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3D Image Deconvolution
Basic Microscopy Requirements
MagnificationHow large can I make something appear?
ResolutionHow small an object can I detect?
ContrastWhat is real and what is artifact?
Magnification
Resolution
DIC image, 100X magnificationA. North, JCB 2006
Contrast
Signal to BackgroundBackground is the systematic addition to the intensity of the signal.
Contrast
Signal to NoiseNoise is any random variation that adds to the uncertainty of the signal
Microtubules (Objects)
Microtubules (Images)
Appearance of a 100 nm (0.1 mm) Fluorescent Bead in a Light Microscope
Images from the beadBead
Point Spread Function (PSF)
y
x
z
x
y
x
Point Spread Function: measurement of a physical phenomenon
Convolution
ActualActual MeasuredMeasured
2 Adjacent Points2 Adjacent Points 4 Adjacent Points4 Adjacent Points
PSF in the Bead
PSF in the Sample
Confocal vs Widefield Microscopy
Pinhole
Pinhole
Confocal Widefield
Illumination
Detector
Object
Condenser
Objective
Confocal vs Widefield Microscopy
Confocal Widefield
Image Image
Deconvolution Microscopy
PSF Image
=
PSFImage
=-1
Object
Object
YFP-α-tubulin in T. gondii
Swedlow et al., PNAS 2002
Confocal Wide-field Deconvolution
Deconvolution
Accounts for “blur” introduced by the optical pathwayImproves contrast
Two types of deconvolution 1. Deblurring/Nearest Neighbor2. Image Restoration/Constrained Iterative
Comparison of Deconvolution Methods
XLK2 Cell: Ab: α-tubulin-Cy5
Deblurring
Uses 3 z planes: focal plane +/- 1 planeTreats a 3D image as a 2D image
ProsFastMay be visually pleasing
ConsInvolves a few assumptions:1. Out-of-focus light from other planes is negligible2. PSFs are equivalentSubtractive methodNot quantitative
Deblurring
Imagen = Objectn ⊗ PSFn + Objectn+1 ⊗ PSFn+1 + Objectn-1 ⊗ PSFn-1
Z
n0
n-1
n+1
Image Restoration
Uses all z planes collectedTreats a 3D image as a 3D image
ProsQuantitativeReassigns light to plane of originImproves signal-to-noise ratio
ConsRequires computational resourcesSensitive to optical imperfections
Image Restoration
• Guess at In• Convolve In with PSF• Compare to On
• Check for negative intensities• Make new guess• Continue until a stable guess is achieved
What is a guess?!?A “guess” is where the signal distribution in x, y, and z can
be calculated to satisfy the above equation
Imagen = Objectn ⊗ PSFn
Image Restoration
Z
Image Restoration Microscopy
Image Restoration Microscopy
Comparison of Deconvolution Methods
XLK2 Cell: Ab: a-tubulin-Cy5
Deconvolution
What are the system requirements for deconvolution?Deconvolution is not just about algorithms!
Blur is introduced by the optical pathwayYou must examine your opticsNot all PSFs are equal because…
Not all lenses are equalPrecise stage movement
Objectives
J. Murray, CSHL 2006.
Objectives
J. Murray, CSHL 2006.
Objectives
CLEAN THEM!
J. Murray, CSHL 2006.
X
Z
PSF Varies by Objective
Coma
P. Goodwin
Axial Skew
CAA B C
A B CA B C
Spherical Aberration
Chromatic Aberration
SDY = 0.041 mmSDZ = 0.037 mm
SDY
= 41 nmSD z = 37 nm
A pixel is 0.064 mmor 64 nm
OpticalResolution
~ 200 nm in XY~ 500 nm in Z
A pixel and the voxel are the smallest units that compose an image or a volume
Stage RepeatabilityDeviation from Median Position
61 Point Visits over 24 minutes
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
-0.5 -0.3 -0.1 0.1 0.3 0.5
Y Position (um)
Z Po
sitio
n (u
m)
OneVoxel 64 nm
Environmental Chamber
Maintain environment for cells: heat and humidity.Provides protection from dust, dirt and grime
Stage Drift• Mechanical Factors
– Overall stage stability– Z drift: motorized stage vs objective
• Environmental Factors– Room temperature– Humidity– Air conditioning and heating fluctuations
Why Bother?
If so many things can affect deconvolution, why bother?
QuantitativeIncreases signal-to-noise ratio: critical in live-cell
imagingHigh resolutionComputer processing speeds continue to improveWide-field imaging is gentle on live cellsMaximize your data collection while minimizing
phototoxicity
HIV-1 Vpr Nuclear Envelope Localization
Vodicka et al., Genes Dev. 1998
Vpr colocalizes to nuclear membrane pores
2.5 µm
Confocal and epifluorescent data indicated nuclear localizationBiochemically Vpr was membrane associated
HIV-1 Vpr Nuclear Envelope Localization
Vodicka et al., Genes Dev. 1998
1.0 µm
mab 414:recognizes subset of nucleoporins
HIV in Dendritic- T-Cell Synapses
McDonald et al., Science 2003
HIV viral particles recruited to synapse
DNA
ActinHIV
Protein trafficking
M. Falk, Scripps Research Institute
YFP-tubulinCx43-CFPlysosomes
One cell nucleus from one field of 40 in a 24 hour time lapse experiment
Histone-2B-GFP
L. Pelletier, Max Planck Institute
Tubulin-GFP
L. Pelletier, Max Planck Institute
Centrin-GFP
L. Pelletier, Max Planck Institute