Transcript 2006Oct18CryoEMCourseCCDs.ppt

Introduction CCD Cameras In
Electron Cryomicroscopy
Christopher Booth
Gatan Inc
October 18, 2006
Overview
 CCD Camera Overview
 CCD Camera Core Concepts
 CCD Characterization
 Practical CCD Questions
 The Future Of CCD Cameras
Digital Cameras Proliferate
There are many types of
CCD Cameras but they work
DIRECTLY with light – NOT
ELECTRONS !
The Similarity with TEM cameras ends
here.
CCD Cameras for TEM
TEM cameras are closer
to
Astronomical cameras
Attaching CCD Camera to TEM Column
TEM Applications
Magnification
on CCD
Less than film
(~ 70-75% less)
Close to film
(~ 30-40% more)
(35 mm Port)
Large field of view
Life science
CCD
CCD
(Bottom)
High resolution
Materials science
Structural Biology
Practical TEM Setup
CCD
Control
Unit
PC &
Monitor
To View
Images
CCD Camera
Practical TEM Setup II
CCD Camera
Generic TEM CCD Architecture
Scintillator
Optical fiber (1:1)
CCD Chip
Thermo-electric
cooling
Scintillators and Phosphors
 Phosphors are powders with particle sizes
typically 1-6 um. Phosphors are delicate and must
be replaced if damaged. They are usually the most
sensitive.
 Scintillators are single crystals such as YAG and
are cleanable. They are often less sensitive than
phosphors but have better optical properties in
some applications.
 Selection of the optimal scintillator is dependent
on both the camera design and the application.
Imaging CCD Sensor
Output
Sensor area
Electrical
connection
Courtesy of Roper Scientific
Light Detection by CCD
Incoming Light
Electrical Connection
Polysilicon Gate
Silicon Dioxide
Silicon
Potential Well
Courtesy of Roper Scientific
Readout Of A CCD Frame
CCD pixel collects light and converts it into packet
of charge
One row of charge is quickly moved across the CCD chip
One pixel of charge is shifted to the output and digitized.
(Courtesy of Roper Scientific)
CCD Architecture
•
Full Frame CCD
High sensitivity, low noise. Beam shutter required.
•
Frame Transfer CCD
High sensitivity, low noise. Can operate in either full frame or
frame transfer mode if equipped with movable mask. Beam
shutter not required in frame transfer mode.
Single and Multi-port CCD Readout
4-port Readout
Serial readout
Serial register
Single port Readout
Serial readout
Parallel shift
Parallel shift
Parallel shift
Serial readout
Serial register
Serial readout
Serial register
Serial readout
Frame Transfer CCD
4-port Readout
Serial readout
Serial register
Single port Readout
Serial readout
Parallel shift
Masked Area
Parallel shift
Parallel shift
Masked Area
Masked Area
Serial readout
Serial register
Serial readout
Serial register
Serial readout
CCD Normalization
• Standard Normalization
I final ( x) 
I acquired ( x)  I dark _ reference ( x)
I gain_ reference ( x)
• Quadrant Normalization
Dark Subtraction
Unprocessed
Dark Reference
Courtesy of University of Southern California Signal & Image Processing Institute
Gain Normalization
Dark Subtracted
Gain Reference
Courtesy of University of Southern California Signal & Image Processing Institute
Gain Normalization
Dark Subtracted
Gain Normalized
Courtesy of University of Southern California Signal & Image Processing Institute
Quadrant Normalization
Core CCD Concepts
• Dynamic Range
• Quantum Efficiency
• Linearity
• Nyquist Frequency
• Point Spread Function
• Modulation Transfer Function
Dynamic Range
• The ratio between the smallest and largest
possible detectable values.
• Very important for imaging diffraction patterns to
detect weak spots and very intense spots in the
same image
Quantum Efficiency
• The Quantum Efficiency of a detector is the ratio
of the number of photons detected to the number
of photons incident
Linearity
• Linearity is a measure of how consistently the
CCD responds to light over its well depth.
• For example, if a 1-second exposure to a stable
light source produces 1000 electrons of charge, 10
seconds should produce 10,000 electrons of
charge
Calculating The Fourier Transform Of an
Image
Image Of Carbon Film
• amorphous specimen
• not beam sensitive
• common
Also called the power
spectrum of the image
Nyquist Frequency
periodicity = D
Real Space
periodicity = D
CCD pixels D
1
2 3 4
5 6 7
8
1
2
Reciprocal
Space
¼ Nyquist limit
1/(8D)
Nyquist limit
1/(2D)
Point Spread Function (PSF)
• The blurring of an imaginary point as it passes through
an optical system
• Convolution of the input function with a
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
5
10
15
20
25
0
0
5
10
15
20
25
Modulation Transfer Function (MTF)
• A representation of the point spread function in
Fourier space
1.2
3
3
1
2.5
2.5
0.8
2
2
x
0.6
=
1.5
1.5
0.4
1
1
0.2
0.5
0.5
0
0
0
5
10
15
20
25
0
0
5
10
15
20
25
0
5
10
15
20
25
Electron Path After Striking The
Scintillator
100 kV
200 kV
300 kV
400 kV
MTF Attenuates Higher Frequencies
Courtesy of University of Southern California Signal & Image Processing Institute
Zero Noise Image
Input
MTF = 0.125
2
2
1.8
1.8
1.6
1.6
1.4
1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
Rescaling
Input
Rescaled
2
2
1.8
1.8
1.6
1.6
1.4
1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
Noise in an Imaging System
 All images do have noise that can be divided into
three categories:
 Primary Noise:
Inherent noise in the electron beam and additional noise
due to electron tracks in the scintillator and gain variations
within the scintillator.
 Secondary Noise:
Noise due to scattering, blurring or failure to detect photons.
 Readout Noise:
Thermal and electrical noise present in the CCD camera.
With Noise
MTF = 1
MTF = 0.125
1.8
1.8
1.3
1.3
0.8
0.8
0.3
0.3
-0.2
-0.2
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
Rescaling Cannot Restore a Noisy
Image
MTF = 1
Rescaled
1.8
1.8
1.3
1.3
0.8
0.8
0.3
0.3
-0.2
-0.2
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
DQE
 DQE is the ratio:
DQE = (SNRout)2 / (SNRin)2
 In a perfect imaging system, the DQE = 1. The
image detection process does not reduce the
SNR. The only noise is the noise in the electron
beam.
 DQE is a method of comparing the performance of
imaging systems that is not specimen or
microscope dependent.
DQE
 Using Fourier analysis it can be shown that
DQE(K, d) = [ (MTF)2 / NPS ] x dose
 The MTF is measured with the knife edge method
and does not include the microscope MTF or CTF.
 The noise power spectrum (NPS) is measured
from uniform illumination images.
Calculating Spectral Signal To Noise Ratio
• Signal To Noise Ratio is more meaningful if we
think in Fourier Space
PowerSpectrum( s)  Noise ( s)
SNR( s) 
Noise( s)
Power Spectrum Of Amorphous Carbon
On Film and CCD
Comparing The Signal To Noise Ratio
From Film and CCD at 200 kV
Confirming A 9 Å Structure
Recent Comparison OF SNR
between Film and CCD at 300 kV
CCD Image of T7Phage
110,000 X Magnfication
1.10 µm defocus
7Å
600 Å
T7 Phage Reconstruction
β
α
β
β
α
α
Practical Questions
Magnification Of CCD relative to Film
• 2010F Film Mag x 1.38 = 2010F CCD Mag
• 3000SFF Film Mag x 1.41 = 3000SFF CCD Mag
• This has to be calibrated for each microscope detector.
How Do I Calculate Angstroms/Pixel?
•
Å/pixel = Detector Pixel Size/Magnification
•
•
•
For a microscope magnification of 60,000 on the 3000SFF:
Å /pixel = 150,000 Å / (microscope magnification x 1.41)
Å /pixel = 150,000 Å / (60,000 x 1.41)
Å /pixel = 1.77
How Do I Tell If Something Is Wrong?
Conclusion
• Understand what you are trying to achieve and
use the detector that will make your job the easiest
• Check Your Own Data!
Acknowledgements
• BCM
– Wah Chiu
– Joanita Jakana
• Purdue
– Wen Jiang
• MIT
– Peter Weigele
– Jonathan King
• Gatan Inc.
– Paul Money
– Brent Bailey
• UTH
– Hong Zhou