Fluorescence imaging with two-photon evanescent wave excitation

Florian Schapper, José Tiago Gonçalves, Martin Oheim

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidinactin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.

Original languageEnglish (US)
Pages (from-to)635-643
Number of pages9
JournalEuropean Biophysics Journal
Issue number7
StatePublished - Oct 2003
Externally publishedYes


  • Evanescent wave excitation
  • Microscopy
  • Non-linear excitation
  • Total internal reflection fluorescence microscopy
  • Two-photon excitation fluorescence

ASJC Scopus subject areas

  • Biophysics


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