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Quasi-two-dimensional (2D) metal halide perovskites (MHPs) are promising photovoltaic (PV) materials because of their impressive optical and optoelectronic properties and improved stability compared to their 3D counterparts. The presence of domains with varying numbers of inorganic layers between the organic spacers ( n -phases), each with different bandgaps, makes the photoinduced carrier dynamics in films of these materials complex and intriguing. Existing interpretations of the ultrafast femto- or picosecond spectroscopy data have been inconsistent, most of them focusing either on exciton/charge transfer from low- n to high- n phases or on hot carrier cooling, but not combined. Here, we present a comprehensive study of the carrier dynamics in the Dion-Jacobson type (PDMA)(MA) ( n -1) Pb n I (3 n +1) (PDMA = 1,4-phenylenedimethylammonium, MA = methylammonium) perovskite, stoichiometrically prepared as ⟨ n ⟩ = 5. Within the film, a coexistence of various n -phases is observed instead of solely the n = 5 phase, resulting in an interesting energy landscape for the motion of excitons and charge carriers. We disentangle hot carrier cooling from exciton transfer between low- n and high- n phases using ultrafast time-resolved photoluminescence and transient absorption spectroscopy. Photophysical modeling by target analysis shows that carrier cooling occurring on a subpicosecond time scale is followed by exciton transfer from low- n into high- n phases in ca. 35 ps when the film is excited by 532 or 490 nm light. Carriers in the high- n phase are much longer lived and decay in a ns time window. Overall, our results provide a comprehensive understanding of the photophysics of this material, which helps to optimize quasi-2D MHP materials for a new generation of PV devices., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins ( f dark ) in the ensemble. To date, f dark has rarely been quantified, and different methods to determine f dark have not been compared. Here, we use and compare two different methods to determine the f dark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain f dark . Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine f dark values between 30 and 60% for the different VFPs. The high values for f dark of these commonly used VFPs highlight that f dark has to be accounted for in quantitative microscopy and spectroscopy.

Monitoring the performance of polymer-functionalized surfaces that aim at removing and inactivating viruses is typically labor-intensive and time-consuming. This hampers the development and optimization of such surfaces. Here we present experiments of low complexity that can be used to characterize and quantify the antiviral properties of polymer-functionalized surfaces. We showcase our approach on polyethylenimine (PEI)-coated poly(ether sulfone) (PES) microfiltration membranes. We use a fluorescently labeled model virus to quantify both virus removal and inactivation. We directly quantify the log removal of intact viruses by this membrane using single particle counting. Additionally, we exploit the change in photophysical properties upon disassembly of the virus to show that viruses are inactivated by the PEI coating. Although only a small fraction of intact viruses can pass the membrane, a considerable fraction of inactivated, disassembled viruses are found in the filtrate. Fluorescence microscopy experiments show that most of the viruses left behind on the microfiltration membrane are in the inactivated, disassembled state. Combined, our fluorescence microscopy and spectroscopy experiments show that not only does the model virus adsorb to the PEI coating on the membrane but also the interaction with PEI results in the disassembly of the virus capsid., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins.

Models for bacterial adhesion to substratum surfaces all include uncertainty with respect to the (ir)reversibility of adhesion. In a model, based on vibrations exhibited by adhering bacteria parallel to a surface, adhesion was described as a result of reversible binding of multiple bacterial tethers that detach from and successively reattach to a surface, eventually making bacterial adhesion irreversible. Here, we use total internal reflection microscopy to determine whether adhering bacteria also exhibit variations over time in their perpendicular distance above surfaces. Streptococci with fibrillar surface tethers showed perpendicular vibrations with amplitudes of around 5 nm, regardless of surface hydrophobicity. Adhering, nonfibrillated streptococci vibrated with amplitudes around 20 nm above a hydrophobic surface. Amplitudes did not depend on ionic strength for either strain. Calculations of bacterial energies from their distances above the surfaces using the Boltzman equation showed that bacteria with fibrillar tethers vibrated as a harmonic oscillator. The energy of bacteria without fibrillar tethers varied with distance in a comparable fashion as the DLVO (Derjaguin, Landau, Verwey, and Overbeek)-interaction energy. Distance variations above the surface over time of bacteria with fibrillar tethers are suggested to be governed by the harmonic oscillations, allowed by elasticity of the tethers, piercing through the potential energy barrier. Bacteria without fibrillar tethers "float" above a surface in the secondary energy minimum, with their perpendicular displacement restricted by their thermal energy and the width of the secondary minimum. The distinction between "tether-coupled" and "floating" adhesion is new, and may have implications for bacterial detachment strategies.

We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the biomolecule can be switched from inside to outside the brush and vice versa by a change in pH. For this, we grafted ultrathin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a bis-N-succinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble, and it is swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface under basic pH conditions, and fluorescence recurred (in-state) when the chains collapsed under acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single-molecule level, and we demonstrate that this switching is fast but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush.