Three-dimensional differentiation of photo-autotrophic biofilm constituents by multi-channel laser scanning microscopy (single-photon and two-photon excitation).

A simple microscopic method to three-dimensionally differentiate between various members in photo-autotrophic biofilm systems is described. By dual-channel single-photon (confocal) and two-photon laser scanning microscopy, the signals in the red and far red channels as well as their combination can be simultaneously recorded. The method takes advantage of the autofluorescent signal of cyanobacteria-recorded in the red and far red channel and the autofluorescent signal of the green algae-recorded in the far red channel only. The differentiation is based on the specific pigment composition of cyanobacteria and green algae in combination with the appropriate filter settings for detection of the autofluorescent emission signals. The method allows the non-destructive, three-dimensional examination of fully hydrated interfacial microbial communities at high resolution as well as the clear separation between autofluorescent signals of cyanobacteria and green algae. Furthermore, there is a third option to record additional signals simultaneously such as nucleic acid stained bacteria, bacteria labeled with phylogenetic probes or glycoconjugates stained by using lectins. With state of the art laser scanning microscopes, even a fourth channel is available for recording yet another parameter, e.g. in the reflection (single-photon only) or fluorescence (single- and two-photon) mode. Thus the approach represents a convenient tool to study multiple parameters of complex photo-autotrophic biofilm systems.