Phosphorescence microscope

Marriott et al. [15] constructed a phosphorescence microscope, in which a laser or an arc lamp was used. The excitation light was focused onto a rotating excitation chopper blade so that the on-off transit time of the excitation pulse was kept at a minimum. A quartz optic fiber was used to lead the light into the microscope. The emission chopper and slit assembly were placed as close as possible to the CCD camera. The two choppers were rotated at the same speed and phase locked. Note that in all setups a small aperture plane of excitation light is created for fast and effective chopping (Fig. 3). 

Fig. 3 Scheme of a phosphorescence microscope [10, 15], Fluorescence excitation is performed by chopping a laser (section A) or an arc lamp (section B)... 

Fig. 2. Schematic drawing of one form of the field emission microscope. E, glass envelope S phosphorescent screen M, metal backing A, anode lead-in T, emitter tip C, tip support structure V, vacuum lead.

The emphasis of our studies to date has been to investigate the use of the phosphorescence technique as an adjunct to microscopic examination (28). Hairs from light-haired individuals, all approximately the same color, were examined microscopically. Hair from eight individuals that could not be differentiated on the basis of color, diameter, morphology of the hair root, presence or lack of medulla, and cuticular scale pattern was selected. 

The optical absorption, measurable via the excitation spectra of secondary emissions (delayed fluorescence,143 phosphorescence,142 EPR triplet spectra,166 etc.). The absorption is the response of the crystal to an excitation spatially homogeneous at the microscopic scale ... 

It would be desirable to insert a probe into the polymer to ascertain the local environmental conditions. In addition to having microscopic dimensions, the probe must act as a timing device which specifies the time-scale of the observation. Such a probe is a fluorescent molecule. Its dimensions are about the size of a monomer residue, namely of the order of 10 A, and the lifetime of fluorescence, r, varies between about 10-9— 10"7 sec., depending on the fluorescent compound and the medium (9). Still longer time-scales, namely, 10"4—10 sec., are achieved with organic molecules in the phosphorescent state (21). 

In addition to the spontaneous emission of excited molecules, fluorescence and phosphorescence (Section 2.1.1), the interaction of electromagnetic radiation with excited molecules gives rise to stimulated emission, the microscopic counterpart of (stimulated) absorption. Albert Einstein derived the existence of a close relationship between the rates of absorption and emission in 1917, before the advent of quantum mechanics (see Special Topic 2.1). 

This emission can be classified as fluorescence, which has a very rapid decrease in intensity or phosphorescence, where emission decay is much slower. The difference between the two is characterized by the value of the constant k, which for fluorescence is much greater than for phosphorescence. The lifetime of fluorescence Tq is defined, using the rate constant k, by Tq = l/k. At the instant Tq, the intensity f will become, according to expression 11.1, 36.8 per cent of the initial intensity 7q. In other words a fluorescent compound corresponds, on the microscopic scale, to a population of individual species of which 63.2 per cent have relaxed to an non-emissive state after this brief period of time. 

The first technique for detecting properties of individual atoms on metallic surfaces was developed by Erwin Muller in 1936 (Chen 1993, 412). (For a history of this invention, see Drechsler 1978). This instrument, known as a field ion microscope, has a simple design comprising a vacuum system, a needle tip, and a phosphorescent screen. Muller s diagrams and discussion enable readers to visualize the technique he developed (Muller and Tsong 1969, 99) (Figure 1). 

Hennink EJ, de Haas R, Verwoerd NP et al (1996) Evaluation of a time-resolved fluorescence microscope using a phosphorescent Pt-Porphine model system. Cytometry 24 312-320... 

Low-volume micro cells are available for situations in which sample volumes are limited. Several companies make flow cells for fluorescence detection in chromatography and in continuous flow analysis. Samplehandling accessories include micro-plate readers, microscope attachments, and fiber-optic probes. Low-volume cells are often used for room-temperature phosphorescence and for chemiluminescence. Special cells and sample handling are needed for low-temperature phosphorescence measurements. 

All mps were taken with a microscope hot stage and are uncorrected. UV-visible spectra were recorded on a Shimadzu UV-200, fluorescence/phosphorescence spectra on a Hitachi MPF-2A, and GLC on a Yanagimoto Yanaco G-80F (FID). The GLC conditions used Column A (3.0 mm X 2.0 m, 5% PEG-20M on 60-80 mesh Gasport in a stainless steel column) was used for product analyses. Column B (3.8 mm x 3.0 m, 25% Apiezon L on 30-60 mesh fire brick in a glass column) was used to analyse the degradation of 1. 

If techniques based upon fluorescence and phosphorescence spectroscopy are going to gain acceptance as routine tools in the industrial laboratory, they must prove their merit by establishing that they can provide important information about typical polymeric industrial materials. Such materials are frequently prepared from recipes, where the recipes themselves have been optimized for product performance and not for structural simplicity. This means that one is dealing with complex materials composed of mixtures of homopolymers and various kinds of copolymers. These may generate microphase structures, interfaces and interphase regions. One normally believes that the microscopic structure of the material and its dynamic response are somehow responsible for its desirable properties. 

---