Digital Imaging - BIOP -Homepagebiop.epfl.ch/pdf/DigitalImaging2010.pdf · Dr. Arne Seitz PT-BIOP...
Transcript of Digital Imaging - BIOP -Homepagebiop.epfl.ch/pdf/DigitalImaging2010.pdf · Dr. Arne Seitz PT-BIOP...
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Dr. Arne SeitzSwiss Institute of Technology (EPFL)
Faculty of Life Sciences Head of BIOIMAGING AND OPTICS – BIOP
Digital Imaging
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Digital imagingOutlook
1600 1700 1800 1900 2000
Digital Camera(Kodak, 1975)
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Human Eye
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
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
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Digital imagingOutlook
Principle of digital imagingDifferent detection devices
– Photomultiplier– CCD Camera– etc.
Application in light microscopy– Do‘s and dont‘s– Pitfalls
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
What is lightparticle/wave
• 1669 NewtonEmanations theory (particle)
• 1677 Huygenswave theory
• 1802 Younginterference
• 1871 Maxwellelectromagnetic light theory
• 1886 Hertzexperimental proof of Maxwells theory
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
What is lightPhotoelectric effect
Albert EinsteinNobel price 1921
Experiments by Lennart 1902– 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
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
What is lightparticle/wave
Light can be described as– Electromagnetic wave
Amplitude, Phase, wavelength, polarization
– Particle (Photons)
Photoelectric effect can be used to detect photons.
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Analog ImagingPhotographic plate
Detection Principle– Ag+ is reduced to Ag0
(complex photochemical reaction)
– development = amplification =more Ag+ is reduced to Ag0
– Fixationremoval of Ag+ ions
– NegativeLight exposed=blacknot exposed=transparent
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Analog ImagingPhotographic plate
Advantages– Easy to handle.– High spatial resolution,
e.g. Fuji Velvia 50: 100 lines/mm.Disadvantages
– Low quantum efficiencyca. 0.8 % for photons butalmost 100% for electrons.
– “slow”, i.e. time consuming– Irreversible– Poor linearity
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Digital imagingOutlook
Principle of digital imagingDifferent detection devices
– Photomultiplier– Photodiode, CCD Camera– etc.
Application in light microscopy– Do‘s and don'ts– Pitfalls
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Photomultiplier
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Photomultiplier
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
PhotomultiplierSummary
• 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
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Digital imagingDefinition
• A digital image is a representation of a two-dimensional image using ones and zeros (binary). (Wikipedia)
• Analog = continuous values• Digital = discrete steps
1
0
01
01
1 10 01
1 110 0
11 01 11 1
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Digital imagingDefinition
• 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
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CamerasPrinciple
E
conductor(metal)
Semi conductor Isolator
valenceband
conductionband
band gap
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Semi conductor
E
CCD Cameras
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-p+ p+ p+ p+
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
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)
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraBlooming
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraBlooming
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CamerasBasic
Principle/Architecture
Types– Back illuminated, front illuminated
„Data“ Processing– Frame transfer, interline transfer, full frame
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraQuantum Efficiency
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD Architecture
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD Architecture
1
0
01
01
1 10 01
1 110 0
11 01 11 111 0
1
0
01
01
1 10 01
1 110 0
1 11 111 0
1
0
01
01
1 10 01
1 110 0
1 11 111 0
1
0
01
01
1 10 01
1 110 0
1 11
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD Architecture
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD Architecture
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Spectral Detection
• Complementary metal oxide semiconductor (CMOS)
• Unacceptable performance until 1990
• Advantages•Low power consumption•Single voltage power supply
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Digital imagingOutlook
Principle of digital imagingDifferent detection devices
– Photomultiplier– Photodiode, CCD Camera– etc.
Application in light microscopy– Do‘s and don'ts– Pitfalls
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Resolution/Pixel Size
Nyquist theorem
Undersampling/oversampling
Same areaLess pixels
Larger pixel size(in the image)
Less sampling frequency
Lateral resolution
NA61.0 λδ =R
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
Objective(numerical aperture)
ResolutionLimit
(microns)
ProjectedSize on CCD
(microns)
Required PixelSize
(microns)
4x (0.20) 1.5 5.8 2.910x (0.45) 0.64 6.4 3.220x (0.75) 0.39 7.7 3.940x (0.85) 0.34 13.6 6.840x (1.30) 0.22 8.9 4.560x (0.95) 0.31 18.3 9.260x (1.40) 0.21 12.4 6.2
100x (0.90) 0.32 32.0 16.0100x (1.25) 0.23 23.0 11.5100x (1.40) 0.21 21.0 10.5
Resolution/Pixel Size
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD cameraBinning
Binning:- increases effective pixel size-Decreases sampling frequency-Increases read-out speed
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD cameraNoise/Background
Noise=signal fluctuation
background
Sources of noise:•Detector noise
•Dark noise•Read noise
•Photon noise (shot noise)2Re
22
2
22
adDarkCam
Sig
CamSigtot
NNN
PQEN
NNN
+=
∗=
+=
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD cameraSignal to Noise Ratio
Photonflux: 104 photons/s Exposure time: 0.1 sSNR: ~ 5 for a typical CCD camera
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD cameraSignal to Noise Ratio
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD cameraNoise
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraDynamic Range/Noise
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraDynamic Range
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraDynamic Range
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
EM CCD
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
EM CCD
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
43
Readout noiseCamera Relative
gainAbsolute gain
Absolute noise, e-
Relative noise, e-
Camera offset
0 1 6.3 6.3 200CCD 128 5 33.2 6.6 200
255 10 54.8 5.5 2000 1 30.2 30.2 460
EMCCD 128 15 64.8 4.3 460255 550 887.9 1.6 460
Absolute readout noise increase with gain
Relative readout noise:CCD - does not depend on gainEMCCD - decrease with gain
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
44
SNR of CCD and EMCCD
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
CCD CameraSummary
• 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
Dr. Arne Seitz PT-BIOP course, Digital Imaging, EPFL 2010
BioImaging &Optics Platform
More about digital imaging
1. Lecture Biomicroscopy I + II, Prof. Theo Lasser, EPFL
2. Interneta) http://micro.magnet.fsu.edub) 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/