procesamiento de imagenes 3d sistemas de captura 3d conceptos de resolucion axial y lateral concepto...
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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
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