George W. H. Wurpel

Chemical specificity in three-dimensional imaging with multiplex coherent anti-Stokes Raman scattering microscopy

We demonstrate the three-dimensional (3D) imaging capabilities and chemical specificity of multiplex coherent anti-Stokes Raman scattering microscopy. The simultaneous acquisition of a significant part of the vibrational spectrum at each specimen position permits straightforward differentiation among chemical species. 3D imaging is illustrated with a lipid multilamellar vesicle, and lateral and axial resolutions are determined.

Imaging orientational order and lipid density in multilamellar vesicles with multiplex CARS microscopy

Multiplex coherent anti‐Stokes Raman scattering (CARS) microscopy is used to measure the width of the orientational order distribution of lipid acyl chains within a three‐dimensionally confined microscopic probing volume. A theoretical model is developed to describe and simulate the polarization‐dependent measurements. We observe that the orientational order in phosphatidylcholine multilammellar vesicles increases significantly upon addition of small amounts (≤ 15 mol%) of cholesterol and is significantly reduced for unsaturated lipids. Based on these measurements and using the quantitative nature of multiplex CARS microscopy the exact local concentration of lipid molecules within the vesicles can be measured in terms of the number of lipid bilayers present in the microscopic probing volume.

Photoinduced dynamics in hydrogen-bonded rotaxanes.

Two classes of rotaxanes are described in which photoinduced processes modulate a large-amplitude motion. In the first type, E–Z-isomerization of a fumaric diamide unit to a maleic diamide leads to a substantial weakening of the hydrogen bonds between the diamide and the macrocyclic ring that surrounds it. As a result, the rate of the pirouetting motion is increased approximately by six orders of magnitude. In the second type, intermolecular photoinduced electron transfer is used to induce a reversible shuttling motion on a time scale of microseconds. Medium effects on the rate of shuttling are presented.

Shot-noise limited detection sensitivity in multiplex CARS microscopy

In multiplex CARS microscopy the generated anti-Stokes signal is generated and detected simultaneously over a significant part of the vibrational spectrum. The signal-to-noise ratio of the thus detected spectra is limited only by shot-noise. This principle is demonstrated using a dilution series of 2-propanol in water. It is derived theoretically and shown experimentally that for low solute concentrations - in contrast to methods that suppress the non-resonant background - the CARS signal strength from a particular vibrational mode depends linearly on its concentration. Furthermore, excellent agreement is shown between the experimental data and fits to the theory. It is shown that this approach permits rapid (20 ms acquisition) detection of a single lipid mono-layer, with sufficient signal-to-noise to determine the order parameter for the acyl chain packing. Also it is demonstrated that this detection scheme provides an absolute measure of the solute concentration.

Chapter 4 Visualization and Characterization of Domains in Supported Model Membranes

Abstract Lateral segregation of lipid and protein components within biomembranes can lead to the formation of biologically functional domains. This chapter reviews microdomain formation in supported model membranes, induced by lipid–lipid interactions. The discussed techniques used for bilayer imaging are atomic force microscopy and fluorescence microscopy. Furthermore, we describe the promising capabilities and thus far obtained results of multiplex coherent anti-stokes Raman scattering microscopy with respect to functional imaging of microdomains in lipid model systems.