Fluorescence spectroscopy of normal, SV40-transformed human keratinocytes, and carcinoma cells.

Native fluorescence emission and excitation spectra of SV40 infected human keratinocytes, A431 and SCC324 carcinoma cells, and normal human keratinocytes were measured and compared. A difference in the intracellular metabolic state of NADH was found between the normal cells and the cancer or virus-transformed cells. The observed difference, namely an increased proportion of bound, mitochondrial NADH in the cancer and virus-infected cells, manifests as a blue spectral shift in the emission spectra.

Correction of the internal absorption effect in fluorescence emission and excitation spectra from absorbing and highly scattering media: theory and experiment.

Fluorescence spectra measured from biological samples, such as tissues or cell suspensions, are usually distorted due to the light absorption by intrinsic chromophores. These distortions are aggravated by strong scattering of light inside the samples. A new method is described for a fast correction of these spectral distortions, using only steady-state spectroscopic measurements. The method is based on the formulas derived from a simplified photon diffusion model, in the isotropic one-dimensional approximation applied to a semi-infinite, highly scattering, and moderately absorbing medium with a refractive-index-matched boundary. The formulas describe the spectral distortions of the fluorescence emission and excitation spectra, together with the diffuse reflectance spectrum, as the functions of one spectral characteristic of the medium, the darkness, which is the ratio of absorption coefficient and reduced scattering coefficient. The algorithm does not involve any iterative procedures, and offers a direct, simple, and fast method for real-time spectral correction. The true fluorescence emission or excitation spectrum is directly calculated from a pair of experimental spectra: the fluorescence emission or excitation spectrum and the diffuse reflectance spectrum, measured from the same position on a sample. The correction produces the profile of the true fluorescence spectrum, the same as the one measured from the corresponding sample with an infinitely low absorption and no scattering. The restoration of the spectral profiles of true fluorescence emission and excitation spectra was tested experimentally, using highly scattering phantoms with a fluorescent dye and a deliberately added nonfluorescent dye producing strong inner-filter distortions. © 1998 Society of Photo-Optical Instrumentation Engineers.

[The effect of acridine dyes on the molecular structure of DNA]. / Issledovanie vliianiia akridinovykh krasitelei na molekuliarnuiu strukturu DNK

DNA--acriflavin complexes have been investigated by the methods of flow birefringence and viscometry. The intrinsic viscosity and the optic anysotropy of the complex increase with the increasing quantities of binding dye. Experimental data are treated on the basis of different models of binding. At high ionic strength (mu = 0,1) one type of binding takes place which is described by the intercalation model. In this case the thermodynamic rigidity of DNA-molecule within the complex is proportional to "r". In solutions of low ionic strength (mu = 0,001), two types of DNA-acriflavin binding occur: intercalation and external binding. At low ionic strength, the spectrophotometric titration technique is shown to give a reduced value of "r".

[Spectral manifestations of different types of acriflavine binding to DNA in ultraviolet and visible region]. / Spektral'nye proiavleniia raznykh tipov sviazi akriflavina s DNK v ul'trafioletrovoi i vidimoi oblastiakh

Difference absorption spectra (complex-sum of the initial reagents) are obtained in the visible and longwave UV region for the system of actiflavine and DNA in a number of cases differing in initial and final degrees of DNA filling by the dye, in particular separately for two types of dye binding to DNA. For these binding types conventional absorption spectra are calculated. In the visible region for the first binding type ("strong" binding) red shift of the absorption band is observed; for the second type ("weak" binding) we observed splitting of the band, short wavelength component being highly prevailing, and hypochromism. In the UV region for both binding types the spectra changed in approximately similar way; a slight blue shift and a rather remarkable hypochromism are observed. It is shown that the dye brings the main contribution into the spectral changes in the UV region. If to take into account the spectral properties of molecular aggregates the data obtained are compatible with the intercalation model for "strong" binding and dye stacking on DNA for "weak" binding.