Abstract
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 language | English (US) |
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Pages (from-to) | 635-643 |
Number of pages | 9 |
Journal | European Biophysics Journal |
Volume | 32 |
Issue number | 7 |
DOIs | |
State | Published - Oct 1 2003 |
Externally published | Yes |
Keywords
- Evanescent wave excitation
- Microscopy
- Non-linear excitation
- Total internal reflection fluorescence microscopy
- Two-photon excitation fluorescence
ASJC Scopus subject areas
- Biophysics