Photobleaching in two-photon excitation microscopy

Area of Science:

• Microscopy
• Biophysics
• Photochemistry

Background:

• Two-photon excitation microscopy (TPM) offers focal confinement and reduced photobleaching in thick samples due to its intensity-squared dependence.
• High photon flux in TPM can induce higher-order photon interactions, potentially affecting sample integrity.

Purpose of the Study:

• To investigate the excitation power dependence of fluorescence intensity and photobleaching rates in thin samples under one- and two-photon excitation.
• To determine if higher-order photobleaching occurs in TPM and identify its prevalence across different fluorophores.

Main Methods:

• Examined thin fluorescence samples (approx. 1 micrometer) using one- and two-photon excitation microscopy.
• Analyzed the excitation power dependence of fluorescence intensity and photobleaching rates using log-log plots.
• Tested common fluorophores including fluorescein, Indo-1, NADH, and aminocoumarin.

Main Results:

• One-photon excitation showed linear power dependence (slope ~1) for both fluorescence intensity and photobleaching.
• Two-photon excitation demonstrated a quadratic dependence (slope ~2) for fluorescence intensity.
• Two-photon excitation exhibited a photobleaching rate with a slope greater than or equal to 3, indicating higher-order photon interactions.

Conclusions:

• Higher-order photobleaching is a common phenomenon in two-photon excitation microscopy, even in thin samples.
• This effect, driven by high photon flux, can significantly increase photobleaching compared to linear excitation.
• The increased photobleaching may limit the utility of multi-photon microscopy for detailed studies of thin biological specimens.