Crystalline or paracrystalline tobacco mosaic virus (TMV) inclusions are known to be composed mainly of densely packed TMV-rods. These inclusions were studied in situ within infected tobacco leaf hair cells by scanning UV-microspectrophotometry. Comparative measurements in the macroand micro ranges of the instruments were carried out on purified TMV at low and high concentrations, on its separated and reconstituted RNA and protein parts and on isolated TMV-crystals, to permit the interpretation of the optical properties of TMV inclusions in situ. The optical absorbance of TMV in solution and in dried concentrates at room temperature could in part be attributed to distribution inhomogeneities caused by local particle aggregations being oriented to differing degrees. The resulting non-uniform electric fields around and within such unevenly distributed particle complexes caused local jumps of the refraction index and thereby unspecific light losses, chiefly through scattering. The apparent deviation from Beers law was found to be greatest at particle concentrations of 1 - 2 per cent. At higher concentrations the contribution of scatter to light losses was found to decrease again, probably because of increasing order of particles within the aggregates. On the other hand the specific absorbance of the chromophores of TMV over a wide range of concentration was not affected to a measurable degree by changing the distances between the rods. There was no indication that the charged groups of the RNA-cores within intact particles interacted with charged groups of other particles however great their proximity. The ribose phosphate backbone of the RNA strand, deeply embedded within the protein helix, may account for this phenomenon. However, isolated TMV-RNA reacted strongly to changes of the surrounding electric fields when concentrations were varied, and showed pronounced hypochromicity at higher concentrations and following prolonged irridation by x-rays and UV-light. RNA in dried and irradiated droplets was characterized by up to 55% lower extinction coefficients than freshly prepared RNA in solution. The hyprochromic effects caused by irradiation were shown to be almost, but not completely reversible. Hypochromicity increased towards the shorter wavelengths, diminishing the ratio E260/E280 from about 2.0 for diluted RNA to 1.4 for concentrated specimens. Mixing TMV-protein subunits with RNA before drying, leading to partial reconstitution of TMV particles, diminished the hypochromic effect resulting from irradiation of the concentrate. High UV-radiation doses applied to concentrated TMV solutions led to a marked splitting of RNA from protein, as revealed by UV-spectrophotometry of the supernatants and pellets of centrifuged irradiated specimens. Model measurements of intracellular and subsequently isolated TMV crystals combined with empirically derived parameters led to the construction of a correction curve, permitting interpretations of optical measurements on in situ TMV inclusion bodies.
Designing a miniaturised heated stage for in situ optical measurements of solid oxide fuel cell electrode surfaces, and probing the oxidation of solid oxide fuel cell anodes using in situ Raman spectroscopy