70 W. T. Anderson et al. signature have found that Staphylococcus aureus, a biofilm-forming bacterial species, is a common microbe aboard the ISS [4–6]. NASA has recognized a “Risk of Adverse Health Effects Due to Host-Microorganism Interactions” [7] based on past experiments in space, with a suggested high-level research approach that includes “phenotypic evaluation of microorganisms (including virulence studies) and temporal changes for these parameters under both spaceflight and spaceflight analogue conditions” [7]. Biofilm experiments in space (Table 1) are a subset of the microbial experiments that have been completed in space. However, a major focus of the biofilm studies has been genetic analysis, with less emphasis on structural and morphological changes in which SPT techniques excel. Additionally, conclusions drawn from the experiments have been hindered by unreproducible findings [8]. Ground-based simulations of the space environment play an important role in bioastronautics research because of the time and financial limitations associated with in-space research. They may also be required before an on-orbit experiment is approved. On Earth, microgravity can be simulated with temporal dependence by clinostats which continuously rotate biological specimens to balance out gravity’s force to near zero. Analogs must also accurately model the diffusiondriven fluidic environment produced by microgravity, meaning platforms must avoid machine-induced stirring/motion of fluids. To enable single particle tracking in simulated microgravity-grown biofilms, the following materials and techniques are needed: appropriate and well-characterized particles, the ability to accurately track them, and a microgravity simulation platform for biofilms. The focus of this report is a brief review of previous biofilm spaceflight experiments, biofilm SPT experiments and techniques, and current work to merge the two. Table 1 Previous space biofilm experiments. Adapted and expanded from [9] Mission/Launch Vehicle Location Bacterial Species Project Name Results Ref. STS-81 Space Shuttle Burkholderia cepacia N/A [10] STS-95 Space Shuttle Pseudomonas aeruginosa N/A [11] STS-132 Space Shuttle Pseudomonas aeruginosa N/A [12] STS-135 NG-12 ISS Pseudomonas aeruginosa Space Biofilms, CU Boulder [13] SpaceX-22 ISS Unknown oral bacteria Oral Biofilms in Space, UNLV, Colgate N/A—private funding SpaceX-23 ISS Staphylococcus capitis BIOFILMS Experiment, ESA N/A as of 02/2025 N/A ISS Cupriavidus metallidurans ORAcinetobacter radioresistens SpaceX-27 ISS SPT in Biofilms Particle tracking techniques have been employed in various biofilm species with several particle types and sizes to investigate diffusive and microrheological properties. As the motion of a particle is influenced by its environment, analysis of this motion through SPT techniques can elucidate the properties of the environment. SPT can broadly be categorized into two categories: one in which particles actively bind to specific targets to track (proteins, molecules, etc.), and one in which particles do not actively bind and are the focus of tracking. The latter is the primary technique for biofilm studies. Fluorescent polystyrene beads ranging from 40-2000 nm are a common particle choice. Larger particles, on the order of the biofilm mesh size, are used for mechanical property measurements like viscoelasticity, while smaller particles are used for diffusion-based metrics—although there is no consensus for a size cutoff. Particles are expected to exhibit confined behavior indicated by a decrease in diffusion coefficients with respect to values in water, and nonlinear mean squared displacements (MSD). Results show that particle size, charge, and coating impact the measured values, with some results displayed in Table 2.
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