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Detecting Electrons: CCD vs Film
Practical CryoEM Course
July 26, 2005
Christopher Booth
Overview
• Basic Concepts
• Detector Quality Concepts
• How Do Detectors Work?
• Practical Evaluation Of Data Quality
• Final Practical Things To Remember
Basic Concepts
• Fourier Transform and Fourier Space
• Convolution
• Transfer Functions– Point Spread Function– Modulation Transfer Function
• Low Pass Filter
Fourier Transform
The co-ordinate (ω) in Fourier space is often referred to as spatial frequency or just frequency
Graphical Representation Of The Fourier Transform
Convolution
Convolution In Fourier Space
• Convolution in Real Space is Multiplication in Fourier Space
• It is a big advantage to think in Fourier Space
Low Pass Filter
• Reducing or removing the high frequency components
• Only the low frequency components are able to “pass” the filter
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Transfer Functions
• A transfer function is a representation of the relation between the input and output of a linear time-invariant system
• Represented as a convolution between an input and a transfer function
dyyxtyfxf
xtxfxf
inputoutput
inputoutput
)()()(
)()()(
Transfer Functions
• In Fourier Space this representation is simplified
)()()( sTsXsX inputoutput
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Point Spread Function (PSF)
• The blurring of an imaginary point as it passes through an optical system
• Convolution of the input function with a
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Modulation Transfer Function (MTF)
• A representation of the point spread function in Fourier space
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Summarize Basic Concepts
• Fourier Transform and Fourier Space• Convolution describes many real processes • Convolution is intuitive in Fourier Space• Transfer Functions are multiplication in Fourier
Space• MTF is the Fourier Transform Of the PSF• MTF is a Transfer Function• Some Filters are easiest to think about in Fourier
Space
Detector Specific Concepts
• Nyquist Frequency
• Dynamic Range
• Linearity
• Dark Noise
Nyquist Frequency
• Nyquist-Shannon Sampling Theorem
• You must sample at a minimum of 2 times the highest frequency of the image
• This is very important when digitizing continuous functions such as images
Example Of Sampling Below Nyquist Frequency
Quantum Efficiency
• The Quantum Efficiency of a detector is the ratio of the number of photons detected to the number of photons incident
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
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
Summarize CCD Specific Terms
• Nyquist Frequency, must sample image at 2x the highest frequency you want to recover
Quantum Efficiency (%)
Dynamic Range
Linearity
CCD 50 – 90 10,000 Very linear
Film 5 – 20 100 Limited linearity
So Why Does Anyone Use Film?
• For High Voltage Electron Microscopes, the MTF of Film is in general better than that of CCD at high spatial frequencies.
• If you have an MTF that acts like a low pass filter, you may not be able to recover the high resolution information
How a CCD Detects electrons
Electron Path After Striking The Scintillator
100 kV 200 kV 300 kV 400 kV
How Readout Of the CCD Occurs
How Film Detects Electrons
Silver Emulsion
Film
Incident electrons
Silver Grain Emulsion At Various Magnification
How Film Is Scanned
Developed Silver Emulsion
Film
Incident Light
Scanner CCD Array
Options For Digitizing Film
Summary Of Detection Methods
• Scintillator and fiber optics introduce some degredation in high resolution signal in CCD cameras
• Film + scanner optics introduce a negligible amount of degredation of high resolution signal
Practical Evaluation Of The CCD Camera
Decomposing Graphite Signal
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Calculating Spectral Signal To Noise Ratio
• Signal To Noise Ratio is more meaningful if we think in Fourier Space
)(
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sNoise
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Calculating The Fourier Transform Of an Image
Also called the power spectrum of the image
Image Of Carbon Film• amorphous (non crystalline) specimen• not beam sensitive• common
Power Spectrum Of Amorphous Carbon On Film and CCD
Comparing The Signal To Noise Ratio From Film and CCD
Film Vs CCD Head-To-Head
CCD Film
Linearity
Quantum Efficiency
Dynamic Range
MTF
Calculating SNR for Ice Embedded Cytoplasmic Polyhedrosis Virus
Reconstruction To 9 Å Resolution
Confirming A 9 Å Structure
Relating SNR(s) To Resolution
2/5 Nyquist Frequency
Further Experimental Confirmation Of 2/5 Nyquist
Table 2: Comparison of Reconstruction Statistics between Several Different Ice Embedded Single Particles Collected On the Gatan 4kx4k CCD at 200 kV at the Indicated Nominal Magnification
ComplexNumber Of Particles
Nominal Microscope Magnification
Expected Resolution (Å) at
2/5 Nyquist
Final Resolution (0.5 FSC cutoff, Å)
Software Package For Reconstructi
on
CPV 5,000 60,000 9 9 SAVR
GroEL 8,000 80,000 6.8 7-8 EMAN
Ryr1 29,000 60,000 9 9.5 EMAN
Epsilon Phage 15,000 40,000 13.6 13 EMAN/SAVR
Evaluate Your Data To Estimate The Quality Of Your Imaging
• You can use ctfit from EMAN to calculate a spectral signal to noise ratio– Built In Method– Alternate Method Presented Here
)(
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sNoise
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Final Practical Things to Remember…
• Good Normalization Means Good Data– Dark Reference– Gain Normalization– Quadrant Normalization
• Magnification Of CCD relative to Film
• Angstroms/Pixel
Normalization
• Standard Normalization
• Quadrant Normalization
)(
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_
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xI
xIxIxI
referencegain
referencedarkacquiredfinal
Quadrant Normalization
Dark Reference
Gain Normalization
How Do I Tell If Something Is Wrong?
Magnification Of CCD relative to Film
• 2010F Mag x 1.38 = 2010F CCD Mag• 3000SFF 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 Step-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
Conclusion
• Understand what you are trying to achieve and use the detector that will make your job the easiest
• Check Your Own Data!