Super-resolution microscopy of lipid bilayer phases

Sub-diffraction optical imaging with nanometer resolution of lipid phase-separated regions is reported. Merocyanine 540, a probe whose fluorescence is sensitive to the lipid phase, is combined with super-resolution imaging to distinguish the liquid- and gel-phase nanoscale domains of lipid bilayers supported on glass. The monomer-dimer equilibrium of MC540 in membranes is deemed responsible for the population difference of single-molecule fluorescence bursts in the different phase regions. The extension of this method to other binary or ternary lipid models or natural systems provides a promising new super-resolution strategy.     

Two-dimensional infrared spectral signature and hydration of the oxalate dianion

Ultrafast vibrational spectra of the aqueous oxalate ion in the region of its carboxylate asymmetric stretch modes show novel relaxation processes. Two-dimensional infrared vibrational echo spectra and the vibrational dynamics obtained from them along with measurements of the anisotropy decay provide a picture in which the localization of the oxalate vibrational excitation onto the carboxylate groups occurs in ~450 fs. Molecular dynamics simulations are used to characterize the vibrational dynamics in terms of dihedral angle motion between the two carboxylate planes and solvation dynamics. The localization of the oxalate vibrational excitation onto the carboxylates is induced by the fluctuations in the carboxylate vibrational frequencies which are shown by theory and experiment to have a similar correlation time as the anisotropy decay.     

Dynamic structures of aqueous oxalate and the effects of counterions seen by 2D IR

Two dimensional vibrational echo spectra of oxalate in the carboxylate asymmetric stretch region in D(2)O show two transitions having anomalously slow spectral diffusion and a third transition having relaxation properties typical of the free carboxylate ion. Quantitative analysis of the frequency shifts of the carboxylate asymmetric stretch modes caused by a singly charged cation in the oxalate hydration shell supports that ion pairs can be responsible for these new transitions. Experimental evidence and DFT calculations are consistent with oxalate forming a mixture of "side-on" and "end on" contact ion pairs wherein the carboxylate groups are protected from mobile heavy water molecules.     

Simultaneous quantification of ten cytotoxic drugs by a validated LC–ESI–MS/MS method

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2 ng mL(-1) for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces.     

Controlled bimolecular collisions allow sub-diffraction limited microscopy of lipid vesicles

The concentration and vesicle size-controlled collisions of single molecules with target biological assemblies allow sub-diffraction limited optical images to be obtained that are not subject to the usual photobleaching problems with single molecule experiments. For example, single molecules of the probe Nile Red in aqueous solution emit a burst of fluorescence when they collide with a 50 nm hydrophobic vesicle situated on the surface in the laser focus. The bimolecular kinetics of the bursts is defined by their on- and off-time distribution functions which depend on the concentration and diffusion of the probe and the vesicle size. The mean burst frequency changes much more sharply than does the fluorescence intensity when a vesicle is raster scanned through the laser focus. This sharpness allows the spatial resolution of two objects to be improved and separations less than the diffraction limited resolution of the conventional optical microscope to be measured. The principle of this method of trajectory time distribution optical microscopy (TTDOM) could be used in a far field optical microscopic system with a resolution of several nanometers.     

High-resolution optical imaging from trajectory time distributions

Trajectory time distribution optical microscopy (TTDOM), which records the mean off-times of single molecular fluorescent indicators that light up when they collide with vesicles, is extended to record fluorescence durations or on-times. TTDOM can distinguish shapes of objects that are smaller than the diffraction limited resolution. The fluorescence duration time image can also provide high-resolution information. The effects of the threshold that separates fluorescent bursts from background signals and of two or more probes visiting the vesicles simultaneously have been investigated systematically. New experimental results along with simulations indicate that TTDOM is capable of providing the size and shape of objects and information on probe-vesicle binding.     

Comparison of linear and 2D IR spectra in the presence of fast exchange

The few picosecond time scale H-bond making and breaking in the system acetonitrile-methanol dominates the mechanism of vibrational coherence transfer that is evident in the shapes of both the linear and nonlinear IR spectra of the CN group.     

Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.     

Observation of collision-free spectral relaxation in p-difluorobenzene

Collision-free evolution of the fluorescence spectrum of p-difluorobenzene was observed directly by means of picosecond time gating. A rapid evolution (6–10 ps) of the structured portion of the emission was attributed to near resonant light scattering, while a slower (≈ 103 ps) decay of the ratio of the structured to unstructured parts of the spectrum was attributed to intramolecular vibrational relaxation. This result compares well with the IVR time measured by O₂ quenching methods