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Detecting Electrons: CCD vs Film

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Detecting Electrons: CCD vs Film

Practical CryoEM Course

July 26, 2005

Christopher Booth

- Basic Concepts
- Detector Quality Concepts
- How Do Detectors Work?
- Practical Evaluation Of Data Quality
- Final Practical Things To Remember

- Fourier Transform and Fourier Space
- Convolution
- Transfer Functions
- Point Spread Function
- Modulation Transfer Function

- Low Pass Filter

The co-ordinate (ω) in Fourier space is often referred to as spatial frequency or just frequency

- Convolution in Real Space is Multiplication in Fourier Space
- It is a big advantage to think in Fourier Space

- Reducing or removing the high frequency components
- Only the low frequency components are able to “pass” the filter

x

=

- 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

- In Fourier Space this representation is simplified

=

x

- The blurring of an imaginary point as it passes through an optical system
- Convolution of the input function with a

- A representation of the point spread function in Fourier space

=

x

- 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

- Nyquist Frequency
- Dynamic Range
- Linearity
- Dark Noise

- 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

- The Quantum Efficiency of a detector is the ratio of the number of photons detected to the number of photons incident

- 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 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

- Nyquist Frequency, must sample image at 2x the highest frequency you want to recover

- 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

100 kV

200 kV

300 kV

400 kV

Incident electrons

Silver Emulsion

Film

Incident Light

Developed Silver Emulsion

Film

Scanner CCD Array

- 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

x

x

- Signal To Noise Ratio is more meaningful if we think in Fourier Space

- Image Of Carbon Film
- amorphous (non crystalline) specimen
- not beam sensitive
- common

Also called the power

spectrum of the image

2/5 Nyquist Frequency

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

- You can use ctfit from EMAN to calculate a spectral signal to noise ratio
- Built In Method
- Alternate Method Presented Here

- Good Normalization Means Good Data
- Dark Reference
- Gain Normalization
- Quadrant Normalization

- Magnification Of CCD relative to Film
- Angstroms/Pixel

- Standard Normalization
- Quadrant Normalization

- 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.

- 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

- Understand what you are trying to achieve and use the detector that will make your job the easiest
- Check Your Own Data!