Digital Imaging Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP arne.seitz@epfl.ch
Digital imaging Outlook 1600 1700 1800 1900 2000 Digital Camera (Kodak, 1975)
Human Eye
Human Eye Visual Pigments Protein + Retinal 13 cis/trans Photoreaction (Isomerization) Signal transduction pathway Amplification steps Hyperpolarization Stimulated cells release less Neurotransmitter System is noiseless
Digital imaging Outlook Principle of digital imaging Different detection devices Photomultiplier CCD Camera etc. Application in light microscopy Do s and dont s Pitfalls
What is light particle/wave 1669 Newton Emanations theory (particle) 1677 Huygens wave theory 1802 Young interference 1871 Maxwell electromagnetic light theory 1886 Hertz experimental proof of Maxwells theory
Albert Einstein Nobel price 1921 Experiments by Lennart 1902 BioImaging &Optics Platform What is light Photoelectric effect Maximal energy of the photo electrons is independent of the intensity of the light Slope is the same for different cathode materials Light can be described as particles called photons or light quants. Detection of Photons
What is light particle/wave Light can be described as Electromagnetic wave Amplitude, Phase, wavelength, polarization Particle (Photons) Photoelectric effect can be used to detect photons.
Detection Principle BioImaging &Optics Platform Analog Imaging Photographic plate Ag + is reduced to Ag 0 (complex photochemical reaction) development = amplification = more Ag + is reduced to Ag 0 Fixation removal of Ag + ions Negative Light exposed=black not exposed=transparent
BioImaging &Optics Platform Analog Imaging Photographic plate Advantages Easy to handle. High spatial resolution, e.g. Fuji Velvia 50: 100 lines/mm. Disadvantages Low quantum efficiency ca. 0.8 % for photons but almost 100% for electrons. slow, i.e. time consuming Irreversible Poor linearity
Digital imaging Outlook Principle of digital imaging Different detection devices Photomultiplier Photodiode, CCD Camera etc. Application in light microscopy Do s and don'ts Pitfalls
Photomultiplier
BioImaging &Optics Platform Photomultiplier
Photomultiplier Summary Photons produce electrons which are multiplied by acceleration and detected afterwards. Gain (=multiplication factor) can be varied. Large linear range. Quantum efficiency of the photocathode up to 30% in the visible range. no spatial resolution, i.e. photomultipliers can only be used as point (scanning) detectors
BioImaging &Optics Platform Digital imaging Definition A digital image is a representation of a twodimensional image using ones and zeros (binary). (Wikipedia) Analog = continuous values Digital = discrete steps 1 1 1 1 1 0 0 0 1 1 1 0 1 0 0 0 1 1 01 1
Digital imaging Definition Array of photosensitive elements Signal is dependent on the number of detected photons; ideally linearly dependent= high dynamic range Easy read-out procedure Reusable Appropriate size, comparable to an analogue film
CCD Cameras Principle conduction band E band gap conductor (metal) Semi conductor Isolator valence band
CCD Cameras Semi conductor Principle Internal photoelectric effect. Energy of the photon is used to transfer an electron from the valence band to the conduction band (semi conductors). Photodiodes E e - e - e - e - e - e - e - e - p + p + p + p +
CCD Architecture Silicon based integrated circuits Dense matrix of photodiodes Electrons are stored in a potential well Electrons can be transported across the chip (=read out)
CCD Camera Blooming
Principle/Architecture Types BioImaging &Optics Platform CCD Cameras Basic Back illuminated, front illuminated Data Processing Frame transfer, interline transfer, full frame
CCD Camera Quantum Efficiency
CCD Architecture
CCD Architecture 1 1 1 1 1 10 0 1 0 1 1 1 0 1 0 0 1 10 0 1 0 1 1 0 1 0 1 1 0 1 1 1 1
CCD Architecture
CCD Architecture
Spectral Detection Complementary metal oxide semiconductor (CMOS) Unacceptable performance until 1990 Advantages Low power consumption Single voltage power supply
Digital imaging Outlook Principle of digital imaging Different detection devices Photomultiplier Photodiode, CCD Camera etc. Application in light microscopy Do s and don'ts Pitfalls
Resolution/Pixel Size Same area Less pixels Larger pixel size (in the image) Less sampling frequency Lateral resolution Nyquist theorem Undersampling/oversampling R δ = λ 0.61 NA
Objective (numerical aperture) BioImaging &Optics Platform Resolution/Pixel Size Resolution Limit (microns) Projected Size on CCD (microns) Required Pixel Size (microns) 4x (0.20) 1.5 5.8 2.9 10x (0.45) 0.64 6.4 3.2 20x (0.75) 0.39 7.7 3.9 40x (0.85) 0.34 13.6 6.8 40x (1.30) 0.22 8.9 4.5 60x (0.95) 0.31 18.3 9.2 60x (1.40) 0.21 12.4 6.2 100x (0.90) 0.32 32.0 16.0 100x (1.25) 0.23 23.0 11.5 100x (1.40) 0.21 21.0 10.5
CCD camera Binning Binning: - increases effective pixel size -Decreases sampling frequency -Increases read-out speed
CCD camera Noise/Background Sources of noise: Detector noise Dark noise Read noise Photon noise (shot noise) N N N tot 2 Sig 2 Cam = N 2 Sig = QE P = N + 2 Dark N + 2 Cam N 2 Read Noise=signal fluctuation background
CCD camera Signal to Noise Ratio Photonflux: 10 4 photons/s SNR: ~ 5 for a typical CCD camera Exposure time: 0.1 s
CCD camera Signal to Noise Ratio
CCD camera Noise
CCD Camera Dynamic Range/Noise
CCD Camera Dynamic Range
CCD Camera Dynamic Range
BioImaging &Optics Platform EM CCD
EM CCD
Camera Relative gain BioImaging &Optics Platform Readout noise Absolute gain Absolute noise, e - Relative noise, e - 0 1 6.3 6.3 200 CCD 128 5 33.2 6.6 200 255 10 54.8 5.5 200 0 1 30.2 30.2 460 EMCCD 128 15 64.8 4.3 460 255 550 887.9 1.6 460 Absolute readout noise increase with gain Relative readout noise: CCD - does not depend on gain EMCCD - decrease with gain Camera offset 42
SNR of CCD and EMCCD 43
CCD Camera Summary Photons elevate electrons from the valence to the conduction band High Quantum efficiency Large linear range. Spatial resolution is influenced by the pixel size of the camera EM CCD cameras for low light applications
More about digital imaging 1. Lecture Biomicroscopy I + II, Prof. Theo Lasser, EPFL 2. Internet a) http://micro.magnet.fsu.edu b) b) Web sites of camera vendors, e.g. Hamamatsu Rooper Andor 3. PT-BIOP EPFL, SV-AI 0241, SV-AI 0140 http://biop.epfl.ch/