Metal‐Enhanced Fluorescence to Quantify Bacterial Adhesion

Abstract

Bacterial adhesion, the fi rst step of biofi lm formation, is of fundamental signifi cance for multiple industries (e.g., petroleum recovery, food processing, drinking water, medicine and healthcare, shipping, or pulp and paper production) due to the huge economic costs associated with biofi lm formation. Assays to monitor bacterial adhesion are the key to elucidate mechanisms of colonization and biofi lm formation. However, the existing microscopy tools typically used to monitor bacterial adhesion are based on observations of bacterial colony formation. In particular, the current optical toolboxes qualitatively defi ne cell adhesion as a simply physically stable association. They neither allow for studying the three-dimensional distribution of bacteria associated with a surface nor for molecular-level analysis of the bacterial interactions that mediate close contact. While some effort has been made in recent years to monitor the adhesion of bacteria populations in real-time using surface plasmon resonance, those techniques do not allow for individual cells to be tracked and provide no quantitative information regarding contact distance so far. [ 1 ] Here, we demonstrate the possibility of obtaining precise information on the three-dimensional distribution of bacteria coming into contact with a surface and propose this new optical technique as a method to monitor and probe individual bacterial adhesion.In our experimental approach ( Figure 1 a), a fl at gold surface of 35 nm thickness was used as the basic substrate for cell adhesion and was prepared by a standard electron beam lithographic technique. Previous reports showed that fi eld enhancement is maximal for 30-60-nm-thick gold layers, and the thickness (35 nm) used in our studies was in this range. [ 2 ] The morphologies of the glass substrate and gold surfaces were investigated using atomic force microscopy (AFM), and the root-mean-square (RMS) roughness of the gold-coated surface was similar to the one of the glass substrate ( Figure S1 in the Supporting Information). The membranes of Pseudomonas aeruginosa strains (PA14 wild type, PA14 pilB , or PA14 fl gK ::Tn5Tet) were labeled with FM4-64, a red fl uorescent dye, as it provided the most uniform and even staining of the various dyes we examined (Table S1 in the Supporting Information). [ 3 ] Wildtype PA14 in close contact with a gold substrate showed a strong increase of FM4-64 fl uorescence compared to cells in contact with the adjacent glass substrate (Figure 1 b and demonstration movies in the Supporting Information). The fl uorescence enhancement was hypothesized to mirror the distance between the cell and the gold surface and to provide a tool to quantitatively monitor bacterial proximity to surfaces. The behavior of individual bacteria on gold surfaces was analyzed next. First, the residence time of the bacteria, defi ned by the length of time the bacteria exhibited enhanced fl uorescence, was analyzed, and a strong correlation was noted between the fl uorescence intensity of the wild-type cells a...

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