Fluorescence line narrowing-nonphotochemical hole burning spectrometry: femtomole detection and high selectivity for intact DNA-PAH adducts

Abstract

A new fluorescence line narrowing (FLN) apparatus is described and evaluated through experiments on intact DNA-PAH (polycyclic aromatic hydrocarbon) and globin-PAH adducts, as well as polar PAH metabolites. A detection limit of approximately 3 modified bases in 10(8) for a DNA adduct formed with a diol-epoxide of benzo[a]pyrene (BPDE-DNA) is reported for 20 micrograms of DNA at a spectral resolution of approximately 8 cm-1. The methodology employed avoids or minimizes spectral degradation and loss of sensitivity due to photooxidation and nonphotochemical hole burning (NPHB). A new double selection technique that employs both FLN and NPHB is described and found to lead to a significant improvement in selectivity over that obtained with conventional FLN.

Accurate modeling of fluorescence line narrowing difference spectra: Direct measurement of the single-site fluorescence spectrum

“…[12][13][14][15][16][17][18][19][20][21] The essential idea behind this technique is that the contribution of the pseudo-PSB to the measured FLN signal can be effectively eliminated by taking the difference between two consecutively measured FLN spectra separated by a period of HB. [12][13][14][15][16][17][18][19][20][21] The essential idea behind this technique is that the contribution of the pseudo-PSB to the measured FLN signal can be effectively eliminated by taking the difference between two consecutively measured FLN spectra separated by a period of HB.…”

“…This process was originally described theoretically by Jaaniso 13 whose findings were corroborated by Fünfschilling and co-workers 15 using model calculations; in both cases it was demonstrated that for low burn fluences the contribution of the pseudo-PSB to the measured difference spectrum should be all but eliminated. [16][17][18][19] A similar approach has been used for eliminating fluorescence from nonresonant molecules in single-molecule experiments. [16][17][18][19] A similar approach has been used for eliminating fluorescence from nonresonant molecules in single-molecule experiments.…”

Laser excited fluorescence spectroscopic studies of cellular macromolecular damage from chemical carcinogens

“…Utilization of vibronic excitation has several advantages over the origin band excitation from the standpoints of characterization and selectivity. The improved selectivity with vibronic excitation is based upon the fact that the vibronic features in SQ absorption spectrum are often more sensitive to structure perturbations than the fluorescence spectrum (71,72). Vibrational relaxation is indicated by the downward wiggly arrows which show that fluorescence is due to two different isochromats linked to the two vibronic levels initially excited.…”

Aluminum Phthalocyanine Tetrasulfonate in MCF-10F, Human Breast Epithelial Cells: A Hole Burning Study

“…Thus high-resolution optical techniques such as fluorescence line narrowing (FLN) (Jankowiak and Small, 1991) and hole burning , which have been developed to probe the inhomogeneously broadened absorption spectra of impurity molecules in glasses, are increasingly being used to investigate biological samples. FLN, for example, has been used to study DNA-carcinogen complexes at physiological damage levels (Jankowiak et al, 1988;Ariese et al, 1996) and proteinchromophore interactions (Kaposi et al, 1993). Hole burning has been used extensively to study electron and energy transfer processes in photosynthetic systems Reddy et al, 1992;Small, 1995).…”