European Biophysics Journal

, Volume 32, Issue 7, pp 635–643 | Cite as

Fluorescence imaging with two-photon evanescent wave excitation

  • Florian Schapper
  • José Tiago Gonçalves
  • Martin Oheim


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-phalloidin-actin/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.


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



one-photon excited fluorescence


two-photon excited fluorescence


avalanche photo diode


Chinese hamster ovary


Dulbecco's modified Eagle's medium


enhanced green fluorescent protein


evanescent wave


fetal calf serum




total internal reflection



We thank T. Pons and G. Bunt for help with some experiments, J.-S. Schonn and C. Chapuis for chromaffin cell preparation and B. Babour for comments on the manuscript. Supported by the French Ministry of Research and Technology (M.N.R.E.T.) (ACI "jeunes chercheurs" no. 5242, to M.O.) and a Studienstiftung fellowship to F.S.


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Copyright information

© EBSA 2003

Authors and Affiliations

  • Florian Schapper
    • 1
  • José Tiago Gonçalves
    • 1
    • 2
  • Martin Oheim
    • 1
  1. 1.Neurophysiology and New MicroscopiesEcole Supérieure de Physique et Chimie Industrielles (ESPCI)ParisFrance
  2. 2.Molecular Biology of Neuronal SignalsMax-Planck-Institute for Experimental MedicineGöttingenGermany

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