Identification and restoration in 3D fluorescence microscopy

Alain Dieterlen; Chengqi Xu; Olivier Haeberle; Nicolas Hueber; R. Malfara; B. Colicchio; Serge Jacquey

doi:10.1117/12.559754

4 June 2004 Identification and restoration in 3D fluorescence microscopy

Alain Dieterlen, Chengqi Xu, Olivier Haeberle, Nicolas Hueber, R. Malfara, B. Colicchio, Serge Jacquey

Proceedings Volume 5477, Sixth International Conference on Correlation Optics; (2004) https://doi.org/10.1117/12.559754
Event: Sixth International Conference on Correlation Optics, 2003, Chernivsti, Ukraine

Abstract

3-D optical fluorescent microscopy becomes now an efficient tool for volumic investigation of living biological samples. The 3-D data can be acquired by Optical Sectioning Microscopy which is performed by axial stepping of the object versus the objective. For any instrument, each recorded image can be described by a convolution equation between the original object and the Point Spread Function (PSF) of the acquisition system. To assess performance and ensure the data reproducibility, as for any 3-D quantitative analysis, the system indentification is mandatory. The PSF explains the properties of the image acquisition system; it can be computed or acquired experimentally. Statistical tools and Zernike moments are shown appropriate and complementary to describe a 3-D system PSF and to quantify the variation of the PSF as function of the optical parameters. Some critical experimental parameters can be identified with these tools. This is helpful for biologist to define an aquisition protocol optimizing the use of the system. Reduction of out-of-focus light is the task of 3-D microscopy; it is carried out computationally by deconvolution process. Pre-filtering the images improves the stability of deconvolution results, now less dependent on the regularization parameter; this helps the biologists to use restoration process.

Citation Download Citation

Alain Dieterlen, Chengqi Xu, Olivier Haeberle, Nicolas Hueber, R. Malfara, B. Colicchio, and Serge Jacquey "Identification and restoration in 3D fluorescence microscopy", Proc. SPIE 5477, Sixth International Conference on Correlation Optics, (4 June 2004); https://doi.org/10.1117/12.559754

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