Influence of image acquisition parameters on quantitative measurements of biofilms using confocal laser scanning microscopy

The influence of confocal laser scanning microscope (CLSM) image acquisition on quantitative measurements of entrapped fluorescent microbeads and biofilms was studied. The effects of acquisition settings such as pinhole size, detector gain, amplifier offset/gain, scan speed/average, zoom, and laser intensity on microbead area and fluorescence intensity were measured. Prior to the acquisition of biofilm images, microbeads were immobilized within biofilm samples as calibration controls. Pinhole size, detector gain, and amplifier offset/gain had significant influence on microbead area and intensity measurements, while scan speed/average, zoom, and laser intensity influenced intensity only. An optimal setting was selected based on the experiment in order to obtain the measurements with minimum error. Quantification of biofilm components was performed under optimal conditions and two non-optimal conditions (with respect to pinhole size, detector gain and amplifier gain), along with microbeads. Significant difference in percentage coverage of biofilm and microbeads were observed among these three conditions (P < 0.0001). The non-optimal settings resulted in an increase in biofilm percentage area coverage of 43-88% as compared to optimal settings. The immobilized bead error was minimum (9.2%) under optimal conditions when compared to non-optimal conditions (55-96%). The results clearly show that immobilized microbeads serve as a useful control to determine the error in biofilm quantification caused by sub-optimal image acquisition settings. It is suggested that microscopic evaluations of biofilm parameters should be accompanied by calibration control using microbeads.