BioImaging &Optics Platform. Digital Imaging. Dr. Arne Seitz

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1 Digital Imaging Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP

2 Digital imaging Outlook Digital Camera (Kodak, 1975)

3 Human Eye

4 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

5 Digital imaging Outlook Principle of digital imaging Different detection devices Photomultiplier CCD Camera etc. Application in light microscopy Do s and dont s Pitfalls

6 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

7 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

8 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.

9 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

10 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

11 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

12 Photomultiplier

13 BioImaging &Optics Platform Photomultiplier

14 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

15 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

16 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

17 CCD Cameras Principle conduction band E band gap conductor (metal) Semi conductor Isolator valence band

18 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 +

19 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)

20 CCD Camera Blooming

21 Principle/Architecture Types BioImaging &Optics Platform CCD Cameras Basic Back illuminated, front illuminated Data Processing Frame transfer, interline transfer, full frame

22 CCD Camera Quantum Efficiency

23 CCD Architecture

24 CCD Architecture

25 CCD Architecture

26 CCD Architecture

27 Spectral Detection Complementary metal oxide semiconductor (CMOS) Unacceptable performance until 1990 Advantages Low power consumption Single voltage power supply

28 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

29 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

30 Objective (numerical aperture) BioImaging &Optics Platform Resolution/Pixel Size Resolution Limit (microns) Projected Size on CCD (microns) Required Pixel Size (microns) 4x (0.20) x (0.45) x (0.75) x (0.85) x (1.30) x (0.95) x (1.40) x (0.90) x (1.25) x (1.40)

31 CCD camera Binning Binning: - increases effective pixel size -Decreases sampling frequency -Increases read-out speed

32 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

33 CCD camera Signal to Noise Ratio Photonflux: 10 4 photons/s SNR: ~ 5 for a typical CCD camera Exposure time: 0.1 s

34 CCD camera Signal to Noise Ratio

35 CCD camera Noise

36 CCD Camera Dynamic Range/Noise

37 CCD Camera Dynamic Range

38 CCD Camera Dynamic Range

39 BioImaging &Optics Platform EM CCD

40 EM CCD

41 Camera Relative gain BioImaging &Optics Platform Readout noise Absolute gain Absolute noise, e - Relative noise, e CCD EMCCD Absolute readout noise increase with gain Relative readout noise: CCD - does not depend on gain EMCCD - decrease with gain Camera offset 42

42 SNR of CCD and EMCCD 43

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

44 More about digital imaging 1. Lecture Biomicroscopy I + II, Prof. Theo Lasser, EPFL 2. Internet a) b) b) Web sites of camera vendors, e.g. Hamamatsu Rooper Andor 3. PT-BIOP EPFL, SV-AI 0241, SV-AI