Methods: Biofilms were grown in glass capillary biofilm reactors. Images were collected using a Leica TCS-SP2 AOBS confocal microscope with or without the use of a two-photon excitation option (Leica/Spectra Physics MaiTai 2-photon system: Ti/Sapphire 780- to 920-nm infrared laser). To examine photobleaching and photodamaging, three-species (Streptococcus oralis, Streptococcus gordonii and Actinomyces naeslundii) biofilms were stained with Calcein-AM (CAM), and average fluorescence intensity of the same field irradiated every 30 s was calculated. Three-dimensional reconstruction was carried out using MetaMorph software for biofilms of a gfp-expressing strain of Pseudomonas aeruginosa on which fluorescent beads (15μm) were immobilized, and Staphylococcus epidermidis biofilms stained with BacLight LiveDead.
Results: After 20 min irradiation, average fluorescence intensity showed a 20.4% decrease with the single-photon system, whereas only an 8% decrease was recorded with the two-photon system. Thus, the intensity was significantly high when the two-photon system was used (p<0.05, Greenhouse-Geisser test). Three-dimensional reconstruction demonstrated that deformation artifact of the beads on the P. aeruginosa biofilms was minimal for the two-photon system. Moreover, some parts of S. epidermidis biofilms that appeared hollow using the single-photon system were proved to be solid when observed with the two-photon system. Thus, the overall resolution appeared better for the two-photon system.
Conclusion: The two-photon laser scanning microscopy provided better three-dimentional resolution with deeper light penetration and reduced photobleaching and photodamaging, and thus may open new possibilities for biofilm imaging.