title: Spatiotemporally Resolved Tracking of Bacterial Responses to ROS-Mediated Damage at the Single-Cell Level with Quantitative Functional Microscopy.
abstract: Herein we report on the implementation of photofunctional microparticles in combination with optical tweezers for the investigation of bacterial responses to oxidative stress by means of quantitative functional microscopy. A combination of a strongly hydrophobic axially substituted Si(IV) phthalocyanine adsorbed onto silica microparticles was developed, and the structural and photophysical characterization was carried out. The microparticles are able to produce reactive oxygen species under the fluorescence microscope upon irradiation with red light, and the behavior of individual bacteria can be consequently investigated in situ and in real time at the single cell level. For this purpose, a methodology was introduced to monitor phototriggered changes with spatiotemporal resolution. The defined distance between the photoactive particles and individual bacteria can be fixed under the microscope before the photosensitization process is started, and the photoinduced damage can be monitored by tracing the time-dependent fluorescence turn-on of a suitable marker. The results showed a distance-dependent photoinduced death time, defined as the onset of the incorporation of propidium iodide. Our methodology constitutes a new tool for the in vitro design and evaluation of photosensitizers for the treatment of cancer and infectious diseases with the aid of functional optical microscopy, as it enables a quantitative response evaluation of living systems toward oxidative stress. More generally, it provides a way to understand the response of an ensemble of living entities to reactive oxygen species by analyzing the behavior of a set of individual organisms.
authors.affiliation: Institut für Angewandte Physik and Center for Nonlinear Science (CeNoS), Westfälische Wilhelms-Universität Münster , Corrensstraße 2, D-48149 Münster, Germany.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Barroso
authors.affiliation: Physikalisches Institut and Center for Nanotechnology (CeNTech), Westfälische Wilhelms-Universität Münster , Heisenbergstraße 11, D-48149 Münster, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Grüner
authors.affiliation: Institut für Angewandte Physik and Center for Nonlinear Science (CeNoS), Westfälische Wilhelms-Universität Münster , Corrensstraße 2, D-48149 Münster, Germany.
authors.forename: Taylor
authors.initials: T
authors.lastname: Forbes
authors.affiliation: Institut für Angewandte Physik and Center for Nonlinear Science (CeNoS), Westfälische Wilhelms-Universität Münster , Corrensstraße 2, D-48149 Münster, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Denz
authors.affiliation: Physikalisches Institut and Center for Nanotechnology (CeNTech), Westfälische Wilhelms-Universität Münster , Heisenbergstraße 11, D-48149 Münster, Germany.
authors.forename: Cristian A
authors.initials: CA
authors.lastname: Strassert
mesh_terms: D018407:Bacterial Physiological Phenomena / Q000528:radiation effects*; D008027:Light; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D017319:Photosensitizing Agents / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism* / Q000494:pharmacology; D059010:Single-Cell Analysis
chemical_list: D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species
keywords: bacteria; optical tweezers; photoactive particles; phototherapy; quantitative functional microscopy; reactive oxygen species