5.2.5. Confocal Laser Scanning Microscopy (CLSM)

Confocal laser scanning microscopy (CLSM) is an optical microscope equipped with a laser beam that is particularly useful for examining thick samples like microbial biofilms. Samples are stained with specific fluorescent dye insofar as the fluorescent light from the illuminated spot is collected on the objective and transformed by a photodiode into an electrical signal to be computer-processed [160] given the complexity of the microbial biofilm’s extracellular matrix formed by heterogeneous compounds: polysaccharide, lipids, enzymes, extracellular DNA, and proteins [186].

However, no fluorescence labelling method is currently available for visualising the whole biofilm matrix owing to its different compositions, which depend on each bacterium and environmental condition, which means that each matrix component must be individually stained. Unfortunately, however, a general stain for polysaccharides does not exist because the chemical structure of matrix polysaccharides differs between distinct bacteria: Gram + and Gram− [186].

Extracellular DNA has been related to bacterial attachment and early biofilm formation stages in many species across the phylogenetic tree. These findings were discovered by employing combined stains, such as PicoGreen^® and SYTOX^®, PI, 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridinone (DDAO), TOTO^®-1, TO-PRO^® 3. Most reports employed DDAO for staining eDNA in biofilms after the first publications by Allesen-Holm et al. [187] and Conover et al. [188]. Excellent efficacy has been reported for TOTO^®-1, SYTOX^® Green, while PI provides the most reliable results. TO-PRO^®-3 and DDAO are not completely cell-impermeant [189].

With biofilm proteins, which may sometimes be more important than polysaccharides, this occurs in cell wall-anchored proteins in Staphylococcus aureus and S. epidermidis, and contributes to aggregation by homophilic interactions [190], or interacts with matrix components that originate from the host, such as fibronectin, collagen, or fibrin [191]. These biofilm proteins can be visualised with strains FilmTracer™ SyPro^® [192]. Several proteins also play a key role in the P. aeruginosa biofilm matrix, such as CdrA and others, perform functions that range from nutrient acquisition to protection from oxidative stress [193]. Moreover, serine-protease inhibitor ecotin has been identified as a matrix protein that binds to Psl [194].

Nowadays, confocal microscopy is a relevant tool for studying the structure of biofilms thanks to its excellent real-time visualisation capability of fully hydrated living samples. The limitation of light microscopy’s spatial resolution is improved by a fluorescence technique and by coupling CLSM with other imaging techniques [157]. The PNA FISH and CLSM combination allows the spatial organisation of and changes in specific members of complex microbial populations to be studied without disturbing the biofilm structure [195,196].