title: The Switching Behaviour of Single Photochromic Triads studied with Fluorescence Microscopy
Creators: name: Maier, Johannes
Publisher: name: University of Bayreuth
Year: 2021
Subjects: subject: Physics
subject: Fluorescence
subject: 530

description: Photochromism is the phenomenon that mocules can undergo reversible changes in their structure with light of a specific wavelength. Among a number of photochromic molecules, dithienylcyclopentene (DCP) is a molecular switch which undergoes a cyclization (ring-closing) reaction upon illumination with light in the ultraviolet (UV) spectral range. The cycloreversion (ring-opening) reaction is accomplished by illumination with light in the visible range. Such photochromic reactions can be useful for the application in two major fields. The first field is the application in molecular electronics, where their function as a switch can be exploited by designing transistors, gates, logic circuits, or even devices assembled thereof. The second big field of application is super-resolution microscopy. Special techniques are relying on the digital bright/dark- or on-/off-toggling of the fluorescence of single molecules in the sample in order to identify and localize single molecule fluorescence as single bright spots. Currently, most techniques rely on stochastic molecular processes causing intermittency in single molecule fluorescence. Photochromic switches with their ability to be deliberately switched feature a clear advantage. In this dissertation, photochromic triads consisting of one DCP and two covalently linked fluorophores, are examined. The triads are tailor-made macromolecules designed in such a way that the the state of the switch is monitored by the fluorescence of the fluorophores, which is quenched if the DCP is in its closed state due to an energy transfer from the fluorophores to the photochromic switch. Monitoring of the fluorescence means the use of confocal fluorescence microscopy in order to reveal the characteristics of the switching processes for ensembles and single triads. As tens or hundreds of thousand of photochromic triads are present in ensemble samples where the triads are immobilised in a polymer matrix, the fluorescence will decrease exponentially upon UV illumination, and increase with a saturated exponential growth when the UV illumination is ceased and only visible illumination excites the triads to fluorescence, each described by rate equations. This is in stark contrast for the switching of single triads, where a digital change between a highly fluorescent “On”-state and a weakly fluorescent “Off”-state is expected. A summary of those concepts is given, and additionally an experimental strategy for the switching of single photochromic triads is presented, where the focus lies on a slowed switching reaction. This is achieved making use of special triads and the conduction of experiments at low temperatures. The slowed reaction at low temperatures is of crucial interest for the experiments presented in two chapters, where the experimental focus lies on the detection of single molecule switching. In the first of these chapters, triads composed of a DCP switch and two strong perylene bisimide (PBI) fluorophores were used. The triads were immobilised in a polymer matrix and cooled down to 10K. The single triads are initialized in their closed, non-fluorescent state with UV illumination, which is then probed by visible illumination. As the triad will cycle excitation and radiationless decay in its closed form and then, after a statistically distributed time, undergo the cycloreversion reaction, the fluorescence will be delayed with respect to the onset of probe illumination. One measurement contained 400 switching cycles. However, also unavoidable stochastic blinking processes lead to a quenching of the fluorescence of the triads, and obscure the distribution of fluorescence delay times. This is why a control experiment of the same length was conducted and a statistical model of conditional probabilities was applied in order to distinguish between deliberate switching and stochastic blinking presenting the positive predictive value (PPV). It was found that on the average over 15 single triads, the PPV amounts to 0.8±0.1, the probability which can be assigned to deliberate switching rather than blinking for the triads measured. In the second chapter dealing with single molecule switching, the single molecule measurements were continued with the known triads with PBI moieties as fluorophores, and additionally triads that use boron dipyrromethene (BODIPY) as fluorophores were examined, and as many on-/off-cycles were probed as possible. As a result, the corresponding average PPV for 20 DCP-PBI and 18 DCP-BODIPY triads of 0.7±0.1 is - on the average - the same for both kinds of triads. Furthermore, this PPV was confirmed to be in the same range as for the one in the preceding chapter . In addition, the many repetitions of the measurement pairs switching experiment/control experiment lead to the conclusion that several thousands of switching cycles per triad are possible, showing a remarkably high fatigue resistance of the switching process.