Acquisition of a fluorescence microscope with confocal, total internal reflection, and high-content analysis capabilities to understand how microbes interact with charged surfaces
Abstract
Microbial interactions with surfaces via formation of biofilms have important implications in bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Despite this, our understanding of these interactions is remarkably incomplete. An important property of any surface is its intrinsic charge, and electrostatic forces represent the earliest interactions of microbes with surfaces. Most natural surfaces and microbes carry a net negative charge. Yet microbes readily interact with such surfaces. Most of what we know about the ability of microbes to interact with charged surfaces comes from the microbial fuel cell field where people have studied in some depth how Geobacter and Shewanella interact with positively charged surfaces while using them as electron acceptors via a process called reductive extracellular electron transfer (reductive EET). The interaction of microbes with negatively charged surfaces where microbes use them as electron donors directly (a.k.a. oxidative EET) has come to the fore only recently. As a consequence, very little is known about this process. Importantly, oxidative EET represents an ideal test case to study the ability of microbes to interact with negatively charged surfaces. Key questions that we intend to answer are (1) What are the dynamics of short and long term microbial interactions with negatively charged surfaces? (2) What mechanisms underlie these interactions? We choose the organism Rhodopseudomonas palustris TIE-1 as a model for oxidative EET. TIE-1 was observed to accumulate unknown proteins at the point of contact with negatively charged surfaces. However, three major challenges prevent us from answering these questions; 1) Our inability to visualize these processes at the microscopic level using the current technology; 2) The lack of throughput in studying microbial interactions with charged surfaces using bioelectrochemical reactors; 3) Our inability to use single molecule live cell imaging with high-throughput to corroborate biochemical and genetic data on the mechanisms of microbe-charged surface interactions. To surmount these challenges, we have developed a miniaturized, microfluidic, bioelectrochemical system compatible with bio-imaging. Our current research will lead to a novel bioelectrochemical device that would aid fundamental cell biological studies in microbiology, cancer biology and neurobiology. Due to its broad applicability, such a device will be of direct value to the Army by furthering research toward Òoptimizing warfighter physical and mental performance capabilities, and a range of revolutionary applications to protect the SoldierÓ. The fundamental knowledge on how microbes interact with charged surfaces that would be revealed by the proposed research would help Òunderstand the underlying properties, principles, and mechanisms governing DNA, RNA, proteins, organelles, cells, organisms, multi-species interactionsÓ. This knowledge will ultimately help provide solutions to potential issues affecting the Soldier such as disease and/or others annoyances caused by microbes that attach to charged surfaces in the human body, and to prosthetics. We have encountered two major hurdles while pursuing our stated goals; Hurdle 1) To achieve high-throughput, we need a fluorescence microscope that has both confocal and high-content analysis (HCA) capabilities. This hurdle affects our ability to address Challenge 2; Hurdle 2) To understand the mechanisms underlying microbe-charged surface interactions, we need single molecule imaging (via total internal reflection fluorescence microscopy; TIRFM) capabilities in a high-throughput platform. This hurdle affects our ability to address Challenge 3. Acquisition of a fluorescence microscope with confocal, TIRFM, and HCA capabilities will allow us to surmount these hurdles. This instrument will also benefit other researchers and train numerous students in techniques of great interest to the U.S. Department of Defense.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 22, 2019
- Source ID
- W911NF1910203
Entities
People
- Arpita Bose
Organizations
- Army Contracting Command
- United States Army
- Washington University in St. Louis