3D resolved two-photon fluorescence microscopy of living cells using a modified confocal laser scanning microscope

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Non-linear microscopy offers advanced 3D imaging capabilities for biological samples.
• Femtosecond lasers provide high peak power for multiphoton excitation of fluorophores.

Purpose of the Study:

• To demonstrate non-linear 3D imaging of fluorophore-labeled vital cells using femtosecond near-infrared (NIR) microscopy.
• To adapt a conventional confocal laser scanning microscope for two-photon microscopy.

Main Methods:

• Utilized a tunable Ti:Sapphire laser emitting 780 nm femtosecond pulses for simultaneous two-photon excitation of intracellular fluorophores.
• Coupled the laser to an upright Zeiss confocal laser scanning microscope.
• Achieved pinhole-free non-linear 3D imaging with 400 nm lateral and ~1 micron axial resolution, further improvable with a detection pinhole.

Main Results:

• Successfully performed non-linear 3D imaging of vital cells, including macrophages during phagocytosis.
• Demonstrated excitation of various fluorophores (Fura-2, Calcium Green, Rhodamine 123) and fluorescent microspheres.
• Determined optimal NIR average power (1-4 mW) and pulse width (150-200 fs) to avoid cell damage, evidenced by preventing photoinduced lysis of human erythrocytes.

Conclusions:

• Femtosecond NIR microscopy is a powerful, universal tool for high-resolution 3D imaging of cellular structures and dynamics.
• The adapted confocal microscope provides versatile two-photon imaging capabilities for biological research.
• This technique allows for detailed visualization of cellular functions in living systems.