The main objective of multi-fluorophoric sample spectroscopy is to find the relative concentrations of individual components. If the spectra of fluorophores have broad complex shapes and significantly overlap, direct analysis is not possible.For various reasons, regression to model spectra attributed to known substances might not be easy either. Here, we describe a method of estimating the spectra of pure fluorophores in complex samples of unknown nature. This method is based on principal component analysis. Principal components themselves cannot be determined uniquely, so the method includes subsequent finding of suitable linear combinations of those principal components. Two physically reasonable assumptions were used to estimate individual fluorophore spectra: their intensities should always be non-negative and with the smallest possible spectral widths. The method's features were tested by numerical simulations using the random generated spectra of three component mixtures, experiments with optical phantom, and analysis of previously collected spectra from real tissue samples. The accuracy of the method depends on the spectral features of the samples. If spectra intersect pairwise, components could be obtained precisely. In other cases reconstructed spectra closely match the original ones, allowing them to be attributed to possible sample components. This approach makes this method convenient for various applied diagnostic tasks. It provides not only quantitative data for sample comparison, as straight principal component analysis does, but also makes data representation more demonstrative, allowing for the creation of qualitative conclusions. The method provides a unique solution dependent only on the shapes of the fluorophores' spectra. K E Y W O R D S fluorophores, laser-induced fluorescence, modeling, principal component analysis 1 | INTRODUCTION Fluorescence spectroscopy is a widely used technique in chemical and biological analysis. When dealing with multicomponent samples, its application faces certain complications. In condensed matter, collisional broadening and electrostatic interactions with surrounding molecules take place. Thus, fluorophores emit relatively broad continuous
Traumatic brain injury (TBI) is a major public health problem. Here, we developed a novel model of non-invasive TBI induced by laser irradiation in the telencephalon of adult zebrafish (Danio rerio) and assessed their behavior and neuromorphology to validate the model and evaluate potential targets for neuroreparative treatment. Overall, TBI induced hypolocomotion and anxiety-like behavior in the novel tank test, strikingly recapitulating responses in mammalian TBI models, hence supporting the face validity of our model. NeuN-positive cell staining was markedly reduced one day, but not seven days, after TBI, suggesting increased neuronal damage immediately after the injury, and its fast recovery. The brain-derived neurotrophic factor (Bdnf) level in the brain dropped immediately after the trauma, but fully recovered seven days later. A marker of microglial activation, Iba1, was elevated in the TBI brain, albeit decreasing from Day 3. The levels of hypoxia-inducible factor 1-alpha (Hif1a) increased 30 min after the injury, and recovered by Day 7, further supporting the construct validity of the model. Collectively, these findings suggest that our model of laser-induced brain injury in zebrafish reproduces mild TBI and can be a useful tool for TBI research and preclinical neuroprotective drug screening.
The paper is devoted to the development of a new methodology of statistical analysis of laser-induced fluorescence excitation-emission matrices. The method allows one to calculate the number of fluorescent components, their excitation and fluorescence spectra in samples under study without using any prior information about their nature. The algorithm is based on the principal component method and is modified for analysis of excitation-emission matrices. Its features were tested on two-component optical phantoms, namely, optically thin mixtures of fluorescent dyes (pyridoxine and fluorescein). The calculated excitation and fluorescence spectra of components coincided with the initial ones with good accuracy (deviation less than 5% in intensity). The weight coefficients proportional to concentrations were calculated by the new algorithm. The comparison of its ratios with those calculated from known concentrations also showed good agreement (deviation of 5%).
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