SISTEMAS 3D/4D.

Presentación del tema: "SISTEMAS 3D/4D."— Transcripción de la presentación:

1 SISTEMAS 3D/4D

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

3 SISTEMAS DE CAPTURA 3D IX81 , IX71 motorizado BX61 , BX51 motorizado
BX61WI , BX51WI motorizado CONFOCAL DSU FV1000 FV300 WIDE FIELD DECONVOLUCION

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

5 CONCEPTO DE PSF 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 Objective Cover slip Sample

6 CONCEPTO DE APERTURA NUMERICA
1.4 1.3 La cantidad de luz detectada (por el ojo, la CCD o el fotomultiplicador) es inversamente proporcional al cuadrado de la magnificación. MX SEÑAL La cantidad de luz detectada (por el ojo, la CCD o el fotomultiplicador) es directamente proporcional a la apertura numérica (NA). N.A. SEÑAL Ergo, para experimentos de baja luminosidad usar lentes de baja magnificación y alta NA (p.ej. 60X aceite 1.42) N.A Z XY Mayor resolucion!!

7 CONFOCAL vs WIDEFIELD

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

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

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

11 QED

12 Multidimensional acquisition and analysis
Count Size Extended Depth of Field

13 WIDE FIELD (X-Y-Z-λ-t) t λ λ t QED Z PCI-bus RS-232C or USB RS-232C
Filter wheel with Shutter Filter wheel Mot stage IX81 QED t λ Z Y x λ t

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

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

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

17 Wide field 2D Blind deconvolution

18 Multiple wavelengths - Widefield

19 2D Blind deconvolution – TIRF
widefield

20 SISTEMA CONFOCAL SISTEMAS FV 300 , FV1000
Maxima eficiencia, minima resolucion AXIAL SISTEMA DISK SCANNING UNIT DSU Maxima VELOCIDAD, minimos requerimientos

21 SISTEMAS CONFOCALES DSU

22 How to choose ‘Disk’ ? Brightness Confocalitty Airy pattern L > D
DAiry=1.2*MagObj*λ/NAObj L D

23 FV1000 FRAP , FRET , FLIP , TIME LIPSE and LIVE CELL PSF for CONFOCAL
SISTEMAS CONFOCALES FV1000 FRAP , FRET , FLIP , TIME LIPSE and LIVE CELL PSF for CONFOCAL

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

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

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

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

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

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

30 SISTEMAD DE RECONSTRUCCION EN 3D
CONFOCAL SYSTEM FV CONFOCAL tienen incluido el modulo 3D para la visualizacion de los STACK Tambien el modulo “Tiempo” permite el analisis de la señal en esta dimension para aplicaciones 4D. FV 1000 con su software FV ASW 1.4 PERMITE DIFERENTES ALGORITMOS DE VISUALIZACION DE IMAGENES 3D Tambien FV ASW 1.4 permite hacer barridos l , llegando a la 5D

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

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

33 3D 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 3D solution for light microscopy when combined with Image-Pro Plus’ Scope-Pro and AFA modules. • Reads most major file formats including: • AutoQuant • TIFF-series • Image-Pro Sequence • Olympus FluoView Confocal

34 3D constructor Visualization Features Include:
Volume Measurement Options Include: • Volume • Surface Area • Sphericity • Feret measurements • Bounding box measurements • Centroid information • Automatic update of measurements from frame-toframe playback of 4D 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.

35 ANALISIS DE 4D 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