Investigation of Microbial Biofilm Structure by Laser Scanning Microscopy

Abstract

Microbial bioaggregates and biofilms are hydrated three-dimensional structures of cells and extracellular polymeric substances (EPS). Microbial communities associated with interfaces and the samples thereof may come from natural, technical, and medical habitats. For imaging such complex microbial communities confocal laser scanning microscopy (CLSM) is the method of choice. CLSM allows flexible mounting and noninvasive three-dimensional sectioning of hydrated, living, as well as fixed samples. For this purpose a broad range of objective lenses is available having different working distance and resolution. By means of CLSM the signals detected may originate from reflection, autofluorescence, reporter genes/fluorescence proteins, fluorochromes binding to specific targets, or other probes conjugated with fluorochromes. Recorded datasets can be used not only for visualization but also for semiquantitative analysis. As a result CLSM represents a very useful tool for imaging of microbiological samples in combination with other analytical techniques.

Critical review on biofilm methods

“…Confocal Laser Scanning Microscopy (CLSM) has emerged in the early 90s as the most versatile powerful microscopic technique to decipher biofilm spatial structure and associated functions (Lawrence et al, 1991;Neu & Lawrence, 2014b). In CLSM, out of focus fluorescent signals are eliminated, and the focal plane is collected with a resolution compatible with single cell visualization (Daddi Oubekka et al, 2012).…”

“…As EPS contribute to the 3-D structuration of biofilms, their visualization from 3-D microscopy revealed the general architecture of the biofilm, their distribution within the biofilm and their dynamics during adhesion, growth, maturation, dispersal and hyper-colonization of surfaces. Being noninvasive, CLSM represents the methods of choice for the distribution and in situ characterization analyses of EPS (Bhardwaj et al, 2013;Neu & Lawrence, 2014b;Watrous & Dorrestein, 2011). Identification of the EPS carbohydrates could be approached using fluorescence lectinbinding analysis (FLBA) which detects glycoconjugates and their distribution within the biofilm (Figure 10(C), and S1) (Marchal et al, 2011;Neu & Lawrence, 2014bTuronova et al, 2016;Zippel & Neu, 2011).…”

“…Being noninvasive, CLSM represents the methods of choice for the distribution and in situ characterization analyses of EPS (Bhardwaj et al, 2013;Neu & Lawrence, 2014b;Watrous & Dorrestein, 2011). Identification of the EPS carbohydrates could be approached using fluorescence lectinbinding analysis (FLBA) which detects glycoconjugates and their distribution within the biofilm (Figure 10(C), and S1) (Marchal et al, 2011;Neu & Lawrence, 2014bTuronova et al, 2016;Zippel & Neu, 2011). Characterization of EPS carbohydrate using FLBA depends on the specificity of the lectins used (Weissbrodt et al, 2013;Zhang et al, 2015a).…”

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms

“…With the success of confocal microscopy, numerous fluorescent dyes have been used to obtain more details on the biofilms organization (Bridier et al 2013;Neu and Lawrence 2014). Targeting both the cellular and extracellular components, the relative distribution of EPS and cells can be visualized, but precise quantification of proteins or polysaccharides appears rather difficult.…”

Fluorescence-based tools for single-cell approaches in food microbiology

“…Laser scanning microscopy (LSM) combines a set of techniques that overcomes this strong optical limitation (Neu and Lawrence, 2014). The basic principle is to acquire a series of two-dimensional (2D) fluorescence images in the focal plane at different altitudes in the sample and to reconstruct the 3D organization of the sample with dedicated softwares such as IMARIS® (Bitplane), AMIRA® (Visage Imaging) and ImageJ for instance.…”