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Register a new userRapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of D2O Metabolic Incorporation in a Single Bacterium
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Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with correct antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, we report a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D2O) metabolism. Metabolic incorporation of D2O into biomass in a single bacterium is probed in as short as 10 minutes after culture in 70% D2O medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 hours from colony to results. The SC-MIC results of 37 sets of samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Towards clinical translation, SRS imaging of D2O metabolic incorporation and SC-MIC determination after 1-h antibiotics treatment and 30-minutes mixture of D2O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.
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Stimulated low-frequency Raman scattering can give essential information about the elastic properties of different nanoparticles systems, in particular, biological nanostructures. In the present study, low-frequency vibrational modes in human and bovine serum albumin were for the first time investigated using stimulated low-frequency Raman scattering. Stimulated low-frequency Raman scattering frequency shifts, corresponding to acoustic eigenfrequencies of the sample, were registered by Fabri-Perot interferometers. Conversion efficiency, threshold and set of eigenfrequencies were also measured. Stimulated low-frequency Raman scattering can be applied for biological objects identification and impact on them.
Stimulated Raman scattering (SRS) in plasma in a non-eigenmode regime is studied theoretically and numerically. Different from normal SRS with the eigen electrostatic mode excited, the non-eigenmode SRS is developed at plasma density $n_e>0.25n_c$ when the laser amplitude is larger than a certain threshold. To satisfy the phase-matching conditions of frequency and wavenumber, the excited electrostatic mode has a constant frequency around half of the incident light frequency $omega_0/2$, which is no longer the eigenmode of electron plasma wave $omega_{pe}$. Both the scattered light and the electrostatic wave are trapped in plasma with their group velocities being zero. Super hot electrons are produced by the non-eigen electrostatic wave. Our theoretical model is validated by particle-in-cell simulations. The SRS driven in this non-eigenmode regime may play a considerable role in the experiments of laser plasma interactions as long as the laser intensity is higher than $10^{15}$W/cm$^2$.
Cellular metabolism, the integrated interconversion of thousands of metabolic substrates through enzyme-catalyzed biochemical reactions, is the most investigated complex intercellular web of molecular interactions. While the topological organization of individual reactions into metabolic networks is increasingly well understood, the principles governing their global functional utilization under different growth conditions pose many open questions. We implement a flux balance analysis of the E. coli MG1655 metabolism, finding that the network utilization is highly uneven: while most metabolic reactions have small fluxes, the metabolisms activity is dominated by several reactions with very high fluxes. E. coli responds to changes in growth conditions by reorganizing the rates of selected fluxes predominantly within this high flux backbone. The identified behavior likely represents a universal feature of metabolic activity in all cells, with potential implications to metabolic engineering.
The global optimum for valence population transfer in the NO$_2$ molecule driven by impulsive x-ray stimulated Raman scattering of one-femtosecond x-ray pulses tuned below the Oxygen K-edge is determined with the Multiconfiguration Time-Dependent Hartree-Fock method, a fully-correlated first-principles treatment that allows for the ionization of every electron in the molecule. Final valence state populations computed in the fixed-nuclei, nonrelativistic approximation are reported as a function of central wavelength and intensity. The convergence of the calculations with respect to their adjustable parameters is fully tested. Fixing the 1fs duration but varying the central frequency and intensity of the pulse, without chirp, orientation-averaged maximum population transfer of 0.7% to the valence B$_1$ state is obtained at an intensity of 3.16$times$10$^{17}$ W cm$^{-2}$, with the central frequency substantially 6eV red-detuned from the 2nd order optimum; 2.39% is obtained at one specific orientation. The behavior near the global optimum, below the Oxygen K-edge, is consistent with the mechanism of nonresonant Raman transitions driven by the near-edge fine structure oscillator strength.
Single particle diffraction imaging experiments at free-electron lasers (FEL) have a great potential for structure determination of reproducible biological specimens that can not be crystallized. One of the challenges in processing the data from such an experiment is to determine correct orientation of each diffraction pattern from samples randomly injected in the FEL beam. We propose an algorithm (see also O. Yefanov et al., Photon Science - HASYLAB Annual Report 2010) that can solve this problem and can be applied to samples from tens of nanometers to microns in size, measured with sub-nanometer resolution in the presence of noise. This is achieved by the simultaneous analysis of a large number of diffraction patterns corresponding to different orientations of the particles. The algorithms efficiency is demonstrated for two biological samples, an artificial protein structure without any symmetry and a virus with icosahedral symmetry. Both structures are few tens of nanometers in size and consist of more than 100 000 non-hydrogen atoms. More than 10 000 diffraction patterns with Poisson noise were simulated and analyzed for each structure. Our simulations indicate the possibility to achieve resolution of about 3.3 {AA} at 3 {AA} wavelength and incoming flux of 10^{12} photons per pulse focused to 100times 100 nm^2.
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