PROCESAMIENTO DE IMAGENES 3DPROCESAMIENTO 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

SISTEMAS DE CAPTURA 3DSISTEMAS DE CAPTURA 3D IX81 , IX71 motorizado BX61 , BX51 motorizado BX61WI , BX51WI motorizado

CONFOCALDSU FV1000 FV300 WIDE FIELD

DECONVOLUCION

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

La resolucion Axial viene dada por la APERTURA NUMERICA DEL OBJETIVO , indice de refraccion, longitud de onda.

zz

X

YY

X

CONCEPTO DE PSFCONCEPTO 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

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)

CONCEPTO DE APERTURA CONCEPTO DE APERTURA NUMERICANUMERICA

1.4

1.3

N.A. Z XY Mayor resolucion!!

CONFOCAL vs WIDEFIELDCONFOCAL vs WIDEFIELD

SISTEMA WIDE FIELDSISTEMA WIDE FIELD

IX o BX , CAMARA , AND???

PSF para WIDE FIELD

OPCIONES paraaplicaciones QED

DIFERENTES SHUTTERS , TIRFM con laser

Contro de multiples FILTROS

SINCRONIZACION Z- filtros

SINCRONIZACION shutters- filtros- Z

QEDQEDCamera 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

QED in vivo =QED in vivo =3D, Deconv. & 3D, Deconv. & MeasurementMeasurement

QED

Multicolor Z and burst Z acquisition

IMAGE PRO PLUS

Multi Color Deconvolution 2D&3D Blind

Advanced 3D analysis and Measurements

QEDQED

Multidimensional acquisition and Multidimensional acquisition and analysisanalysis

Count Size

Extended Depth of Field

RS-232C

RS-232C or USB

PCI-bus

Filter wheel with Shutter

Filter wheel

Mot stage

IX81

(X-Y-Z-λ-t)WIDE FIELDWIDE FIELD

Z

Y

x

QED

λ

λ

t

t

WIDE FIELD DECONVOLUCIONWIDE FIELD DECONVOLUCION

When to use deconvolution?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

WIDE FIELD DECONVOLUCIONWIDE FIELD DECONVOLUCION

When to use deconvolution?When to use deconvolution? Documentation

Images and projections will show more detail and greater contrast

Improvements can be striking

WIDE FIELD DECONVOLUCIONWIDE FIELD DECONVOLUCION

When to use deconvolution?When to use deconvolution? Analysis

Deconvolution retains quantitative accuracy

Improves contrast facilitating analysis Counting Tracking Area measurements Intensity measurements

Wide fieldWide field 2D Blind deconvolution2D Blind deconvolution

Wide fieldWide field

Multiple wavelengths - WidefieldMultiple wavelengths - Widefield

widefieldwidefield2D Blind deconvolution – TIRF2D Blind deconvolution – TIRF

SISTEMAS FV 300 , FV1000 Maxima eficiencia, minima

resolucion AXIAL

SISTEMA DISK SCANNING UNIT DSU Maxima VELOCIDAD, minimos

requerimientos

SISTEMA CONFOCALSISTEMA CONFOCAL

SISTEMAS CONFOCALESSISTEMAS CONFOCALES DSU

How to choose ‘Disk’ ?How to choose ‘Disk’ ? Brightness

L > D Confocalitty

L = 0.6 * D

Airy pattern DAiry=1.2*MagObj*λ/

NAObj D

L

SISTEMAS CONFOCALESSISTEMAS CONFOCALES

FV1000

FRAP , FRET , FLIP , TIME LIPSE and LIVE CELLPSF for CONFOCAL

APERTURA CONFOCAL Y APERTURA CONFOCAL Y RESOLUCION AXIALRESOLUCION AXIAL

RESOLUCION AXIAL

Zr=2*nj*λ/NA^2Obj

CELL200n

m

WIDE FIELD DECONVOLUCIONWIDE FIELD DECONVOLUCION

When to use deconvolution?When to use deconvolution? In confocal microscopy

Improve signal to noise ratios increase dynamic range Improve image grayscale resolution Increase axial resolution

SISTEMAS DE SISTEMAS DE DECONVOLUCOIONDECONVOLUCOION SHARP STACK, Application widefield , brightfiels

SHARP STACK PLUSWidefield , Mejora detalles mediante

algoritmos más poderosos

AUTO DEBLUR, Para deconvolucion de imagenes confocales.

What happens during What happens during deconvolution?deconvolution?

How it’s done (8/8)How it’s done (8/8)

Non-Blind DeconvolutionNon-Blind Deconvolution

ReblurredInitialObject

EstimatePSF Original

Blurred-

Update ErrorImproved

ObjectEstimate

Blind deconvolutionBlind deconvolution

Current PSF

ReblurredCurrent Object

EstimateOriginal Blurred

-

Update ErrorNew ObjectEstimate

Current PSF

ReblurredCurrent Object

EstimateOriginal Blurred

-

Update ErrorNew ObjectEstimate

Current PSF

ReblurredCurrent Object

EstimateOriginal Blurred

-

Update ErrorNew PSFEstimate

Current PSF

ReblurredCurrent Object

EstimateOriginal Blurred

-

Update ErrorNew PSFEstimate

SISTEMAD DE RECONSTRUCCION EN 3DSISTEMAD DE RECONSTRUCCION EN 3DCONFOCAL 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 1.4PERMITE DIFERENTES ALGORITMOS DE VISUALIZACION DE

IMAGENES 3D Tambien FV ASW 1.4 permite hacer

barridos llegando a la 5D

SISTEMAD DE RECONSTRUCCION EN SISTEMAD DE RECONSTRUCCION EN 3D3D

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.

ANALISIS DE 3D WIDEFIELDANALISIS 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.

3D constructor3D 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

3D constructor3D constructorVolume 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.

ANALISIS DE 4DANALISIS DE 4DMeasurement 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