Fluorescence imaging with two-photon evanescent wave excitation.
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.
Schapper
Florian
F
Neurophysiology and New Microscopies, Ecole Supérieure de Physique et Chimie Industrielles (ESPCI), 10 rue Vauquelin, 75005 Paris, France.
Gonçalves
José Tiago
JT
Oheim
Martin
M
D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000166:cytology* / Q000378:metabolism*; D003198:Computer Simulation; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008954:Models, Biological; D015203:Reproducibility of Results; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure*; D012680:Sensitivity and Specificity; D041701:Swiss 3T3 Cells