Comparison and applications to in vivo biology of two different methods of spectral imaging: confocal scanning full spectrum and global illumination spectral band-pass imaging

Paolo Valisa; S. Sharonov; C. Favard; C. Herben; Michel Manfait; Paul Vigny; E. Da Silva

doi:10.1117/12.228886

27 December 1995 Comparison and applications to in vivo biology of two different methods of spectral imaging: confocal scanning full spectrum and global illumination spectral band-pass imaging

Paolo Valisa, S. Sharonov, C. Favard, C. Herben, Michel Manfait, Paul Vigny, E. Da Silva

Author Affiliations +

Proceedings Volume 2627, Optical Biopsies; (1995) https://doi.org/10.1117/12.228886
Event: BiOS Europe '95, 1995, Barcelona, Spain

Abstract

With classical fluorescence microscopes, the sample is illuminated by a monochromatic source and the image is recorded through a bandpass filter that selects a portion of the emitted fluorescence. Recently, the spatial resolution and sensitivity of these devices have been considerably increased with the introduction of confocality and large 2D CCD array detectors and it is now possible to perform even 3D mapping of fluorescent markers in single living cells. Unfortunately, small changes occurring in fluorescence spectra cannot be mapped so easily. A set of colored filters is hardly enough even to separate slightly overlapping fluorescence contributions from multiple marking. The number of biological studies requiring a detailed description of fluorescence spectrum is growing continuously. Spectral changes are induced, for example, by environment (hydrophilic/hydrophobic binding), pH, oxygenation, ion concentration (Ca, Mg, Na), conformation of bound macromolecule (A/B DNA, protein folding), metabolism, dimerization of the probe, etc. . . . The classical approach to these problems is to couple a spectrograph to a microscope and obtain, point after point, a set of significative fluorescence spectra from the sample. This is a long procedure which gives incomplete information. We report in this paper on two methods we have developed to quickly record an array of spectra over the sample and to map spectral features such as bandwidth, maxima shifts, or decomposition in multiple overlapping components. We distinguish between the confocal scanning method and the global illumination (non confocal) method.

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Paolo Valisa, S. Sharonov, C. Favard, C. Herben, Michel Manfait, Paul Vigny, and E. Da Silva "Comparison and applications to in vivo biology of two different methods of spectral imaging: confocal scanning full spectrum and global illumination spectral band-pass imaging", Proc. SPIE 2627, Optical Biopsies, (27 December 1995); https://doi.org/10.1117/12.228886

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