1 Percepción y Deconvolución Dr. Steffen Härtel, CECS, Valdivia, Chile

2 1| La percepción humana 2| Visión de máquinas (machine vision) en la microscopía contemporánea 3| Ejemplos de aplicación en procesos relacionados con la membrana celular

3 1| La percepción humana René Descartes (1596-1650) Epiphysis cerebri / órgano pineal 2| señales de otros sentidos... logramos una representación simbólica del mundo exterior integrando información de... 1| señales directas de objetos 3| circuitos realimentativos

4 2| Visión de máquinas (machine vision) René Descartes (1596-1650) Epiphysis cerebri / órgano pineal NAOS: Óptica Adaptativa

5 Hans Janssen (1595),.... Galileo Galilei (1610)

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7 2|Deconvolución Objeto OriginalObjeto Observado Deconvolución Objeto Reconstruido Observación

8 10 µm 2|Deconvolución Objeto OriginalObjeto Observado Deconvolución Objeto Reconstruido Observación

9 2|Deconvolución Células OriginalesCortes a través de 2 células Deconvolución Reconstrucción 3D Observación

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12 Figure M3: Three-dimensional reconstruction of two cells from the pineal organ of a zebrafish embryo in vivo, 24h after fertilization. The cells express a membrane tagged version of the green fluorescent protein (GFP-Gap43) under the control of a specific promoter. Confocal data was deconvolved with SVI- Software and reconstructed with 3D-SCIAN routines. The reconstruction leads to a high definition of morphological cellular details like filopodia.

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15 Figure M2: Three-Dimensional reconstruction of two HeLa-cells which were transfected with f-EGFP (f-EGFP was kindly provided by HH Gerdes, 48). Confocal data was deconvolved with SVI-Software and reconstructed with 3D-SCIAN routines. In contrast to Fig. M1, cells were grown until they formed 2-3 confluent layers. Since the rate of transfection with f-EGFP is low (5- 10%), the details of single embedded cells can be resolved satisfactorily: filopodia (diameter 200 nm) can still be identified (compare to Fig. M1). Bar = 15 m.

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17 Hydrogen peroxide induced cell swelling in necrotic HeLa cells. Absolute volume was measured by reconstructing 3D confocal microcopy stacks of DiIC 18 -stained HeLa cells as described in Castro et al. (2005). A cluster of cells was imaged before and during exposure to hydrogen peroxide, which started at zero min. For each time, the xy-projection is shown together with a cross section in the xz-plane taken at the indicated y-position (dotted line). Bars represent 20 µm. Right: Normalized volumes for four experiments (0, 15, 30, 45 min) and two experiments (-15 and 60 min). In each experiment 4 to 13 cells were measured. For 24 control cells the volume measured was 4.0 ± 1.0 pl. Context: Image processed confocal microscopy opens the access to diverse structural, morphological, and physiological features in cell biology in a unique manner. The routines recently developed in the scope of the project FONDECYT 3030065 permit to determine time resolved changes in cellular volume precisely on a single cell basis, and to couple the data with novel molecular mechanisms of ion transport during cell death. In the presented context, the data published in Casto et al. (2005) reveals that cell swelling occurs before a delayed Ca 2+ overload in HeLa cells. La figura esta publicada en: Castro, J., Ruminot, I., Porras, O., Flores, C, Hermosilla, T., Verdugo, E., Härtel, S. & Barros, L.P. (2005) ATP steal: a novel mechanism linking Na+ with the onset of necrotic Ca2+ overload. revised version submitted to Cell Death & Differentiation. La técnica de reconstrucción 3D de células a través de procesamiento de imágenes esta incluida en las siguientes presentaciones: 1.MORFOGÉNESIS ASIMÉTRICA DEL ÓRGANO PARAPINEAL EN EL CEREBRO EMBRIONARIO DE PEZ CEBRA. (Asymmetric morphogenesis of the parapineal organ in the embryonic zebrafish brain). Cabrejos M.E., Lemus C.G., Härtel S., Concha M.L. Programa de Anatomía y Biología del Desarrollo, ICBM, Facultad de Medicina, Universidad de Chile. CECS, Valdivia. 2.CROSS CORRELATION AND SCALING INDEX METHODS FOR THE AUTOMATED QUANTIFICATION OF COLOCALIZATION IN FLUORESCENCE MICROSCOPY. Härtel 1 S., Cornejo 1 I., Rojas 2 R. y Barros 1 L.F. 1 Centro de Estudios Científicos, Arturo Prat 514, Valdivia, Chile. 2 Xperts Ltda., Yerbas Buenas 170, Valdivia, Chile.

18 objetivo  400 nm

19   ?