Highly nonlinear photodamage in two-photon fluorescence microscopy

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Two-photon fluorescence excitation is widely used in laser scanning microscopy due to low photodamage.
• Nonlinear photodamage occurs when excitation intensity exceeds a specific threshold.

Purpose of the Study:

• To investigate nonlinear photodamage in bovine adrenal chromaffin cells.
• To determine the relationship between light intensity and photodamage.
• To identify strategies for mitigating photodamage in two-photon microscopy.

Main Methods:

• Studied photodamage in bovine adrenal chromaffin cells.
• Utilized changes in resting calcium ion ([Ca(2+)]) levels as a damage indicator.
• Assessed the degranulation reaction as a second damage indicator.
• Analyzed the relationship between light intensity and damage, finding it proportional to intensity raised to the power of approximately 2.5.

Main Results:

• Photodamage was found to be proportional to the integral of light intensity raised to the power of approximately 2.5.
• Widening laser pulse duration and focal volume at constant average intensity reduced photodamage.
• Despite reduced excitation efficiency, signal loss could be compensated by increasing power.
• At constant damaging potential, longer pulses and larger focal volumes can yield larger signals due to a higher damage exponent than the two-photon signal exponent.

Conclusions:

• Nonlinear photodamage in two-photon microscopy is intensity-dependent.
• Optimizing laser pulse shape and focal volume can minimize photodamage.
• Strategies exist to enhance signal while maintaining low photodamage levels in microscopy applications.