ICBM Curso Electivo TericoPrctico Anlisis Cuantitativo de Colocalizacin

ICBM | Curso Electivo Teórico-Práctico | Análisis Cuantitativo de Colocalización en Microscopía Confocal 10 -17 |08|2007 Steffen Härtel Programa de Anatomía y Biología del Desarollo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile

|-> Seminarios |-> Rodrigo Castillo: viernes, 10. 08. 07 @ 9. 30 -13. 00 Measurement of colocalization of objects in dual-color confocal images Manders E. (1993) Journal of Microscopy 169: 375 -382 |-> Ivan Alfarro: lunes, 13. 08. 07 @ 15 -18. 30 STED-Microscopy: Concepts for nanoscale resolution in fluorescence microscopy Hell S. , Dyba, M. , Jakobs S (2004) Current Opinion in Neurobiology 4: 599 -609 |-> Valentina Parra: lunes, 13. 08. 07 @ 15 -18. 30 Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells Costes et al. 2004 Biophys. J. 86, 3993– 4003 |-> Ariel Contreras: martes, 14. 08. 07 @ 9. 30 -13. 00 A syntaxin 1, Galphao, and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization Li, Q. , Lau, A. , Morris, T. J. , Guo, L. , Fordyce, C. B. & Stanley, E. F. (2004) J. Neurosci. 24, 4070– 4081 |-> Barbra Toro: jueves, 16. 08. 07 @ 9. 30 -13. 00 Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies Lachmanovich, E. , Shvartsman, D. E. , Malka, Y. , Botvin, C. , Henis, Y. I. & Weiss, A. M. (2003) Journal of Microscopy. 212, 122– 131 |-> Nancy Leal: jueves, 16. 08. 07 @ 9. 30 -13. 00 Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons Van Steensel, B. , van Binnendijk, E. , Hornsby, C. , van der Voort, H. , Krozowski, Z. , de Kloet, E. & van Driel, R. (1996) J. Cell Sci. 109, 787– 792 |-> Ximena Verges: viernes, 17. 08. 07 @ 9. 30 -13. 00 Multicolour analysis and local image correlation in confocal microscopy Demandolx, D. & Davoust, J. (1997) Journal of Microscopy 185, 21– 36 |-> Leonel Muñoz: viernes, 17. 08. 07 @ 9. 30 -13. 00 A guided tour into subcellular colocalization analysis in light microscopy Bolte S. & Cordelieres P. (2006) Journal of Microscopy, 224 (3): 213– 232

|-> Auto-correlation ‘Auto-correlation measures self-similarity of a time signal and highlights characteristic time constants of underlying processes. ’

|-> Auto-correlation

|-> Auto-correlation Static detection volume: Auto-correlation function for one freely diffusing species of molecules. Mobile detection volume: Amediek et al. 2002

|-> Auto-correlation r

|-> Auto-correlation

|-> Auto-correlation Signal Density: 5% Mean Sig. Int: 30

|-> Auto-correlation The size of the underlying image structures can be determined by the decay-distance r’ of G(r).

|-> PC-CCRI-JMSI CSI / PC CCRI JMSI Correlation Coefficient of Raw Images Joint Moment of Standardized Images

|-> Kernels H (xi, yj, r) · I(x, y) H (xi, yj, r): Y -> X -> Mean: Simplest Low Pass 1 1 1 H =1/9 · 1 1 1 Simplest High Pass -1 -1 -1 H = -1 8 -1 -1

|-> PC-CCRI-JMSI Imagenes I 1/2(x, y) I 1 CSI(xi, yj, r) I 2 yj -> r=2 CSI = PC = r’ xi -> CSI(xi, yj, r) * I 1/2(x, y) CSI(x, y, r) CSI

|-> PC-CCRI-JMSI

|-> PC-CCRI-JMSI =5 = 10 = 500

|-> PC-CCRI-JMSI = 500

|-> ICA or ICQ ICA / ICQ Intensity Correlation Analysis /Quotient

|-> ICQ Є [± 0. 5] Intensity Correlation Quotient One-Sample and Matched-Pairs tests • Sign Test • Mc. Nemar's Test • Wilcoxon Matched-Pairs Signed-Ranks Test • Student-t test for one sample Sign Test p ≤ 0. 5 Σ ( N!/( i ! (N-i) ! )) i = 1, k (k = min(n+, n-))

|-> ICQ Sign Test p ≤ 0. 5 Σ ( N!/( i ! (N-i) ! )) i = 1, k (k = min(n+, n-)) Characteristics: Crudest and most insensitive test. It is also the most convincing and easiest to apply. The level of significance can almost be estimated without the help of a calculator or table. . . Approximation: For N > 30, the Student t-test can be used. However, the Wilcoxon Matched-Pairs Signed-Ranks test is a better alternative. Remarks: The Sign-test answers the question: How often? , whereas other tests answer the question How much? . It must be kept in mind that these two questions might have different answers. When the problem concerns the sizes of the differences, the Wilcoxon Matched-Pairs Signed. Ranks Test should be preferred. Finally, the Sign-test is an almost ideal quick-and-dirty test that can be used to browse datasets or to check the results of other tests (e. g. , as in: "All six subjects show an increase, so why does your test insist that p > 0. 05? "). http: //www. fon. hum. uva. nl/Service/Statistics/Sign_Test. html

|-> calendario I. Image Adquisition I. b|-> Fundamentos de la fluorescencia Jablonski Diagram: Absorción, Conversión interna Fluorescencia Fosforescencia. Stokes Shift Deconvolution Franck-Condon Principle Segmentation Analisys