Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts.

Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.

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 (

Photoinduction of fast, reversible translational motion in a hydrogen-bonded molecular shuttle.

A rotaxane is described in which a macrocycle moves reversibly between two hydrogen-bonding stations after a nanosecond laser pulse. Observation of transient changes in the optical absorption spectrum after photoexcitation allows direct quantitative monitoring of the submolecular translational process. The rate of shuttling was determined and the influence of the surrounding medium was studied: At room temperature in acetonitrile, the photoinduced movement of the macrocycle to the second station takes about 1 microsecond and, after charge recombination (about 100 microseconds), the macrocycle shuttles back to its original position. The process is reversible and cyclable and has properties characteristic of an energy-driven piston.