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The effect of numerical aperture on signal level from fluorescent substances or solutions in the evanescent zone of a cylindrical waveguide is analyzed. The analysis applies to the case in which the fluorescence is excited by the evanescent wave of a fiber and the fluorescence signal is that which tunnels back into the same fiber. The analysis is for two cases: bulk fluorescence and fluorescence of a thin film layer. Experimental results are also presented.

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The progress of instrumentation and measurement science in the next decade will be marked by three major trends. First, as the average instrument achieves a rather considerable level of intelligence, "dumb" systems will become the exception, and we will eventually begin to become proficient in exploiting the resulting capabilities. Second, more sophisticated understanding of measurement science and of actual measurement needs will drive instrumentation design advances such as miniaturized sensors and yet more "hyphenated" instruments and "mapping" instruments. Third, the combination of sensor-based instrumentation and microminiaturization will make possible distributed measurement by allowing point-of-use measurements by nonexperts.

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Improvements in process control, which determine production efficiency and product quality, are critically dependent upon on-line process analysis. The technology of the required instrumentation will be substantially expanded by advances in sensing devices. In the future, the hardware will consist of sensor arrays and miniaturized instruments fabricated by microlithography and silicon micromachining. Chemometrics will be extensively used in software to provide error detection, selfcalibration, and correction as well as multivariate data analysis for the determination of anticipated and unanticipated species. A number of examples of monolithically fabricated sensors now exist and more will be forthcoming as the new paradigms and new tools are widely adopted. A trend toward not only on-line but even in-product sensors is becoming discernible.

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A number of electrooptical techniques are described that discriminate against background fluorescence in biologic staining, whether from sample background or unbound excess stain. These techniques are based on the fluorescent decay lifetime difference between bound stain and the sample background or between the bound stain its free form. The fluorescence decay lifetimes may be measured either directly or in a combination gated photometry scheme to substantially enhance the sample background contrast. An alternative procedure uses the photochemical bleaching of fluorescent dyes under intense exposure to time discriminate with higher selectivity, sensitivity and in a more convenient fashion between diverse fluorescent molecules.

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Four major trends will characterize the evolution of Fourier transform spectroscopy's analytical laboratory applications in the remaining seventies and early eighties: (1) A more thorough analysis of the nonidealities of real FT-IR systems will allow the quantitative accuracy of FT-IR to approach its SNR capability, or to be corrected to this level. (2) The power of FT methods is severely mismatched to current practice and habits in the infrared analytical laboratory. New operating procedures, combination techniques such as GCIR (a much better match to FT-IR capabilities) and more demanding spectroscopic techniques, will become more common. In the wake of this development, greater emphasis will be placed on automatic sample preparation and interpretation. (3) At the same time, the importance of spectra as the final output of spectroscopic measurements will decrease. Instead, higher order spectroscopic functions, or even final analytical data, will become a frequent instrumental output, with or without high level operator interaction. (4) The domain of application of Fourier transform spectroscopy will tee steady extension into new spectral regions, such as the near infrared and UV-VIS regions, while its extension to Raman spectroscopy still appears elusive. The analysis embodies a strong conservative bias, in that it excludes from consideration the consequences of new technology. This also insures its relevance to currently existing instrumentation.

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An instrument based on fluorescence correlation spectrometry and total reflection fluorescence visually and photoelectrically detects and sizes viruses at moderate concentrations in biologic fluids in minutes. Viruses can be classified using their nucleic acid type and amount determined by new fluorescent staining and data handling techniques.

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Two approaches to the analysis of spectral-temporal phenomena in optics have appeared recently. One utilizes channeling of white-light spectra and the other uses temporally resolved phase-locked pulses. We show that the two approaches are very closely related and that devices utilizing them have the same ultimate limits but quite different immediately practical limits.

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Ordinary optical components may behave in extraordinary ways when illuminated with ultrashort optical pulses. In the cases of lenses, only the flat lenses such as Fresnel lenses, Fresnel zone plates, and holographic lenses display such anomalies. For them, the pulse may be both elongated temporally and spread spatially unless very high focal number lenses are used.

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The possible limiting components on optical communication bandwidth are the source, the modulator, the propagation medium, and the detector. It is easy to show that the source bandwidth is the fundamental limit. The possibility of source bandwidth limited communication is demonstrated theoretically and experimentally. The basic principles involved are readily extendable to more practical partial approaches to this limit.

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A phenomenon akin to higher order spectra in grating spectroscopy has been found in Fourier transform spectroscopy. While its relative intensity is orders of magnitude down from similar effects in gratings, the high sensitivity of Fourier transform ir allows this perturbation to be detected.

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A general-purpose multiparameter flow cytophotometry system has been developed for use in the desgin of flow cytophotometers to perform specific tasks in automated cytology. Five separate measurement stations spaced along the axis of a capillary tube can be used to make up to eight optical measurements of individual cells flowing through the capillary. The system uses a broad-band arc source and can measure light scattered at various angles, light absorption by cell constituents and/or dyes and fluorescence of cell constituents and/or fluorochromes, excited directly and/or by energy transfer from neighboring molecules. High numerical aperture optics are used to maximize light-gathering capacity and minimize the effects of cell orientation and eccentricity of position in the fluid stream on measurements. A hard-wired preprocessor is used to detect the presence of cells and adjust sampling timing for changes in cell velocity; the electronic system also controls the gain of the detector photomultiplier tubes to compensate for background variations. Data acquistion and analysis are controled by a small general-purpose digital computer. The system has been used to develop a method and apparatus for blood cell counting and classification.

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A multiparameter flow cytophotometer was used to count and classify fixed human blood cells fluorochromed with a mixture of ethidium bromide (EB), brilliant sulfaflavine and a blue fluorescent stilbene disulfonic acid derivative (LN). The system measures light scattered by the cells and absorption at 420 nm for all cells. In addition, nuclear EB fluorescence (540 leads to 610 nm) and cytoplasmic fluorescence from LN (366 leads to 470 nm), brilliant sulfaflavine (420 leads to 520 nm) and EB exicted by energy transfer from LN (366 leads to 610 nm) are measured for all nucleated cells. This information is sufficient to perform red and white blood cell counts and to classify leukocytes as lymphocytes, monocytes, basophils, eosinophils or neutrophils. Light scattering and/or nuclear and cytoplasmic fluorescence values may be further analyzed to obtain the ratio of immature to mature neutrophils. Counts produced by the system are in reasonable agreement with those obtained by electronic cells counting and examination of Wright's-stained blood smears; some discrepancies appear to be due to systematic errors in the manual counting method.

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