Magnetic resonance imaging has previously demonstrated its potential for indirectly mapping myelin density, either by relaxometric detection of myelin water or magnetization transfer. Here, we investigated whether myelin can be detected and possibly quantified directly. We identified the spectrum of myelin in the spinal cord in situ as well as in myelin lipids extracted via a sucrose gradient method, and investigated its spectral properties. Highresolution solution NMR spectroscopy showed the extract composition to be in agreement with myelin's known chemical make-up. The 400-MHz 1 H spectrum of the myelin extract, at 20°C (room temperature) and 37°C, consists of a narrow water resonance superimposed on a broad envelope shifted ∼3.5 ppm upfield, suggestive of long-chain methylene protons. Superimposed on this signal are narrow components resulting from functional groups matching the chemical shifts of the constituents making up myelin lipids. The spectrum could be modeled as a sum of super-Lorentzians with a T 2 * distribution covering a wide range of values (0.008-26 ms). Overall, there was a high degree of similarity between the spectral properties of extracted myelin lipids and those found in neural tissue. The normalized difference spectrum had the hallmarks of membrane proteins, not present in the myelin extract. Using 3D radially ramp-sampled proton MRI, with a combination of adiabatic inversion and echo subtraction, the feasibility of direct myelin imaging in situ is demonstrated. Last, the integrated signal from myelin suspensions is shown, both spectroscopically and by imaging, to scale with concentration, suggesting the potential for quantitative determination of myelin density. myelin in situ | myelin NMR spectrum | super-Lorentzian fitting | ultrashort echo time

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The photodissociation dynamics of vinyl cyanide (H(2)CCHCN, acrylonitrile) and deuterated vinyl cyanide (D(2)CCDCN) at 193 nm are examined using time-resolved Fourier transform infrared emission spectroscopy. Prior photofragment translational spectroscopy studies [D. A. Blank et al., J. Chem. Phys. 108, 5784 (1998)] of the dissociation have observed the presence of four main dissociation channels; two molecular and two radical in nature. However, with the exception of a<0.01 quantum yield determined for the CN radical loss channel, the branching ratios of the remaining three elimination channels were not measured. The time-resolved emission spectra, including those from the deuterated samples, revealed the presence of acetylene, hydrogen cyanide (HCN), as well as the energetically less stable isomer hydrogen isocyanide (HNC). Acetylene is found in two distinct energetic distributions, suggesting that both three- and four-centered elimination reactions are occurring significantly in the dissociation. In contrast to prior ab initio studies that have suggested the dominant nature of the three-center elimination of molecular hydrogen (H(2)) and cyanovinylidene (:C=CHCN), we find this reaction channel to be of little importance as there is no evidence to support any significant presence of rovibrationally excited cyanoacetylene. Spectral modeling of the product distributions allows for the first experimental determination of the relative occurrence of the three-centered (resulting in HCN+vinylidene) versus four-centered (HNC+acetylene) elimination channels as 3.34 to 1.00, in contrast to the previously calculated value of 126:1. Rice-Ramsperger-Kassel-Marcus analysis depicts that the transition state energy of the four-centered reaction should be about 10 kcal mole(-1) lower than the three-centered reaction.

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For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

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Purpose Direct assessment of myelin has the potential to reveal central nervous system abnormalities and serve as a means to follow patients with demyelinating disorders during treatment. Here, we investigated the feasibility of direct imaging and quantification of the myelin proton pool, without the many possible confounds inherent to indirect methods, via long-T2 suppressed 3D ultra-short echo-time (UTE) and zero echo-time (ZTE) MRI in ovine spinal cord. Methods ZTE and UTE experiments, with and without inversion-recovery (IR) preparation, were conducted in ovine spinal cords before and after D2O exchange of tissue water, on a 9.4T vertical-bore micro-imaging system, along with some feasibility experiments on a 3T whole-body scanner. Myelin density was quantified relative to reference samples containing various mass fractions of purified myelin lipid, extracted via the sucrose gradient extraction technique, and reconstituted by suspension in water, where they spontaneously self-assemble into an ensemble of multi-lamellar liposomes, analogous to native myelin. Results MR signal amplitudes from reference samples at 9.4T were linearly correlated with myelin concentration (R2 = 0.98–0.99), enabling their use in quantification of myelin fraction in neural tissues. An adiabatic inversion-recovery preparation was found to effectively suppress long-T2 water signal in white matter, leaving short-T2 myelin protons to be imaged. Estimated myelin lipid fractions in white matter were 19.9% to 22.5% in the D2O-exchanged spinal cord, and 18.1% to 23.5% in the non-exchanged spinal cord. Numerical simulations based on the myelin spectrum suggest that approximately 4.59% of the total myelin proton magnetization is observable by IR-ZTE at 3T due to T2 decay and the inability to excite the shortest T2* components. Approximately 380 µm of point-spread function blurring is predicted, and ZTE images of the spinal cord acquired at 3T were consistent with this estimate. Conclusion In the present implementation, IR-UTE at 9.4T produced similar estimates of myelin concentration in D2O-exchanged and non-exchanged spinal cord white matter. 3T data suggest that direct myelin imaging is feasible, but remaining challenging on clinical MR systems.

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 Escherichia coli 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.