the electron microscope: the contrast transfer function (ctf) javier vargas centro nacional de...
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The electron microscope: the contrast transfer function (CTF)
Javier Vargas
Centro Nacional de Biotecnología-CSIC
What is an electron microscope?
Why electron microscopes?
What is an electron microscope?
An electron microscope is a tool for obtaining projection images of very small biological objects
Evolution
Important characteristics of cryoEM images
1) Projection images
2) Phase contrast and WPA
Projection images
Images are formed by phase contrast
projection images
3DEM as an inverse problem3DEM as an inverse problem
Limitations of cryo-electron microscopy
1) Radiation damage.
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.3) The samples are very small.
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.3) The samples are very small.
4) Presence of ice.
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.3) The samples are very small.
4) Presence of ice.5) Charging: non conductive samples charge up and act like lenses.
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.3) The samples are very small.4) Presence of ice.5) Charging: non conductive samples charge up and act like lenses.6) Expensive. Titan Krios around 1000€/day (NeCEN)
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.3) The samples are very small.4) Presence of ice.5) Charging: non conductive samples charge up and act like lenses.6) Expensive. Titan Krios around 1000€/day (NeCEN)
We obtain very noisy images
Limitations of cryo-electron microscopy
1) Radiation damage.2) Electron lenses.3) The samples are very small.4) Presence of ice.5) Charging: non conductive samples charge up and act like lenses.6) Expensive. Titan Krios around 1000€/day (NeCEN)
We require a lot of images
Why use electrons?:
Advantages Disadvantages
Visible light
Why use electrons?:
Advantages Disadvantages
Visible light Not very damaging
Easily focused
Why use electrons?:
Advantages Disadvantages
Visible light Not very damaging
Easily focused
Long wavelengths (~400 nm)
Poor Penetration
X rays
Why use electrons?:
Advantages Disadvantages
Visible light Not very damaging
Easily focused
Long wavelengths (~400 nm)
Poor Penetration
X rays Small wavelength (Angstromgs)
Good penetration
Why use electrons?:
Advantages Disadvantages
Visible light Not very damaging
Easily focused
Long wavelengths (~400 nm)
Poor Penetration
X rays Small wavelength (Angstromgs)
Good penetration
Hard to focus
Damage Samples
Why use electrons?:
Advantages Disadvantages
Visible light Not very damaging
Easily focused
Long wavelengths (~400 nm)
Poor Penetration
X rays Small wavelength (Angstromgs)
Good penetration
Hard to focus
Damage Samples
Electrons Small wavelength (pm)
Can be focused
Why use electrons?:
Advantages Disadvantages
Visible light Not very damaging
Easily focused
Long wavelengths (~400 nm)
Poor Penetration
X rays Small wavelength (Angstromgs)
Good penetration
Hard to focus
Damage Samples
Electrons Small wavelength (pm)
Can be focused
Damage Samples
Poor Penetration
Electrons energy
Electrons wavelength
Why electron microscopes?
electron microscopy
light microscopy
Why electron microscopes?
electron microscopes
Electrons energy
Electrons wavelength
The contrast transfer function (CTF)
Main idea
Perfect system: the image of a point is a point
Real system: the image of a point is a spot
Hubble telescope was myopic !!!
Perfect system: the image of a point is a point
Real system: the image of a point is a spot
Hubble telescope was myopic !!!
Perfect system: the image of a point is a point
Real system: the image of a point is a spot
IntroductionIntroduction
There is no any perfect real system!!
Transfer functions
Frequency increase
Transfer functions
bass treble
Transfer functions
Transfer functions
CTF
Transfer functions
Transfer functions
8 Ǻ
With phase-plate
RRRRRR sin1cos 2AAECTF
RRR
R
RRRRRR
sin
0
sin1cos 2
ECTF
A
AAECTF
RRR
R
RRRRRR
sin
0
sin1cos 2
ECTF
A
AAECTF
Spherical m,Astigmatis Defocus,F
Matlab Script to simulate the CTF
How a real microscope distort the ideal projections?
Assuming a LTI system
PSFII ir
CTFIFTIFT ir ·
Defocus = 0 A.
Astigmatism = 0 A.
Defocus = 0 A.
Astigmatism = 0 A.
Defocus = 0 A.
Astigmatism = 0 A.
Defocus = 1000 A (0.1 um).
Astigmatism = 0
Defocus = 10000 A (1um)
Astigmatism = 0
Defocus = 5500 A (0.55 um)
Astigmatism = 4500 A (0.45 um)
How a real microscope distort the ideal projections?
Assuming a LTI system
PSFII ir
CTFIFTIFT ir ·
CTFIFTFTI ir ·1
A little bit of theory…
CTF is important because:
1. Image restoration (deconvolution)
2. Micrograph screening
1. Image restoration (deconvolution)
Problem: CTF have zeros
1CTF Is not well defined at some points
CTFIFTFTI ir ·1 11 · CTFIFTFTI ri
11 · CTFIFTFTI ri
Wiener filter
22
2
1 1·
KCTF
CTF
CTFIFTFTI ri
No problems in frequencies 0, yxCTF
1. Image restoration (deconvolution)
CTF is important because:
2. Micrograph screening
CTF is important because:
2. Micrograph screening
Questions?