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.Abbreviations 1PEF one-photon excited fluorescence - 2PEF two-photon excited fluorescence - APD avalanche photo diode - CHO Chinese hamster ovary - DMEM Dulbecco's modified Eagle's medium - EGFP enhanced green fluorescent protein - EW evanescent wave - FCS fetal calf serum - GPI glycosylphosphatidylinositol - TIR total internal reflectionThis paper is dedicated to the memory of Prof. Horst Harreis (1940–2002)