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Fluorescence imaging with two-photon evanescent wave excitation
Florian Schapper1, José Tiago Gonçalves, Martin Oheim
1Neurophysiology and New Microscopies, Ecole Supérieure de Physique et Chimie Industrielles (ESPCI), 10 rue Vauquelin, 75005 Paris, France.
European Biophysics Journal : EBJ
|September 5, 2003
Summary
We developed a novel microscopy technique using evanescent wave (EW) two-photon excitation fluorescence (2PEF) for precise cellular imaging. This method enhances signal clarity and reduces background noise for advanced biological research.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Two-photon excitation fluorescence (2PEF) microscopy offers optical sectioning capabilities.
- Excitation near a glass/cell interface often suffers from background noise and limited resolution.
- Non-scanning imaging methods are desirable for faster data acquisition.
Purpose of the Study:
- To demonstrate broad-field, non-scanning two-photon excitation fluorescence (2PEF) microscopy utilizing evanescent waves (EW) for enhanced imaging near a glass/cell interface.
- To develop a model predicting 2PEF intensity and excitation volume based on experimental parameters.
- To showcase the application of EW 2PEF for imaging subcellular structures in live cells.
Main Methods:
- Total internal reflection of a femtosecond-pulsed infrared laser beam at the glass/cell interface.
- Exploiting the quadratic intensity dependence of 2PEF for non-linear EW excitation.
- Utilizing a simple model to predict excitation volume and intensity.
Main Results:
- Demonstrated non-linear EW excitation of acridine orange-labeled secretory granules in live chromaffin cells at 860 nm.
- Showcased excitation of TRITC-phalloidin-actin/GPI-GFP double-labeled fibroblasts at 900 nm.
- Achieved confined excitation volumes with significantly reduced background.
Conclusions:
- EW 2PEF provides a powerful method for high-resolution, quantitative imaging in biological samples.
- The technique is suitable for simultaneous multi-color imaging of various fluorophores.
- EW 2PEF is advantageous for applications such as live-cell biochemistry, biosensing, and high-density screening.