Two-versus one photon excitation laser scanning microscopy: Critical importance of excitation wavelength

It is often anticipated that two-photon excitation ( TPE) laser scanning microscopy should improve cell survival and tissue penetration relative to conventional one-photon excitation ( OPE) confocal scanning laser microscopy ( CLSM). However few studies have directly compared live cell imaging using one- vs two-photon laser scanning microscopy. We have used calcein-loaded in situ chondrocytes within cartilage as a model for quantitatively comparing these techniques. TPE reduced photo-bleaching and improved cell viability compared to OPE. Using improved detection sensitivity coupled with increased tissue penetration of the near infra-red TPE laser, it was possible to capture images deeper within the cartilage. However, the advantages of TPE vs OPE were strongly dependent on excitation wavelength. We conclude that optimising TPE conditions is essential for realizing the full benefits of this approach.