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

Non-linear 3D imaging of fluorophore-labelled vital cells has been performed by femtosecond near infrared (NIR) microscopy. Ultraviolet and visible transitions of intracellular fluorophores, such as Fura-2, Calcium Green, Rhodamine 123 and fluorescent microspheres, were excited via simultaneous absorption of two 780 nm photons provided by a tunable Ti:Sapphire laser. The femtosecond laser was coupled to a conventional upright Zeiss confocal laser scanning microscope expanding its one-photon capabilities to 3D resolved two-photon microscopy. Pinhole-free non-linear 3D imaging was possible with 400 nm lateral and approximately 1 micron axial resolution. Axial resolution could be further improved by using an additional detection pinhole. The NIR average power and pulse width at the sample were adjusted to be 1-4 m W and 150-200 fs, respectively. Higher power levels resulted in cell damage as demonstrated by photoinduced lysis of human erythrocytes. The powerful capabilities of this universal microscope were demonstrated by 3D imaging of two-photon excited fluorophore-labelled macrophages during phagocytosis of fluorescent microsized beads.