Photobleaching Analysis of Fluorescent Proteins in Two-Photon Microscopy at High Repetition Rates

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Two-photon excitation fluorescence microscopy (TPM) is crucial for live cell imaging.
• Severe photobleaching limits long-term and quantitative TPM studies.
• High-repetition rate excitation is a potential strategy to mitigate photobleaching.

Purpose of the Study:

• To investigate the utility of high-repetition rate excitation for reducing photobleaching in fluorescent proteins.
• To evaluate the impact of excitation wavelength and repetition rate on photobleaching of EGFP and tdTomato in TPM.

Main Methods:

• Photobleaching assays were performed on EGFP and tdTomato.
• Experiments utilized two-photon excitation microscopy with 80 MHz and 640 MHz repetition rates.
• Analysis focused on the relationship between repetition rate, excitation wavelength, and photobleaching.

Main Results:

• Photobleaching reduction by high-repetition rate excitation is highly dependent on excitation wavelength.
• The effect varies significantly between different fluorophores, including EGFP and tdTomato.
• Optimal parameters for minimizing photobleaching are fluorophore and wavelength-specific.

Conclusions:

• High-repetition rate excitation can reduce photobleaching in TPM, but not universally for all fluorescent proteins.
• Careful selection of excitation wavelength and repetition rate is necessary for minimizing photobleaching and photodamage.
• These findings provide guidance for optimizing TPM experimental conditions for live imaging.