Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm

Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores – ranging in emission from 435 to 665 nm – applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent.

► Five fluorophores on a biofilm specimen can be successfully unmixed. ► We examine spectral effects of different mounting media. ► Thorough examination of settings and controls are required for confocal images.