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

I. Image Adquisition II. Deconvolution III. SegmentationIV. Analisys |-> what‘s up

I. Image Adquisition I.a|-> Fundamentos de la microscopía confocal I.b|-> Fundamentos de la fluorescencia II. Deconvolution III. SegmentationIV. Analisys |-> what‘s up

|-> Rodrigo Castillo: viernes, Measurement of colocalization of objects in dual-color confocal images Manders E. (1993) Journal of Microscopy 169: |-> Ivan Alfarro: lunes, STED-Microscopy: Concepts for nanoscale resolution in fluorescence microscopy Hell S., Dyba, M., Jakobs S (2004) Current Opinion in Neurobiology 4: |-> Valentina Parra: lunes, Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells Costes et al Biophys. J. 86, 3993–4003 |-> Ariel Contreras: martes, 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, 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, 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, Multicolour analysis and local image correlation in confocal microscopy Demandolx, D. & Davoust, J. (1997) Journal of Microscopy 185, 21–36 |-> Leonel Muñoz: viernes, A guided tour into subcellular colocalization analysis in light microscopy Bolte S. & Cordelieres P. (2006) Journal of Microscopy, 224 (3): 213–232 |-> Seminarios

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I. Image Adquisition I.a|-> Fundamentos de la microscopía confocal Confocal Microscopy Point Spread Function Refraction Index Mismatch Deconvolution I II. Deconvolution III. SegmentationIV. Analisys |-> Image Adquisition

Richard Feynman ( ) “It is very easy to answer many of these fundamental biological questions. You just look at the thing ! Make microscopes a hundred times more powerful and many problems of biology would be made very much easier.“ |-> I. Image Adquisition

René Descartes ( ) Passions of the soul (  1649)... just look at the thing … ¿ Human visual perception ? Treatise of man (  1637) |-> I. Image Adquisition

glandula pinealis / pineal organ 2| signals from other senses... A combination of... 1| direct signals... 3| feedback loops produce a symbolic representation of an object. |-> I. Image Adquisition

CSI = PC = r’

|-> I. Image Adquisition CSI = PC = r’

Hans Janssen (1595),.... Galileo Galilei (1610) 1. Microscopía |

Interacciones... intra- e inter moleculares... producen cambios... espectrales tiempos de vida polarización intensidad... - Fluorescencia - Fosforescencia Luminescencia:  t ~ s  t ~ s Absorción / Excitación   t  Emissión 1. Basics of Fluorescence

1. Basics of Fluorescence | Photones Energía de un fotón: (~1-5eV) E = h = hc -1 | c =, frecuencia [s -1 ] h, constante de Planck [ Js -1 ], longitud de onda [m] c, velocidad de luz [~ ms -1 ] Energía molecular: E = E rot + E vib + E el 1 : 10 3 : 50·10 3 Energía térmica: E = k T (~ eV, T ~20°C) k = Constante de Bolzmann ( eV/K) pmnmmmmµm 10 cm _ EHzPHzTHzGHz MeVkeV eV E  XUVIRMWRW VibrationRotationValenz Nucleus Internal ElecrtronesMolecules ESR / NMR 300nm 700nm

1. Microscopía | Diffraction limited microscopy

From Geometric Optics to Diffraction Theory: (‚Image Restauration in Fluorescence Microscopy‘, PhD thesis, van Kempen, 1999) Diffraction: The deviation of an electromagnetic wavefront from the path predicted by geometric optics when the wavefront interacts with a physical object such as an opening or an edge. 1. Microscopía | Diffraction limited microscopy

PSF = |U| 2 = f( J 0 ) U, Integral de Difracción de Kirhoff J 0, Serie de funciones de Bessel Óptica no-geométrica / Teoría de difracción 1. Microscopía | Diffraction limited microscopy

x/y z conventional confocal 4-  1. Microscopía | Diffraction limited microscopy

x/y z 2-photon spinning disc STED 1. Microscopía | Diffraction limited microscopy

380 nm 780 nm Fluorescein

1. Microscopía | Diffraction limited microscopy spinning disc confocal

4 π Microscopy 1. Microscopía | Diffraction limited microscopy

STED Microscopy Stimulated Emission Depletion 1. Microscopía | Diffraction limited microscopy

AFM allows the investigation of structural and functional properties of biomolecules in liquid environments, by a unique combination of : subnanometer spatial resolution millisecond temporal resolution piconewton force sensitivity 1. Microscopía | Diffraction limited microscopy

M Grandbois et al. (1998) Biophys J. 1. Microscopía | Diffraction limited microscopy

Z X Y X

exc Stokes: exc < em ? exc Stokes: exc < em 1. Microscopía | Diffraction limited microscopy

em exc Stokes : exc < em n( exc ) > n( em ) 1. Microscopía | Diffraction limited microscopy

Objeto (f) Resultado ~ f Mejor representación de la realidad Deconvolución  PSF Objeto borroso PSF  f + b Imagen con ruido (I) PSF: Point Spread Function f: Object Function b: Offset Function I: Image Matrix N: Noise Function N(PSF(x, y, z)  f(x, y, z) + b(x, y, z)) = I(x, y, z) 1. Microscopía | Diffraction limited microscopy

Z X PSF:  xy ~ 500 nm |  z ~ 1500 nm 1. Microscopía | Diffraction limited microscopy

Snell‘s Law: sin  1 n 1 = sin  2 n [Zeiss Oil] 1.33 [Water] [Air] Index of refraction: n = (  ·  ) 1/2 = c/v,  electric permittivity and  magnetic permeability.  2 ·  2  1 ·  1 1. Microscopía | Diffraction limited microscopy

Refractive Index: RI = n 1 /n 2 = v 2 /v 1 Snell‘s Law: sin  1 n 1 = sin  2 n 2 n = n( ) ! [Zeiss] 1.33 [Water] [Air] (Egner et al., 1998) 1. Microscopía | Diffraction limited microscopy

n 1  n 2 n 1 = n 2 Micro-esfera:  = 6 µm agua/aceite -- aceite/aceite Ley de Snell: n i · sin  i = n k · sin  k n = n( ) ! cover n 3 (150 µm) objective n1n1 n2n2 1. Microscopía | Diffraction limited microscopy

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  I max (255 for 8 bit) ! I(0) > 0 I > I max 1. Microscopía | Diffraction limited microscopy

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