Table 1.

Most widely used non-electron microscopic techniques for biofilm study.

	Light Microscopy	CLSM	AFM
Pros	Simple protocols Cheap and easy to perform Large investigation area	Allows single cell visualization and 3-D imaging	Nondestructive technique that works under physiological-like conditions, allowing living biofilms qualitative and quantitative assessment with few treatments, sample 3-D structure reconstruction at nanometer scale.
Cons	Low resolution and magnification power, need for sample staining, gross morphological differentiation, finest details not visible	Use of fluorophores, limited number of reporter molecules, intrinsic biofilm fluorescence can interfere with probes fluorescence	Small scan area (max 150 × 150 µm), no image of bacterial cells sidewalls, possible surface damage during imaging due to tip interactions.
Applications	Visualization of biofilm formation and quantitative assessment of its biomass	Assessment of biofilm structural parameters, Biofilm 3D structure, identification and localization of living and death cells	Quantitative biofilm analysis, determination of adhesion forces, biofilm topography, in situ imaging.