Direct Measurement of Two-Photon Action Cross Section

Area of Science:

• Photochemistry
• Biophysics
• Microscopy

Background:

• Phototriggers are essential tools in photochemistry and cell biology for light-induced release of active molecules.
• Accurate measurement of two-photon (2P) action cross sections is crucial for optimizing uncaging experiments.
• Existing methods for determining 2P action cross sections can be complex and require specific standards.

Purpose of the Study:

• To present a simplified and rapid methodology for quantifying the 2P action cross section of phototriggers.
• To enable the measurement of 2P action cross sections using readily available 2P microscopy setups.
• To provide a versatile method applicable to a wide range of caged compounds.

Main Methods:

• Utilizing a standard 2P microscopy setup for both uncaging (photolysis) and fluorescence detection.
• Employing custom-designed, lithographically fabricated microcuvettes to confine the excitation volume.
• Reducing the total excitation volume to minimize photolysis time and enhance measurement speed.

Main Results:

• Successfully measured the 2P action cross section of a ruthenium-based caged serotonin.
• Demonstrated the method's applicability without requiring a reference standard like fluorescein.
• Validated the simplicity and speed of the proposed measurement technique.

Conclusions:

• The presented methodology offers a straightforward and efficient way to determine 2P action cross sections.
• This approach is broadly applicable to various caged compounds, irrespective of their emission properties post-uncaging.
• The use of microcuvettes significantly improves the practicality and speed of 2P action cross section measurements.