Interactions and Optical Properties of Microorganisms on Surfaces

Dr Rafaël Sibilo
November 12th, 2021 RAFAËL SIBILO Optoelectronics

Controlling microbial growth is essential to industries such as healthcare, food, pharmacy but also for ship hulls and water systems. Interactions of bacteria with surfaces are highly dynamic and complex. Once a single cell transitions to a persistent multicellular microbial community (biofilm), elimination becomes complicated. Bacterial adherence, growth, and detachment are regulated by biological, chemical, physical, mechanical, and electrical properties of the bacterial cell, the surface, and the surrounding medium. Comprehensive studies in this field therefore require a multidisciplinary approach involving experts from different branches of science and appropriate choices of equipment depending on the question to address. This thesis focuses on the interaction and optical properties of bacteria on surfaces. More specifically, it investigates novel methods for enhanced bacteria detection, growth monitoring and presents an in-depth study of interaction mechanisms of bacteria and surface nano-structures.

In the first part of the thesis, we validate a newly in-house built bio-sensing device to detect cells and their growth on surfaces. The proposed surface cytometer is compared with two standard laboratory methods, spectrophotometry and fluorescence microscopy. The results obtained with the three different techniques show similar trends, confirming the suitability of the surface cytometer as a compact, fast and low-cost device for measuring bacterial growth. Distinctively, the surface cytometer possesses both a large field-of view (~200 mm2) and depth of focus (~2 mm), these being particularly interesting for in-situ measurements and point-of-care testing.

In order to enhance cell imaging, we propose a new type of surface, ultrathin (<10nm) gold films on a transparent substrate, such as glass. Such a surface has the capability to quench background fluorescence, improving microscopy and imaging. This is demonstrated through both numerical simulations and experiments. The physical mechanism at the basis of our design is that metals can reduce the lifetime of a fluorophore in its proximity. On the contrary, fluorophores further from the surface, because of their separation from the metal due to cell body, will maintain a much higher level of signal, i.e. they are less quenched. The higher signal-to-noise ratio (SNR) compared with a glass bare substrate is observed in both air and water. An improved SNR promotes the collection of a higher number of photons leading to more accurate localization precision, while reducing background, thanks to lower laser powers and shorter acquisition times. The enhanced imaging mediated through an ultrathin metal has potential in single-molecule localization super-resolution microscopy and live-cell imaging applications, especially under controlled conditions to minimize photodamage.

In another study of the thesis, we demonstrate that bacterial growth can be regulated by tuning surface wettability. In contrast to commonly used indirect methods such as bacterial colony counting and scanning electron microscopy, we investigated a direct approach for assessment. First, we used molecular dynamics simulations to predict bacterial behavior on flat and nanostructured glass substrates, with wetting characteristics further modulated by chemical coatings. Then, we experimentally assessed these findings using E. coli bacteria and time-lapse fluorescence microscopy. Obtained data confirmed that nanostructured glass simultaneously hydrophobic, repelling water, and oleophilic, attracting fat, is most destructive, avoids cell adherence and promotes total cell disruption. These direct observations reflect a more accurate spatial- and time evolution of the interactions and bactericidal effects due to surface morphology and wettability. The results provide guidelines to design antimicrobial surfaces using simple nano-structuring and chemistry.

PhD Thesis Defense, November 12, 2020, 17:00. Online (Teams)

Thesis Director: Prof Dr. Valerio Pruneri