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
|-> Image Adquisition I. a|-> Fundamentos de la microscopía confocal Confocal Microscopy Point Spread Function Refraction Index Mismatch Deconvolution I II. Deconvolution III. Segmentation IV. Analisys
|-> what‘s up I. Image Adquisition I. a|-> Fundamentos de la microscopía confocal I. b|-> Fundamentos de la fluorescencia II. Deconvolution III. Segmentation IV. Analisys
|-> 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
|-> intensity The observation volume (femtoliter) defined by the Point Spread Function must be considered as a mini-sprectrofluorimeter. 1. You need to consider the Offset I(0) in order to calibrate your signal I(0) 0 ! 2. Never saturate the signal: I Imax (255 for 8 bit) ! I > Imax I(0) > 0
|-> refractive index Use the right inmersion setup ! Keep refractive index / index of refraction constant !
|-> refractive index Index of refraction / refractive index: n = ( · )1/2 = c/v, electric permittivity and magnetic permeability. Snell‘s Law: sin 1 = sin 2 1· 1 Examples for n: • 1. 518 [Zeiss Oil] • 1. 33 [Water] • 1. 0008 [Air] 2· 2
|-> refractive index Refractive Index: RI = n 1/n 2 = v 2/v 1 Snell‘s Law: sin 1 = sin 2 n = n( ) ! (Egner et al. , 1998)
|-> deconvolution PSF: Point Spread Function N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z) f: Object Function b: Offset Function I: Image Matrix Objeto (f) PSF Objeto borroso PSF f + b N: Noise Function Deconvolución Resultado ~ f Mejor representación de la realidad Imagen con ruido (I)
|-> PSF calculator PSF: Point Spread Function N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z) f: Object Function b: Offset Function I: Image Matrix N: Noise Function http: //support. svi. nl/wiki/Nyquist. Calculator
|-> PSF calculator PSF: Point Spread Function f: Object Function b: Offset Function N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z) Backprojected confocal pinhole I: Image Matrix N: Noise Function http: //support. svi. nl/wiki/Nyquist. Calculator
|-> PSF calculator PSF: Point Spread Function N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z) f: Object Function b: Offset Function I: Image Matrix N: Noise Function http: //support. svi. nl/wiki/Nyquist. Calculator
|-> Noise Statistics Noise Information Theory Statistical Physics & Fluctuation Probability Stochastic Theory Processes Literature: eg. Noise Theory and Application to Physics: Philippe Réfrégier, Springer
|-> Noise PSF: Point Spread Function f: Object Function b: Offset Function I: Image Matrix N: Noise Function - Black Body Irrdiation (Poisson) - Detector Noise (Gauss) N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z)
|-> Noise
|-> Noise Exponential Gauss Uniform Gamma Rayleigh
|-> Noise
|-> Nyquist /Shannon Theorem - Undersampling looses structures. - Oversampling waists memory/computation time. The ‘Nyquist /Shannon Theorem‘ or ‘Sampling Theorem‘ for the digital sampling of analogue signals suggests a Nyquist rate NR 2 ? ! Diffraction theory calculates lateral NR ~ 20 pixel/µm(~50 nm/pixel) !. . . laxial NR ~ (~150 nm/pixel)
|-> PSF calculator PSF: Point Spread Function N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z) f: Object Function b: Offset Function I: Image Matrix N: Noise Function http: //support. svi. nl/wiki/Nyquist. Calculator
|-> PSF calculator PSF: Point Spread Function N(PSF(x, y, z) f(x, y, z) + b(x, y, z)) = I(x, y, z) f: Object Function b: Offset Function I: Image Matrix N: Noise Function http: //support. svi. nl/wiki/Nyquist. Calculator
I|-> saturation The observation volume (femtoliter) defined by the Point Spread Function must be considered as a mini-sprectrofluorimeter. 1. You need to consider the Offset I(0) in order to calibrate your signal I(0) 0 ! 2. Never saturate the signal: I Imax (255 for 8 bit) ! I > Imax I(0) > 0
II|-> Nyquist The sampling frequency (or sampling distance) is a function of the observation volume (femtoliter) defined by the Point Spread Function (PSF): 3. You need to consider sampling distances in x and y 50 nm and z 150 -300 nm for later deconvolution, or calculate the explicit sample distances at http: //support. svi. nl/wiki/Nyquist. Calculator
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