Detecting electrons ccd vs film
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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.

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Detecting electrons ccd vs film

Detecting Electrons: CCD vs Film

Practical CryoEM Course

July 26, 2005

Christopher Booth


Overview

Overview

  • Basic Concepts

  • Detector Quality Concepts

  • How Do Detectors Work?

  • Practical Evaluation Of Data Quality

  • Final Practical Things To Remember


Basic concepts

Basic Concepts

  • Fourier Transform and Fourier Space

  • Convolution

  • Transfer Functions

    • Point Spread Function

    • Modulation Transfer Function

  • Low Pass Filter


Fourier transform

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

Graphical Representation Of The Fourier Transform


Convolution

Convolution


Convolution in fourier space

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

Low Pass Filter

  • Reducing or removing the high frequency components

  • Only the low frequency components are able to “pass” the filter

x

=


Transfer functions

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


Transfer functions1

Transfer Functions

  • In Fourier Space this representation is simplified

=

x


Point spread function psf

Point Spread Function (PSF)

  • The blurring of an imaginary point as it passes through an optical system

  • Convolution of the input function with a


Modulation transfer function mtf

Modulation Transfer Function (MTF)

  • A representation of the point spread function in Fourier space

=

x


Summarize basic concepts

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

Detector Specific Concepts

  • Nyquist Frequency

  • Dynamic Range

  • Linearity

  • Dark Noise


Nyquist frequency

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

Example Of Sampling Below Nyquist Frequency


Quantum efficiency

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

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

  • 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

Summarize CCD Specific Terms

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


So why does anyone use film

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

How a CCD Detects electrons


Electron path after striking the scintillator

Electron Path After Striking The Scintillator

100 kV

200 kV

300 kV

400 kV


How readout of the ccd occurs

How Readout Of the CCD Occurs


How film detects electrons

How Film Detects Electrons

Incident electrons

Silver Emulsion

Film


Silver grain emulsion at various magnification

Silver Grain Emulsion At Various Magnification


How film is scanned

How Film Is Scanned

Incident Light

Developed Silver Emulsion

Film

Scanner CCD Array


Options for digitizing film

Options For Digitizing Film


Summary of detection methods

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

Practical Evaluation Of The CCD Camera


Decomposing graphite signal

Decomposing Graphite Signal

x

x


Calculating spectral signal to noise ratio

Calculating Spectral Signal To Noise Ratio

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


Calculating the fourier transform of an image

Calculating The Fourier Transform Of an Image

  • Image Of Carbon Film

  • amorphous (non crystalline) specimen

  • not beam sensitive

  • common

Also called the power

spectrum of the image


Power spectrum of amorphous carbon on film and ccd

Power Spectrum Of Amorphous Carbon On Film and CCD


Comparing the signal to noise ratio from film and ccd

Comparing The Signal To Noise Ratio From Film and CCD


Film vs ccd head to head

Film Vs CCD Head-To-Head


Calculating snr for ice embedded cytoplasmic polyhedrosis virus

Calculating SNR for Ice Embedded Cytoplasmic Polyhedrosis Virus


Reconstruction to 9 resolution

Reconstruction To 9 Å Resolution


Confirming a 9 structure

Confirming A 9 Å Structure


Relating snr s to resolution

Relating SNR(s) To Resolution

2/5 Nyquist Frequency


Further experimental confirmation of 2 5 nyquist

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


Evaluate your data to estimate the quality of your imaging

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


Final practical things to remember

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

Normalization

  • Standard Normalization

  • Quadrant Normalization


Quadrant normalization

Quadrant Normalization


Dark reference

Dark Reference


Gain normalization

Gain Normalization


How do i tell if something is wrong

How Do I Tell If Something Is Wrong?


Magnification of ccd relative to film

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

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

Conclusion

  • Understand what you are trying to achieve and use the detector that will make your job the easiest

  • Check Your Own Data!


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