POLARISED CONFOCAL

Applied Spectroscopy 52, No.12, Dec.1998, p.1498-504 MEASUREMENT OF ORIENTATION AND CRYSTALLINITY IN UNIAXIALLY DRAWN POLY(ETHYLENE TEREPHTHALATE) USING POLARISED CONFOCAL RAMAN MICROSCOPY Everall N J... 

Polarised confocal Raman microscopy was used to measure molecular orientation in uniaxially drawn PETP films, prepared with draw ratios from 1 to 3.5. The orientation of both polarised Raman microscopy and polarised attenuated total reflection IR spectroscopy. Crystallinity profiles were measured through the thickness of the film samples and compared with the orientation gradients existing in the films. This procedure was to determine whether the intuitive assumption that orientation and crystallinity would be positively correlated actually holds true on the microscopic scale for these samples. 18 refs. 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Fluorescence Polarisation coupled with confocal spectroscopy... 

In cases where the light cannot be collected from both sides of the sample, i.e. in a confocal microscope, a 1 1 beamsplitter and two polarisers or a polarising beamsplitter can be used to separate Ip and h. 

The optieal systems used for both techniques are essentially the same. A small sample volume is obtained by confocal deteetion or two-photon excitation in a microscope. Several detectors are used to deteet the fluorescence in different spectral ranges or under different polarisation angles. Therefore correlation techniques ean be combined with fluorescence lifetime deteetion, and the typical time-resolved single-molecule techniques may use eorrelation of the photon data. The paragraphs below focus on single-molecule experiments that not only use, but are primarily based on pulsed excitation and time-resolved detection. 

Confocal scanning dark-field polarisation microscopy, S. Kimura and T. Wilson, Appl. Optics, 1994, 33, 1274. 

---