High-resolution nonlinear optical imaging of live cells by second harmonic generation

Area of Science:

• Nonlinear optical microscopy
• Biophysics
• Cell biology

Background:

• Second Harmonic Generation (SHG) is a nonlinear optical phenomenon.
• Nonlinear optical microscopy offers high resolution and reduced phototoxicity.
• Live cell imaging requires methods that minimize photodamage.

Purpose of the Study:

• To adapt laser scanning microscopy for Second Harmonic Imaging (SHIM) of live cells.
• To investigate the role of dye chirality in SHIM contrast generation.
• To assess the sensitivity of SHIM to changes in membrane potential.

Main Methods:

• Utilizing a femtosecond pulsed laser with a laser scanning microscope.
• Employing membrane-staining dyes with large nonlinear optical coefficients.
• Imaging three cell lines with both achiral and chiral dyes.

Main Results:

• Achieved live cell imaging using SHIM with high resolution and minimal photobleaching/phototoxicity.
• Demonstrated that dye chirality significantly influences contrast generation in SHIM.
• Showed SHIM is highly sensitive to membrane potential, with depolarization reducing signal intensity.

Conclusions:

• SHIM is a viable technique for high-resolution live cell membrane imaging.
• Dye properties, specifically chirality, are critical for SHIM contrast.
• SHIM can serve as a sensitive probe for cellular membrane potential.