Welcome to: LMBR Imaging Workshop. Imaging Fundamentals Mike Meade, Photometrics
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1 Welcome to: LMBR Imaging Workshop Imaging Fundamentals Mike Meade, Photometrics
2 Introduction CCD Fundamentals
3 Typical Cooled CCD Camera Configuration Shutter Optic Sealed Window DC Voltage Serial Clock Driver Parallel Clock Driver Camera Control Logic and Power Supply Camera Command Input Camera Status Output Incoming Light Cooling Fins HCCD (-45 O C) Thermo- Electric Cooler Low Noise Preamplifier Analog Processing and ADC Digital Pixel Data Output (12-16 bits)
4 ROPER SCIENTIFIC The Key to our Systems!
5 The Charge-Coupled Device (CCD) Invented in 1970 at Bell Labs A silicon chip that converts an image to an electrical signal Image is focused directly onto the silicon chip! Widely used in TV cameras and consumer camcorders Special high-performance CCDs made by Eastman Kodak (Rochester, NY) Thomson CSF (France) Marconi (formerly EEV England) SITe (Beaverton, OR) Sony Others
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7 MOS Photodetector
8 Quantum Efficiency Ability of CCD to convert photons to electrons ROPER SCIENTIFIC Usually reported as a percentage
9 Front vs Backside Illuminated
10 Indium Tin Oxide (ITO ) Technology
11 Enhancing UV Sensitivity with Proprietary CCD Coatings
12 standard frontside-illuminated (Kodak KAF-1400) thinned, backside-illuminated (SITe SI502AB) Metachrome lens-on-chip (Sony interline) ITO (Kodak KAF-1401E) Wavelength (nm) Typical Quantum Efficiencies
13 Preamplifier Serial Register Parallel Register Output Node
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18 Typical CCD Structure
19 Introduction CCD Architecture
20 Serial Clocks Serial Clocks Serial Clocks Serial Register Serial Register Serial Register Parallel Clocks for Storage Array Storage Array (masked) Full Array Direction of Parallel Shift Parallel Clocks for Image Array Image Array Direction of Parallel Shift Full Array Direction of Parallel Shift Full- Frame CCD Frame- Transfer CCD Interline- Transfer CCD
21 Full Frame Frame Transfer (EMCCD) Interline Transfer
22 Full Frame Preamplifier Serial Register Active Array Output Node
23 Preamplifier Serial Register Active Array Output Node
24 Preamplifier Serial Register Active Array Output Node
25 Preamplifier Serial Register Active Array Output Node ADC
26 Preamplifier Serial Register Active Array Output Node
27 Preamplifier Serial Register Active Array Output Node ADC
28 Preamplifier Serial Register Active Array Output Node
29 Preamplifier Serial Register Active Array Output Node ADC
30 Preamplifier Serial Register Active Array Output Node
31 Preamplifier Serial Register Active Array Output Node ADC
32 Preamplifier Serial Register Active Array Output Node
33 Frame Transfer Preamplifier Serial Register Active Array Output Node
34 Preamplifier Serial Register Active Array Output Node
35 Preamplifier Serial Register Active Array Output Node
36 Preamplifier Serial Register Active Array Output Node
37 Preamplifier Serial Register Active Array Output Node
38 Preamplifier Serial Register Active Array Output Node
39 Preamplifier Serial Register Active Array Output Node
40 Preamplifier Serial Register Active Array Output Node
41 Interline Transfer Preamplifier Serial Register Active Array Output Node
42 Preamplifier Serial Register Active Array Output Node
43 Preamplifier Serial Register Active Array Output Node
44 Preamplifier Serial Register Active Array Output Node
45 Preamplifier Serial Register Active Array Output Node
46 Preamplifier Serial Register Active Array Output Node
47 Preamplifier Serial Register Active Array Output Node
48 Preamplifier Serial Register Active Array Output Node
49 Preamplifier Serial Register Active Array Output Node
50 Full Frame Frame Transfer Interline Transfer High Spatial Resolution High Spatial Resolution Large Number of Pixels Large Number of Pixels Greater Selection of Greater Selection of CCD Formats CCD Formats 100% Fill Factor for 100% Fill Factor for Entire Array Entire Array Integrate while Clocking Integrate while Clocking High-Speed Operation High-Speed Operation Well Suited for Complex Well Suited for Complex Readout Schemes Readout Schemes 100% Fill Factor in 100% Fill Factor in Active Array Active Array Integrate while Clocking Integrate while Clocking High-Speed Operation (Video) High-Speed Operation (Video) Lens-on-Chip Technology Lens-on-Chip Technology
51 Ideal CCD Characteristics High quantum efficiency Wide spectral response Low dark current Ability to integrate charge Sufficient resolution
52 Performance Considerations
53 CCD Linearity Illumination Level (Arbitrary)
54 Noise Sources in CCDs Photon-induced shot noise Readout noise Dark current noise ktc reset switch noise Spurious charge Total System Noise = all noise sources added in quadrature
55 Photon (Shot Noise) - Law of physics - Law of physics - Square root relationship between signal and noise - Square root relationship between signal and noise noise = square root of number of electrons noise = square root of number of electrons - Poisson distribution - Poisson distribution - When photon noise exceeds system noise, data is - When photon noise exceeds system noise, data is photon (shot) noise limited photon (shot) noise limited
56 Read Noise (preamplifier noise) - Minimized by careful electronic design - Minimized by careful electronic design - Under low-light/low-signal conditions where - Under low-light/low-signal conditions where read noise exceeds photon noise, data is read read noise exceeds photon noise, data is read noise limited noise limited - Read noise not as relevant in high-signal - Read noise not as relevant in high-signal applications applications
57 Analog Gain Preamplifier Serial Register Active Array Output Node ADC
58 Dark Current Electrons created by thermal emission Increases with time and temperature
59 Dark current subtracts, dark current noise remains - dark current noise = dark current - dark current (dark noise) can lower SNR
60 Reduced by cooling the CCD - Dark current is cut in half as the CCD temperature drops approximately every 6.7 C Reduced by utilizing multi-pinned-phase (MPP) technology
61 Limit of cooling effectiveness - - MPP MPP CCDs CCDs already already have have low low dark dark current current - - Poor Poor CTE CTE (charge (charge transfer transfer efficiency) efficiency) at at <-120 C <-120 C
62 Noise Reduction in CCDs Photon Noise - A law of physics! Readout Noise - Reduced by careful electronics design Dark Current Noise - Reduced by cooling and MPP ktc Noise - Reduced by using correlated double sampling Spurious Charge - Reduced by careful shaping of clock waveforms Ultimately, a High-Performance CCD camera is limited only by Readout Noise and Photon Noise.
63 Introduction Dynamic Range
64 Dynamic Range = Full Well/Read Noise - Full Well = A/D converter bit depth 1x gain
65 Dynamic Range of CCD is matched to A/D Converter 12, 14, 16 bit
66 Intrascene Dynamic Range
67 Introduction Binning
68 Binning - Higher Dynamic Range - Higher Signal-to-Noise Ratio - Faster Readout - Dynamically Change Pixel Size/Aspect Ratio Above all gained at the expense of Spatial Resolution!!
69 Binning Preamplifier Serial Register Active Array Output Node
70 Preamplifier Serial Register Active Array Output Node
71 Preamplifier Serial Register Active Array Output Node
72 Preamplifier Serial Register Active Array Output Node
73 Preamplifier Serial Register Active Array Output Node
74 Preamplifier Serial Register Active Array Output Node ADC
75 Preamplifier Serial Register Active Array Output Node
76 Preamplifier Serial Register Active Array Output Node
77 Preamplifier Serial Register Active Array Output Node ADC
78 High-light, full-resolution image
79 Low-light, full-resolution image
80 2x8 binned image of low-light image
81 CCD Performance What does your experiment require?
82 Performance Applications Benefits of Cooled CCD Technology Low-light sensitivity (ASA 100,000) Ultra-low low noise and dark current Real-time image capture with digital output Dynamic range up to 16 bits (65,000 gray levels) High quantum efficiency over wide spectral range Programmable readout modes - Binning - Subarray readout
83 Performance Applications Matching Camera System Requirements to an Application Camera parameters to consider - Spatial resolution - Dynamic range and linearity - Temporal resolution - Low-light sensitivity - Adapting to instruments: lenses, microscopes, spectrometers, etc. - Image analysis / application-specific software
84 Common Applications Spinning Disk Confocal FRET Low Light Timelapse Deconvolution
85 Camera Performance Aspects Resolution Speed Intensity
86 Performance Limitations Resolution = Many Pixels, small size Speed = Signal/noise? Intensity
87 Spatial Resolution The maximum resolution required depends on the type of data analysis performed. Many applications do not require an optically matched (high) resolution. Ratiometric Imaging ( Fura/Indo) Other calcium or isobestic indicators. Motion tracking Fewer Digital conversions (less pixels) = Increase in frame rate. Larger Pixel Size = Higher Sensitivity
88 Speed Does speed refer to fast framerate or short exposure? Speed requires high sensitivity and fast readout (Light is the limit!) l Is other hardware required to achieve the speed you want? (Dualview( Dualview, piezo focus, AOTF)
89 Intensity (sensitivity) Light collection determines the length of integration. Some experiments demand high frame rates: Live Cell Multi-Dimensional Acquisition (3-D) Motion tracking What are the intensity requirements of the experiment in comparison to the temporal/spatial limitations?
90 Intensity (Dynamic Range) The dynamic range required depends on the experiment and sample. Dynamic Range 8-Bit 8 (255) 12-Bit (4,095) 16-Bit (65,535) Many applications benefit from higher dynamic range. FRET (Fluorescence Resonance Energy Transfer) Ratiometric Imaging FLIM (Fluorescence Lifetime Intensity Measurement) Deconvolution Quiet Digitization makes best use of 16-Bit systems.
91 Common CCD Types Interline Transfer - High Resolution (7um per pixel) - Moderate Frame Rate - Moderate Sensitivity - 12 Bit System (4,095 Greylevels) Intensified (On-Chip Gain) - Moderate Resolution - High Speed and Sensitivity - 16 Bit System (65,535 Greylevels) Short Exposure (BT Camera) - High Sensitivity - Slow, Quiet digitization of Data - Various Resolution Options - 16 Bit System (65,535 Greylevels) Interline EMCCD s Slow Scan Back Illuminated
92 Cameras Resolution Interline Speed Back illuminated EMCCD Intensity
93 Resolution and speed Interline Transfer Most versatile type of camera! High Resolution / Moderate Sensitivity. 10 Frames Per 6.45um Pixel Res. ~60% Light Collecting Ability Balanced System Applications Fixed Fluorescence Timelapse Imaging Colocalization Live Cell Fluorescence
94 Resolution and Intensity Various Resolution / Very High Sensitivity. High Sensitivity Systems Dual or Quad view 95% Light Collecting Ability Very low read noise Deeply cooled Back illuminated Applications Long Term Timelapse FRET Co Localization Bioluminescence
95 Intensified / On-Chip Gain EMCCD Moderate Resolution / Very High Sensitivity. High Speed System 30 Frames Per 16um Pixel Res. 92% Light Collecting Ability Applications Confocal Imaging TIRF FRET Calcium Motion Tracking
96 Do I really need an EMCCD? `Images courtesy of Michael Davidson; FSU
97 Is the application low light fast? `Images courtesy of Michael Davidson; FSU
98 On-Chip Multiplication Gain Technology Based Based on conventional CCD Frame Transfer - fast frame rates, no shutter Back illuminated - highest Q.E. Small pixel size - high spatial resolution Dual Amplifier Capability slow scan, low noise option Solid State detector No damage due to bright light Multiplication in the solid state domain Minimum excess noise No need for external amplifier hardware (e.g., photocathode) Easy to vary the multiplication level
99 Theory of Operation On-chip multiplication gain CCD Active array Masked array Serial register Preamplifier Extended serial register Output node ADC Pre-amp/electronics noise is effectively overcome by multiplying the signal
100 Analog Gain Preamplifier Serial Register Active Array Output Node ADC
101 Signal-to to-noise Ratio Signal-to to-noise (SNR) is an important consideration in low-light light level imaging applications
102 The Key Terms Signal Created when the incoming Photons (S) are detected as electrons. Don t t forget QE! (Quantum Efficiency) Noise (σ)( Three main sources #1: Light itself: Photon Shot Noise S - Law of Physics - Given by square root of the signal #2: Detector and Electronics: Read Noise #3: Thermal Energy: Dark Noise ( D)( -Modern CCD detectors are cooled -not the limiting factor at high frame rates
103 SNR: The Classic equation Standard CCD SNR Equation: SNR = S [Sn2+Rn2+Dn2] High read noise is the limitation for low-light detection
104 The Read Noise limitation Low-light level applications were often read noise limited i.e., signals below the read noise could not be detected Read noise limited Read noise minimized
105 Nothing is free; EMCCD s are low light fast!!
106 SNR: The new equation On-Chip Multiplication Gain CCD SNR: SNR = [S*QE] [S*QE*F 2 + D*F 2 + (σ R /G) 2 ] Note: F is the excess noise factor. For more information, refer to the Technical Note: On-chip multiplication gain Acrobat Documen
107 Excess Noise Factor Excess Excess noise (F) is generated in the multiplication process Increase Increase in the pulse height distribution of the input signal Measured to be between 1.0 and 1.4
108 Two-In In-One Camera Dual readout amplifiers 1. EM Gain amplifier for high speed and low- light applications, operates at 10 MHz and 5 MHz 2. Traditional amplifier for slow scan, low-noise applications, Operates at 5 MHz and 1 MHz
109 EMCCD Applications Single-molecule fluorescence High-speed motility studies High-speed FRET/Ion imaging Bio-Luminescence (NO WAY!)
110 When to use an EMCCD in Life Science Applications Less excitation light reduces phototoxicity Live cell fluorescence Fast bleaching dyes Single Molecule Fluorescence Fast Fast kinetics studies Motility Ratio imaging for Calcium, ph, FRET
111 Common Applications (a recap) Spinning Disk Confocal FRET Low Light Timelapse Deconvolution
112 Spinning Disk Confocal High Frame Speed & Low Light Can be Synchronized with focusing hardware Dynamic Range helpful for decon/processing later. Operated at x Mag, large Pixel Appropriate.
113 FRET Low Light Large Dynamic Range key Could be Fast Framerate or slow. Varying Magnifications used
114 Low Light Timelapse Low Light No Framerate requirement Lowest camera noise possible Varying Magnifications used
115 Deconvolution Adequate Illumination Varying frame rate Maximum resolution
116 What is the right system for your lab? One Lab may have several different needs for an imaging system. Can one system perform all experiments well? Is a bundled system appropriate? What limitations do the microscope systems place on the imaging hardware? Will a dual-view, piezo or high speed excitation system change your camera choice? What system will provide the best performance for future experiments? As experiments develop demands change.
117 Thank you!
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