blocks Rayleigh and specular laser scatter and a band-pass-filter blocks most Raman light scatter that originates from the solvent. Single molecules that have diffused into the evanescent zone at the time of laser exposure yield near-diffraction-limited Airy disk images with diameters of ∼5 pixels. While most molecules diffuse out of the evanescent zone before the next laser exposure, stationary or slowly moving molecules persisting over several frames, and blinking of such molecules, are occasionally observed.

Original languageEnglish (US)
Pages (from-to)301-311
Number of pages11
JournalProceedings of SPIE - The International Society for Optical Engineering
StatePublished - 2001
Externally publishedYes


  • Single-molecule detection
  • Single-molecule imaging
  • Single-molecule spectroscopy

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering


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