TY - JOUR
T1 - Fluorescence imaging with two-photon evanescent wave excitation
AU - Schapper, Florian
AU - Gonçalves, José Tiago
AU - Oheim, Martin
N1 - Funding Information:
Acknowledgements 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.
PY - 2003/10
Y1 - 2003/10
N2 - 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.
AB - 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.
KW - Evanescent wave excitation
KW - Microscopy
KW - Non-linear excitation
KW - Total internal reflection fluorescence microscopy
KW - Two-photon excitation fluorescence
UR - http://www.scopus.com/inward/record.url?scp=0242695823&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0242695823&partnerID=8YFLogxK
U2 - 10.1007/s00249-003-0326-7
DO - 10.1007/s00249-003-0326-7
M3 - Article
C2 - 12955359
AN - SCOPUS:0242695823
SN - 0175-7571
VL - 32
SP - 635
EP - 643
JO - European Biophysics Journal
JF - European Biophysics Journal
IS - 7
ER -