PROCESAMIENTO DE IMAGENES 3 D n n n

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PROCESAMIENTO DE IMAGENES 3 D n n n n n SISTEMAS DE CAPTURA 3

PROCESAMIENTO DE IMAGENES 3 D n n n n n SISTEMAS DE CAPTURA 3 D CONCEPTOS DE RESOLUCION AXIAL Y LATERAL CONCEPTO DE APERTURA NUMERICA SISTEMA WIDE FIELD SISTEMA CONFOCAL SISTEMAS DE DECONVOLUCOION SISTEMAD DE RECONSTRUCCION EN 3 D ANALISIS DE 4 D

SISTEMAS DE CAPTURA 3 D n n n IX 81 , IX 71 motorizado

SISTEMAS DE CAPTURA 3 D n n n IX 81 , IX 71 motorizado BX 61 , BX 51 motorizado BX 61 WI , BX 51 WI motorizado CONFOCAL DSU FV 1000 FV 300 n n n DECONVOLUCION WIDE FIELD

CONCEPTOS DE RESOLUCION AXIAL (Z) Y LATERAL (XY) z z Y Y X X

CONCEPTOS DE RESOLUCION AXIAL (Z) Y LATERAL (XY) z z Y Y X X La resolucion Axial viene dada por la APERTURA NUMERICA DEL OBJETIVO , indice de refraccion, longitud de onda.

CONCEPTO DE PSF n n n Point Spread Function, Función de Difuminación del Punto

CONCEPTO DE PSF n n n Point Spread Function, Función de Difuminación del Punto , es la forma de reproducir un punto para una muestra optica, dicho punto se deformara dependiendo del sistema. Dicha funcion seria un PUNTO para un sistema optico perfecto. TENEMOS UNA PSF PARA CADA SISTEMA OPTICO Cada elemento en el camino optico la modifica n Objective n Cover slip n Sample

CONCEPTO DE APERTURA NUMERICA La cantidad de luz detectada (por el ojo, la CCD

CONCEPTO DE APERTURA NUMERICA La cantidad de luz detectada (por el ojo, la CCD o el fotomultiplicador) es inversamente proporcional al cuadrado de la magnificación. 1. 4 MX SEÑAL n La cantidad de luz detectada (por el ojo, la CCD o el fotomultiplicador) es directamente proporcional a la apertura numérica (NA). 1. 3 N. A. SEÑAL n n Ergo, para experimentos de baja luminosidad usar lentes de baja magnificación y alta NA (p. ej. 60 X aceite 1. 42) N. A. Z XY Mayor resolucion!!

CONFOCAL vs WIDEFIELD

CONFOCAL vs WIDEFIELD

SISTEMA WIDE FIELD n IX o BX , CAMARA , AND? ? ? OPCIONES

SISTEMA WIDE FIELD n IX o BX , CAMARA , AND? ? ? OPCIONES paraaplicaciones QED §DIFERENTES SHUTTERS , TIRFM con laser §Contro de multiples FILTROS PSF para WIDE FIELD §SINCRONIZACION Z- filtros §SINCRONIZACION shuttersfiltros- Z

QED Camera Control Fully Automated Microscope Control Multi Color Imaging, Time Lapse Imaging, Burst

QED Camera Control Fully Automated Microscope Control Multi Color Imaging, Time Lapse Imaging, Burst acquisition 3 D, z-series, burst 3 D with piezo Tiling, Mosiac, Slide and Well scanning

QED in vivo =3 D, Deconv. & Measurement QED Multicolor Z and burst Z

QED in vivo =3 D, Deconv. & Measurement QED Multicolor Z and burst Z acquisition IMAGE PRO PLUS Multi Color Deconvolution 2 D&3 D Blind Advanced 3 D analysis and Measurements

QED

QED

Multidimensional acquisition and analysis Count Size Extended Depth of Field

Multidimensional acquisition and analysis Count Size Extended Depth of Field

WIDE FIELD (X-Y-Z-λ-t) t Filter wheel QED PCI-bus λ RS-232 C or USB IX

WIDE FIELD (X-Y-Z-λ-t) t Filter wheel QED PCI-bus λ RS-232 C or USB IX 81 Mot Y stage Filter wheel with Shutter x Z λ t RS-232 C

WIDE FIELD DECONVOLUCION When to use deconvolution? n In widefield fluorescent Microscopy n n

WIDE FIELD DECONVOLUCION When to use deconvolution? n In widefield fluorescent Microscopy n n n Improve contrast of 2 D and 3 D images Improve resolution in three dimensions Improve 3 D rendering and visualization

WIDE FIELD DECONVOLUCION When to use deconvolution? n Documentation n n Images and projections

WIDE FIELD DECONVOLUCION When to use deconvolution? n Documentation n n Images and projections will show more detail and greater contrast Improvements can be striking

WIDE FIELD DECONVOLUCION When to use deconvolution? n Analysis n n Deconvolution retains quantitative

WIDE FIELD DECONVOLUCION When to use deconvolution? n Analysis n n Deconvolution retains quantitative accuracy Improves contrast facilitating analysis n n Counting Tracking Area measurements Intensity measurements

Wide field 2 D Blind deconvolution

Wide field 2 D Blind deconvolution

Multiple wavelengths - Widefield Wide field

Multiple wavelengths - Widefield Wide field

2 D Blind deconvolution – TIRF widefield

2 D Blind deconvolution – TIRF widefield

SISTEMA CONFOCAL n § SISTEMAS FV 300 , FV 1000 Maxima eficiencia, minima resolucion

SISTEMA CONFOCAL n § SISTEMAS FV 300 , FV 1000 Maxima eficiencia, minima resolucion AXIAL SISTEMA DISK SCANNING UNIT DSU Maxima VELOCIDAD, minimos requerimientos

SISTEMAS CONFOCALES n DSU

SISTEMAS CONFOCALES n DSU

How to choose ‘Disk’ ? n Brightness n n L Confocalitty n n L>D

How to choose ‘Disk’ ? n Brightness n n L Confocalitty n n L>D L = 0. 6 * D Airy pattern n DAiry=1. 2*Mag. Obj*λ/NAO bj D

SISTEMAS CONFOCALES n FV 1000 FRAP , FRET , FLIP , TIME LIPSE and

SISTEMAS CONFOCALES n FV 1000 FRAP , FRET , FLIP , TIME LIPSE and LIVE CELL PSF for CONFOCAL

APERTURA CONFOCAL Y RESOLUCION AXIAL 200 nm n Zr=2*nj*λ/NA^2 Obj CELL n

APERTURA CONFOCAL Y RESOLUCION AXIAL 200 nm n Zr=2*nj*λ/NA^2 Obj CELL n

WIDE FIELD DECONVOLUCION When to use deconvolution? n In confocal microscopy n n Improve

WIDE FIELD DECONVOLUCION When to use deconvolution? n In confocal microscopy n n Improve signal to noise ratios increase dynamic range Improve image grayscale resolution Increase axial resolution

SISTEMAS DE DECONVOLUCOION SHARP STACK, Application widefield , brightfiels n SHARP STACK PLUS Widefield

SISTEMAS DE DECONVOLUCOION SHARP STACK, Application widefield , brightfiels n SHARP STACK PLUS Widefield , Mejora detalles mediante algoritmos más poderosos n n AUTO DEBLUR, Para deconvolucion de imagenes confocales.

What happens during deconvolution? How it’s done (8/8)

What happens during deconvolution? How it’s done (8/8)

Non-Blind Deconvolution Initial Object Estimate PSF Reblurred - Improved Object Estimate Update Error Original

Non-Blind Deconvolution Initial Object Estimate PSF Reblurred - Improved Object Estimate Update Error Original Blurred

Blind deconvolution Current Object Current Estimate PSF New PSF Estimate Current Object Current Estimate

Blind deconvolution Current Object Current Estimate PSF New PSF Estimate Current Object Current Estimate PSF New Object Estimate Reblurred - Update Error Original Blurred

SISTEMAD DE RECONSTRUCCION EN 3 D CONFOCAL SYSTEM n FV CONFOCAL tienen incluido el

SISTEMAD DE RECONSTRUCCION EN 3 D CONFOCAL SYSTEM n FV CONFOCAL tienen incluido el modulo 3 D para la visualizacion de los STACK n Tambien el modulo “Tiempo” permite el analisis de la señal en esta dimension para aplicaciones 4 D. n FV 1000 con su software FV ASW 1. 4 PERMITE DIFERENTES ALGORITMOS DE VISUALIZACION DE IMAGENES 3 D n Tambien FV ASW 1. 4 permite hacer barridos l , llegando a la 5 D

SISTEMAD DE RECONSTRUCCION EN 3 D CONFOCAL DSU and WIDEFIELD n n Estos sistemas

SISTEMAD DE RECONSTRUCCION EN 3 D CONFOCAL DSU and WIDEFIELD n n Estos sistemas utilizan el QED como sistema de CAPTURA 3 D y el IPP con los modulos de 3 D Constructor como sistema de reconstruccion en 3 D. Estos sistemas tambien permiten hacer SEGUIMIENTOS EN EL TIEMPO , analisis de MULTIFLUOROFOROS y reconstrucciones en 3 D.

ANALISIS DE 3 D WIDEFIELD n n 3 D Constructor contains the following modules:

ANALISIS DE 3 D WIDEFIELD n n 3 D Constructor contains the following modules: 3 D Viewer. The 3 D Viewer includes the Volume Renderer, Slicer, Measurement, and Animation features. 3 D Filters. A new item, 3 D Filters, adds convolution and morphological filters to Image-Pro Plus. These filters appear on the Process menu. A new button to activate 3 D Constructor will appear on your Image-Pro Plus toolbar.

3 D constructor Features • Seamless integration with Image-Pro Plus. • Full 32 -bit

3 D constructor Features • Seamless integration with Image-Pro Plus. • Full 32 -bit application available for Microsoft® Windows 2000 and XP Professional. • Multi-language menu selections (upon request). • Fully automated 3 D solution for light microscopy when combined with Image-Pro Plus’ Scope-Pro and AFA modules. • Reads most major file formats including: • Auto. Quant • TIFF-series • Image-Pro Sequence • Olympus Fluo. View Confocal

3 D constructor Volume Measurement Options Include: • Volume • Surface Area • Sphericity

3 D constructor Volume Measurement Options Include: • Volume • Surface Area • Sphericity • Feret measurements • Bounding box measurements • Centroid information • Automatic update of measurements from frame-toframe playback of 4 D sequences • Set filter ranges for any measurement Visualization Features Include: • Calibrated voxel size. • Adjustable lighting controls for enhanced viewing. • Stack Slicer for displaying synchronized, orthogonal views through an image stack. • Animation • Multiple surface renderings • Time-based display of time-lapse volume sequences.

ANALISIS DE 4 D Measurement Features Manual Measurements Include: • Individual point • Point-to-Point

ANALISIS DE 4 D Measurement Features Manual Measurements Include: • Individual point • Point-to-Point distance • Point-to-Surface distance • Line • Angle • Point-to-Line distance • Circumference of an object bisected by a plane • Surface distance between two points • Tracking Data Display and Handling • Individual object measurements