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Proceedings Paper

Recent advances in 3-D fluorescence microscopy: tomography as a source of information
Author(s): A. Dieterlen; M. Debailleul; A. De Meyer; B. Simon; V. Georges; B. Colicchio; O. Haeberlé; V. Lauer
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Paper Abstract

3-D optical fluorescent microscopy becomes now an efficient tool for volume investigation of living biological samples. Developments in instrumentation have permit to beat off the conventional Abbe limit, in any case the recorded image can be described by the convolution equation between the original object and the Point Spread Function (PSF) of the acquisition system. If the goal is 3-D quantitative analysis, whether you improve the instrument capabilities, or (and) you restore the data. These last is until now the main task in our laboratory. Based on the knowledge of the optical Transfer Function of the microscope, deconvolution algorithms were adapted to automatic determine the regularisation threshold in order to give less subjective and more reproducible results. The PSF represents the properties of the image acquisition system; we have proposed the use of statistical tools and Zernike moments to describe a 3-D system PSF and to quantify the variation of the PSF. This first step toward standardization is helpful to define an acquisition protocol optimizing exploitation of the microscope depending on the studied biological sample. We have pointed out that automating the choice of the regularization level; if it facilitates the use, it also greatly improves the reliability of the measurements. Furthermore, to increase the quality and the repeatability of quantitative measurements a pre-filtering of images improves the stability of deconvolution process. In the same way, the PSF pre-filtering stabilizes the deconvolution process. We have shown that Zernike polynomials can be used to reconstruct experimental PSF, preserving system characteristics and removing the noise contained in the PSF. Fluorescent microscopes suffer from limitations; photobleaching and phototoxicity effects, or influence of the sample optical properties to 3-D observation. Amplitude and phase of the object can be reached with optical tomography based on a combination of microholography with a tomographic illumination. So indices cartography of the specimen can be obtained, and combined with fluorescence information it will open new possibilities in 3-D optical microscopy.

Paper Details

Date Published: 22 April 2008
PDF: 8 pages
Proc. SPIE 7008, Eighth International Conference on Correlation Optics, 70080S (22 April 2008); doi: 10.1117/12.796995
Show Author Affiliations
A. Dieterlen, Univ. of Haute Alsace (France)
M. Debailleul, Univ. of Haute Alsace (France)
A. De Meyer, Univ. of Haute Alsace (France)
B. Simon, Univ. of Haute Alsace (France)
V. Georges, Univ. of Haute Alsace (France)
B. Colicchio, Univ. of Haute Alsace (France)
O. Haeberlé, Univ. of Haute Alsace (France)
V. Lauer, Lauer Microscopie (France)

Published in SPIE Proceedings Vol. 7008:
Eighth International Conference on Correlation Optics

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