Light scattering and the absolute characterization of macromolecules

http://dbkgroup.org/Papers/davey_kell_micrev96.pdf

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

Electromagnetic waves from the lower radio frequencies up through the optical spectrum can generate a myriad of effects and responses in biological specimens. Some of these effects can be harmful to man at high radiation intensities, producing burns, cataracts, chemical changes, etc. Biological effects have been reported at lower radiation intensities, but it is not now known if low-level effects are harmful. Even behavioral changes have been reported. Most of the effects are not harmful under controlled conditions, and can thereby be used for therapeutic purposes and to make useful diagnostic measurements. The problem of microwave penetration into the body with resultant internal power absorption is approached from both the theoretical and the experimental viewpoints. The results are discussed in terms of therapeutic warming of tissues and possible hazards caused by internal "hot spots." The absorption and scattering effects of light in biological tissues are reviewed. Molecular absorption peaks in the optical spectrum are useful for making molecular concentration measurements by spectroscopy. Much of the related work in the literature is summarized, some new results are presented, and several useful applications of wave energy and medical instruments are discussed.

Nonionizing electromagnetic wave effects in biological materials and systems

The light attenuation in a photobioreactor is determined using a fully predictive model. The optical properties were first calculated, using a data bank of the literature, from only the knowledge of pigments content, shape, and size distributions of cultivated cells which are a function of the physiology of the current species. The radiative properties of the biological turbid medium were then deduced using the exact Lorenz-Mie theory. This method is experimentally validated using a large-size integrating sphere photometer. The radiative properties are then used in a rectangular, one-dimensional two-flux model to predict radiant light attenuation in a photobioreactor, considering a quasi-collimated field of irradiance. Combination of this radiative model with the predictive determination of optical properties is finally validated by in situ measurement of attenuation profiles in a torus photobioreactor cultivating the microalgae Chlamydomonas reinhardtii, after a complete and proper characterization of the incident light flux provided by the experimental set-up.

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A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor.

Publisher Summary This chapter discusses the methods and applications of flow systems for analysis and sorting of mammalian cells. Flow systems offer several advantages when compared with the various static systems which analyze cells on slides. In most flow instruments, each cell is exposed to the light beam for only a few microseconds; thus, problems with fluorescence decay are minimized. The successful application of flow systems for rapid, single-cell analysis is critically dependent upon preparative techniques which maintain the cells in a monodispersed state during fixation, staining, and measurement. In instances where fluorescent staining techniques are employed, the quality and specificity of cellular staining must also be evaluated to ascertain the reliability of analytical results. Automated analytical systems that are designed to perform rapid and precise measurement of individual cells cannot be totally relied upon to distinguish fluorescent cellular debris or cell clumps from properly stained single cells. Therefore, sample preparation involving both the disaggregation of tissue into single-cell entities and cell staining plays an extremely important role in flow-system methodology.

Chapter 12 Methods and Applications of Flow Systems for Analysis and Sorting of Mammalian Cells1

Differential light scattering techniques appear to represent an attractive physical method for the rapid identification of various microorganisms. Certain general results of inverse scattering theory suggest strongly that characteristic of each distinct microorganism that scatters light is an essentially unique scattering pattern, i.e., unique differential scattered intensity and polarization. Although a mathematically rigorous inversion procedure seems impractical at this time, the use of detailed differential scattered intensity data as an identification fingerprint shows considerable. promise. Published measurements on nonbiological scatterers confirm this possibility. A variety of calculations are presented that contrast the expected scattering characteristics of certain microorganisms such as Bacillus subtilis, B. anthracis, Staphylococcus epidermidis, S. aureus, Escherichia coli, and the spores of B. megaterium and B. cereus. Experimental and instrumentation difficulties and possible procedures are discussed. A review and laboration of some applicable features of Rayleigh-Gans scattering are included as an appendix.

Differential Light Scattering: a Physical Method for Identifying Living Bacterial Cells

The use of single-particle light-scattering measurements to determine the origin of atmospheric hazes has been explored by measurement of laboratory aerosols, field samples, and computer analysis of the light-scattering data. The refractive index of measured spherical particles 800 nm to 1000 nm in diameter was determined within 2%. For particles of diameter less than 500 nm the measurement of absolute scattering intensity is required for complete analysis. Distinctive nonspherical and absorbing particles were observed both in automotive exhaust and atmospheric samples. Electrostatic suspension of atmospheric particulates is demonstrated to provide a practical approach to optical measurement of single particles. The technique may be used to calibrate optical particle counters or identify particles with unique shape or refractive index.

Phillip J. Wyatt

Papers

Differential Light Scattering: a Physical Method for Identifying Living Bacterial Cells

Single-particle light-scattering measurement: photochemical aerosols and atmospheric particulates.