| Pacific Rim Symposium on Surfaces, Coatings and Interfaces (PacSurf 2014) | |
| Biomaterial Interfaces | Monday Sessions |
| Session BI-MoE |
| Session: | Biofouling |
| Presenter: | Xiaoying Yu, Pacific Northwest National Laboratory |
| Authors: | X. Yu, Pacific Northwest National Laboratory M. Marshall, Pacific Northwest National Laboratory X. Hua, Pacific Northwest National Laboratory B. Liu, Pacific Northwest National Laboratory Z. Wang, Pacific Northwest National Laboratory Z. Zhu, Pacific Northwest National Laboratory A. Tucker, Pacific Northwest National Laboratory W. Chrisler, Pacific Northwest National Laboratory T. Thevuthasam, Pacific Northwest National Laboratory |
| Correspondent: | Click to Email |
One of the most important processes in nature involves bacteria forming surface attached microbial communities or biofilms. Biofilms possess a complex structure made of a highly-hydrated milieu containing bacterial cells and self-generated extracellular polymeric substances (EPS). We report a unique approach of molecular imaging of biofilms in their native environments using time-of-flight secondary ion mass spectrometry (ToF-SIMS) to address potentially the grand challenge of complex interfacial dynamics in biogeochemistry. Biofilm is grown on a silicon nitride (SiN) membrane window in a recently developed microfluidic single channel flow reactor. Continuous imaging of complex liquid samples can be performed with high precision and sensitivity using this technique. Direct probing of the biofilm occurs in situ within a windowless detection area of 2 µm in diameter as soon as the hole is drilled through by the SIMS primary ion beam.
The microfluidic reactor consists of a SiN window for biofilm attachment and ToF-SIMS detection. Biofilm formation is conducted by scaling down a known protocol to the microfluidic regime. Shewanella with a green fluorescent protein was used so that biofilm formation can be followed in real time using confocal fluorescence microscopy. Biofilm is generally grown for 6 to 7 days before harvesting. ToF-SIMS analysis is performed immediately upon harvest. A ToF-SIMS V spectrometer (IONToF GmbH, Germany) is used.
Depth profiling is used to drill through the SiN membrane and the biofilm grown on the SiN substrate. Characteristic fatty acids fragments are clearly identified in the m/z spectra. When compared among dried biofilm sample, uninoculated medium solution, and the hydrated biofilm, principal component analysis (PCA) shows distinctions among them. 2D and 3D image reconstructions are conducted. Image PCA is done to further investigate biofilm spatial inhomogeneity. Detailed analysis of dried EPS in bound, loose, and total forms shows distinctions in their chemical makeup. PCA of hydrated biofilm, soluble total EPS, and medium solution provides new insight of the role of EPS in biofilm formation.
We show that molecular imaging of biofilm in the hydrated environment using ToF-SIMS is possible using the unique microfluidic device for the first time. Moreover, probing the natural biofilm microenvironment without drastic sample treatment such as freezing or drying makes it possible to investigate how biofilm develop metabolic and chemical heterogeneities in its hydrated state. The multimodal nature of our microfluidic reactor permits multiplexed in situ chemical imaging and advances mesoscale bioimaging.