Confocal microscopy: an experimental set up for biomolecule structure investigation based on dynamical fluorescence spectroscopy

Amanda Oldani; Maddalena Collini; Giuseppe Chirico; Laura D'Alfonso; Giancarlo Baldini; Filomena Mazzei; Flavia Barone; Luciano Cellai; Cesare Giordano; Marziale Milani; Antonio Pasinetti

doi:10.1117/12.500197

9 October 2003 Confocal microscopy: an experimental set up for biomolecule structure investigation based on dynamical fluorescence spectroscopy

Amanda Oldani, Maddalena Collini, Giuseppe Chirico, Laura D'Alfonso, Giancarlo Baldini, Filomena Mazzei, Flavia Barone, Luciano Cellai, Cesare Giordano, Marziale Milani, Antonio Pasinetti

Author Affiliations +

Proceedings Volume 5139, Confocal, Multiphoton, and Nonlinear Microscopic Imaging; (2003) https://doi.org/10.1117/12.500197
Event: European Conferences on Biomedical Optics 2003, 2003, Munich, Germany

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, Maddalena Collini, Giuseppe Chirico, Laura D'Alfonso, Giancarlo Baldini, Filomena Mazzei, Flavia Barone, Luciano Cellai, Cesare Giordano, Marziale Milani, and 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); https://doi.org/10.1117/12.500197

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KEYWORDS

Modulation

Microscopes

Phase shift keying

Luminescence

Anisotropy

Fluorescence anisotropy

Confocal microscopy

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