9 October 2003 Confocal microscopy: an experimental set up for biomolecule structure investigation based on dynamical fluorescence spectroscopy
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Abstract
Confocal microscopy is one of the most widely used and non-invasive tool for the investigation of biological matter. It improves the performances of the optical microscope by reducing the excitation volume and enhancing the axial resolution due to the use of high numerical aperture lenses. We have adapted an inverted confocal optical microscope to the measurement of fluorescence emission dynamics (lifetime and fluorescence polarization anisotropy). The dynamic spectroscopy measurements are obtained with phase fluorometry and are based on a modulated linearly polarized laser beam which is fed to the epifluorescence port of the microscope. We report the test of the microscope by comparing the lifetime and fluorescence polarization anisotropy decay obtained in cuvettes in the standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor is introduced in the best fit functions used in the data analysis of the decays, the results obtained on microliters volumes is comparable to those obtained in cuvettes in the standard phase fluorometer spectrometer. An example of application is reported on short DNA fragments.
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Amanda Oldani, Amanda Oldani, Maddalena Collini, Maddalena Collini, Giuseppe Chirico, Giuseppe Chirico, Laura D'Alfonso, Laura D'Alfonso, Giancarlo Baldini, Giancarlo Baldini, Filomena Mazzei, Filomena Mazzei, Flavia Barone, Flavia Barone, Luciano Cellai, Luciano Cellai, Cesare Giordano, Cesare Giordano, Marziale Milani, Marziale Milani, Antonio Pasinetti, Antonio Pasinetti, "Confocal microscopy: an experimental set up for biomolecule structure investigation based on dynamical fluorescence spectroscopy", Proc. SPIE 5139, Confocal, Multiphoton, and Nonlinear Microscopic Imaging, (9 October 2003); doi: 10.1117/12.500197; https://doi.org/10.1117/12.500197
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