Chemically Induced Changes to Membrane Permeability in Living Cells Probed with Nonlinear Light Scattering

Second-harmonic light scattering (SHS) permits characterization of membrane-specific molecular transport in living cells. Herein, we demonstrate the use of time-resolved SHS for quantifying chemically induced enhancements in membrane permeability. As proof of concept, we examine the enhanced permeability of the cytoplasmic membrane in living <i>Escherichia coli</i> following addition of extracellular adenosine triphosphate (ATPe). The transport rate of the hydrophobic cation, malachite green, increases nearly an order of magnitude following addition of 0.1 mM ATPe. The absence of an ATPe-enhanced permeability in liposomes strongly suggests the induced effect is protein-mediated. The utility of SHS for elucidating the mechanism of action of antimicrobials is discussed

Label-Free Optical Method for Quantifying Molecular Transport Across Cellular Membranes In Vitro

We demonstrate a nonlinear optical method for the label-free quantification of membrane transport rates of small/medium size molecules in living cells. Specifically, second-harmonic generation (SHG) laser scattering permits surface-specific characterization of transport across membranes. Unfortunately, most biologically relevant molecules are SHG-inactive. In the interest of extending this methodology for characterizing transport of any molecule, we monitor the SHG produced from an SHG-active reference molecule, in the presence of an SHG-inactive target molecule-of-interest as both molecules compete to cross a membrane. Of significance, the SHG-inactive target transport rate can be deduced as a perturbation in the measured transport rate of the reference. As proof-of-principle, we examine competitive transport of the strongly SHG-active cation, malachite green (MG), in the presence of a weakly SHG-active dication, propidium (Pro), across the outer-membrane protein channels in living bacteria. Comparison of the extracted and directly measured Pro transport rates validates the effectiveness of the method

Azithromycin-Induced Changes to Bacterial Membrane Properties Monitored <i>in Vitro</i> by Second-Harmonic Light Scattering

We present a nonlinear light scattering method for monitoring, with real-time resolution and membrane specificity, changes in molecular adsorption, and transport at bacterial membranes induced by an antimicrobial compound. Specifically, time-resolved second-harmonic light scattering (SHS) is used to quantify azithromycin-induced changes to bacterial membrane permeability in colloidal suspensions of living <i>Escherichia coli</i>. Variations in membrane properties are monitored through changes in the adsorption and transport rates of malachite green, a hydrophobic cation that gives SHS signal. Regardless of concentration, instantaneous treatment with azithromycin showed no significant changes in membrane permeability. However, 1 h pretreatment with subminimum inhibitory concentrations of azithromycin induced an order-of-magnitude enhancement in the permeability of both the outer membrane and, through facilitation of a new transport mechanism, the cytoplasmic membrane of the bacteria as well. This study illustrates SHS as a novel tool for monitoring antimicrobial-induced changes to membrane properties in living bacteria

Control of Chemical Reactions through Coherent Excitation of Eigenlevels: A Demonstration via Vibronic Coupling in SO₂

Through coherent excitation of a pair of vibronically coupled eigenlevels, an oscillation of 130 kcal/mol in energy excitation between electronic and vibrational motions (on a time scale of 10–8 s) is created for the triatomic molecule, sulfur dioxide (SO2). The reactivity of the molecule can be influenced depending upon whether the molecule is vibrationally or electronically excited with this substantial amount of energy. The effect of excitation on reactivity is demonstrated through SO2 photodissociation as a function of time following coherent excitation, monitored by multiphoton ionization of the SO product

Collisional Relaxation of Highly Vibrationally Excited Acetylene Mediated by the Vinylidene Isomer

Collisional relaxation of highly vibrationally excited acetylene, generated from the 193 nm photolysis of vinyl bromide with roughly 23,000 cm–1 of nascent vibrational energy, is studied via submicrosecond time-resolved Fourier transform infrared (FTIR) emission spectroscopy. IR emission from vibrationally hot acetylene during collisional relaxation by helium, neon, argon, and krypton rare-gas colliders is recorded and analyzed to deduce the acetylene energy content as a function of time. The average energy lost per collision, ⟨ΔE⟩, is computed using the Lennard-Jones collision frequency. Two distinct vibrational-to-translational (V–T) energy transfer regimes in terms of the acetylene energy are identified. At vibrational energies below 10,000–14,000 cm–1, energy transfer efficiency increases linearly with molecular energy content and is in line with typical V–T behavior in quantity. In contrast, above 10,000–14,000 cm–1, the V–T energy transfer efficiency displays a dramatic and rapid increase. This increase is nearly coincident with the acetylene-vinylidene isomerization limit, which occurs nearly 15,000 cm–1 above the acetylene zero-point energy. Combined quasi-classical trajectory calculations and Schwartz-Slawsky-Herzfeld-Tanczos theory point to a vinylidene contribution being responsible for the large enhancement. This observation illustrates the influence of energetically accessible structural isomers to greatly enhance the energy transfer rates of highly vibrationally excited molecules

Indole Facilitates Antimicrobial Uptake in Bacteria

Indole signaling in bacteria plays an important role in antibiotic resistance, persistence, and tolerance. Here, we used the nonlinear optical technique, second-harmonic light scattering (SHS), to examine the influence of exogenous indole on the bacterial uptake of the antimicrobial quaternary ammonium cation (qac), malachite green. The transport rates of the antimicrobial qac across the individual membranes of Escherichia coli and Pseudomonas aeruginosa, as well as liposomes composed of the polar lipid extract of E. coli, were directly measured using time-resolved SHS. Whereas exogenous indole was shown to induce a 2-fold increase in the transport rate of the qac across the cytoplasmic membranes of the wild-type bacteria, it had no influence on a knockout strain of E. coli lacking the tryptophan-specific transport protein (Δmtr). Likewise, indole did not affect the transport rate of the qac diffusing across the liposome membrane. Our findings suggest that indole increases the bacterial uptake of antimicrobials through an interaction with the Mtr permease

Chemical Activation through Super Energy Transfer Collisions

Can a molecule be efficiently activated with a large amount of energy in a single collision with a fast atom? If so, this type of collision will greatly affect molecular reactivity and equilibrium in systems where abundant hot atoms exist. Conventional expectation of molecular energy transfer (ET) is that the probability decreases exponentially with the amount of energy transferred, hence the probability of what we label “super energy transfer” is negligible. We show, however, that in collisions between an atom and a molecule for which chemical reactions may occur, such as those between a translationally hot H atom and an ambient acetylene (HCCH) or sulfur dioxide, ET of chemically significant amounts of energy commences with surprisingly high efficiency through chemical complex formation. Time-resolved infrared emission observations are supported by quasi-classical trajectory calculations on a global ab initio potential energy surface. Results show that ∼10% of collisions between H atoms moving with ∼60 kcal/mol energy and HCCH result in transfer of up to 70% of this energy to activate internal degrees of freedom