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LMBRLMBRImaging WorkshopImaging Workshop

Imaging Fundamentals Imaging Fundamentals Mike Meade, PhotometricsMike Meade, Photometrics

IntroductionIntroduction

CCD FundamentalsCCD Fundamentals

SealedWindow

DC Voltage

Serial Clock Driver

Parallel Clock Driver

Low Noise Preamplifier

Shutter

IncomingLight

Optic

HCCD (-45OC)

Thermo-ElectricCooler

CoolingFins

Typical Cooled CCD Camera Configuration

CameraControlLogicand

PowerSupply

AnalogProcessingand ADC

CameraCommand

Input

CameraStatusOutput

DigitalPixel Data

Output(12-16 bits)



The Key to our Systems!

ROPER SCIENTIFIC ™

• 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)SonyOthers


The Charge-Coupled Device (CCD)


MOS MOS PhotodetectorPhotodetector


Quantum Efficiency

Ability of CCD to convert photons to electrons

Usually reported as a percentage

ROPER SCIENTIFIC ™


Front vs Backside Illuminated


Indium Tin Oxide (ITO ) Technology


Enhancing UV Sensitivity with Proprietary CCD Coatings

400 500 600 700 800 900 1000Wavelength (nm)

0

10

20

30

40

50

60

90

70

80

ITO(Kodak KAF-1401E)

standardfrontside-illuminated(Kodak KAF-1400)

thinned,backside-illuminated(SITe SI502AB)

lens-on-chip(Sony interline)

300200

Metachrome


Typical Quantum Efficiencies

Serial Register

Preamplifier

Output Node

Par

alle

l R

egis

ter



Typical CCD Structure


CCD ArchitectureCCD Architecture

SerialClocks

SerialClocks

SerialClocks

Direction ofParallel Shift



ParallelClocksforStorageArray

ParallelClocksforImageArray

Full-Frame CCD

Frame-Transfer CCD

Interline-Transfer CCD

SerialRegister

SerialRegister

SerialRegister

Full Array Full ArrayImage Array

Storage Array(masked)


Full Frame Interline TransferFrame Transfer(EMCCD)


Serial Register

Preamplifier

Output NodeActive Array

Full Frame


Serial Register

Preamplifier



Serial Register

Preamplifier


ADC


Serial Register

Preamplifier



Serial Register

Preamplifier


ADC


Serial Register

Preamplifier



ADC

Serial Register

Preamplifier



Serial Register

Preamplifier



ADC

Serial Register

Preamplifier



Serial Register

Preamplifier



Serial Register

Preamplifier


Frame Transfer


Serial Register

Preamplifier



Serial Register

Preamplifier


Interline Transfer


Serial Register

Preamplifier



Full Frame Interline TransferFrame Transfer

High Spatial Resolution

Large Number of Pixels

Greater Selection ofCCD Formats

100% Fill Factor forEntire Array

Integrate while Clocking

High-Speed Operation (Video)

Lens-on-Chip Technology


High-Speed Operation

Well Suited for Complex Readout Schemes

100% Fill Factor in Active Array


High Spatial Resolution

Large Number of Pixels

Greater Selection ofCCD Formats

100% Fill Factor forEntire Array


High-Speed Operation

Well Suited for Complex Readout Schemes

100% Fill Factor in Active Array


High-Speed Operation (Video)

Lens-on-Chip Technology


High quantum efficiencyHigh quantum efficiency

Wide spectral responseWide spectral response

Low dark currentLow dark current

Ability to integrate chargeAbility to integrate charge

Sufficient resolutionSufficient resolution

Ideal CCD Characteristics


Performance Performance ConsiderationsConsiderations

CCD Linearity

20

200

150

100

50

00

Illumination Level (Arbitrary)

250

300

350

40 60 80 100



PhotonPhoton--induced shot noiseinduced shot noiseReadout noiseReadout noiseDark current noiseDark current noisekTCkTC reset switch noisereset switch noiseSpurious chargeSpurious charge

Total System Noise = all noise Total System Noise = all noise sources added sources added

in in quadraturequadrature

Noise Sources in CCDs


Photon (Shot Noise)

- Law of physics- Square root relationship between signal and noise

noise = square root of number of electrons- Poisson distribution- When photon noise exceeds system noise, data is

photon (shot) noise limited

- Law of physics- Square root relationship between signal and noise

noise = square root of number of electrons- Poisson distribution- When photon noise exceeds system noise, data is

photon (shot) noise limited


Read Noise (preamplifier noise)

- Minimized by careful electronic design- Under low-light/low-signal conditions where

read noise exceeds photon noise, data is read noise limited

- Read noise not as relevant in high-signal applications

- Minimized by careful electronic design- Under low-light/low-signal conditions where

read noise exceeds photon noise, data is read noise limited

- Read noise not as relevant in high-signal applications

Serial Register

Preamplifier


ADC

Analog GainAnalog Gain


Dark Current

Electrons created by thermal emission

Increases with time and temperature


Dark current subtracts, dark current noise remains

- dark current noise = dark current- dark current (dark noise) can lower SNR- dark current noise = dark current- dark current (dark noise) can lower SNR


Reduced by cooling the CCD

Reduced by utilizing multi-pinned-phase(MPP) technology

- Dark current is cut in half as the CCD temperaturedrops approximately every 6.7° C


Limit of cooling effectiveness

- MPP CCDs already have low dark current- Poor CTE (charge transfer efficiency) at <-120°C- MPP CCDs already have low dark current- Poor CTE (charge transfer efficiency) at <-120°C

Ultimately, a High-Performance CCD camera is limited only by Readout Noise and Photon Noise.


Photon Noise Photon Noise -- A law of physics!A law of physics!Readout Noise Readout Noise -- Reduced by careful electronics Reduced by careful electronics designdesignDark Current Noise Dark Current Noise -- Reduced by cooling and Reduced by cooling and MPPMPPkTCkTC Noise Noise -- Reduced by using correlated double Reduced by using correlated double samplingsamplingSpurious Charge Spurious Charge -- Reduced by careful shaping Reduced by careful shaping of clock waveformsof clock waveforms

Noise Reduction in CCDs


Dynamic RangeDynamic Range


Dynamic Range = Full Well/Read Noise

- Full Well = A/D converter bit depth × 1x gain

™


Dynamic Range of CCD is matched to A/D Converter

12, 14, 16 bit


Intrascene Dynamic Range


BinningBinning

Above all gained at the expense of Spatial Resolution!!


Binning

- Higher Dynamic Range- Higher Signal-to-Noise Ratio- Faster Readout- Dynamically Change Pixel Size/Aspect Ratio

Serial Register

Preamplifier


Binning


Serial Register

Preamplifier



ADC

Serial Register

Preamplifier



Serial Register

Preamplifier



Serial Register

Preamplifier


ADC



High-light, full-resolution image


Low-light, full-resolution image


2x8 binned image of low-light image

CCD PerformanceCCD Performance

What does your experiment require?What does your experiment require?

Performance ApplicationsPerformance Applications

LowLow--light sensitivity (ASA 100,000)light sensitivity (ASA 100,000)

UltraUltra--low noise and dark currentlow noise and dark current

RealReal--time image capture with digital outputtime image capture with digital output

Dynamic range up to 16 bits (65,000 gray levels)Dynamic range up to 16 bits (65,000 gray levels)

High quantum efficiency over wide spectral rangeHigh quantum efficiency over wide spectral range

Programmable readout modesProgrammable readout modes-- BinningBinning-- SubarraySubarray readoutreadout

Benefits of Cooled CCD Technology

Performance ApplicationsPerformance 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

Common ApplicationsCommon Applications

Spinning Disk ConfocalSpinning Disk ConfocalFRETFRETLow Light TimelapseLow Light TimelapseDeconvolutionDeconvolution

Camera Performance AspectsCamera Performance Aspects

ResolutionResolution SpeedSpeed

IntensityIntensity

Resolution = Many Resolution = Many Pixels, small sizePixels, small size

Speed = Signal/noise?Speed = Signal/noise?

IntensityIntensity

Performance LimitationsPerformance Limitations

Spatial ResolutionSpatial Resolution

The maximum resolution required depends on the type of data The maximum resolution required depends on the type of data analysis performed.analysis performed.

Many applications do not require an Many applications do not require an ““optically matchedoptically matched”” (high) (high) resolution.resolution.

•• Ratiometric Imaging ( Fura/Indo)Ratiometric Imaging ( Fura/Indo)•• Other calcium or isobestic indicators.Other calcium or isobestic indicators.•• Motion trackingMotion tracking

Fewer Digital conversions (less pixels) = Increase in frame ratFewer Digital conversions (less pixels) = Increase in frame rate.e.

Larger Pixel Size = Higher SensitivityLarger Pixel Size = Higher Sensitivity

SpeedSpeed

Does speed refer to fast framerate or short exposure?Does speed refer to fast framerate or short exposure?

Speed requires high sensitivity and fast readout (Light is the lSpeed requires high sensitivity and fast readout (Light is the limit!)imit!)

Is other hardware required to achieve the speed you want? (Is other hardware required to achieve the speed you want? (DualviewDualview, , piezopiezofocus, AOTF)focus, AOTF)

Intensity (sensitivity)Intensity (sensitivity)

Light collection determines the length of integration.Light collection determines the length of integration.

Some experiments demand high frame rates:Some experiments demand high frame rates:•• Live Cell MultiLive Cell Multi--Dimensional Acquisition (3Dimensional Acquisition (3--D)D)•• Motion trackingMotion tracking

What are the intensity requirements of the experiment in compariWhat are the intensity requirements of the experiment in comparison to the son to the temporal/spatial limitations? temporal/spatial limitations?

Intensity (Dynamic Range)Intensity (Dynamic Range)

The dynamic range required depends on the experiment and The dynamic range required depends on the experiment and sample.sample.

Dynamic Range 8Dynamic Range 8--Bit (255) 12Bit (255) 12--Bit (4,095) 16Bit (4,095) 16--Bit (65,535)Bit (65,535)

Many applications benefit from higher dynamic range.Many applications benefit from higher dynamic range.•• FRET (Fluorescence Resonance Energy Transfer)FRET (Fluorescence Resonance Energy Transfer)•• Ratiometric ImagingRatiometric Imaging•• FLIM (Fluorescence Lifetime Intensity Measurement)FLIM (Fluorescence Lifetime Intensity Measurement)•• DeconvolutionDeconvolution

Quiet Digitization makes best use of 16Quiet Digitization makes best use of 16--Bit systems.Bit systems.

Common CCD TypesCommon CCD Types

Interline TransferInterline Transfer-- High Resolution (7um per pixel)High Resolution (7um per pixel)-- Moderate Frame RateModerate Frame Rate-- Moderate SensitivityModerate Sensitivity-- 12 Bit System (4,095 Greylevels)12 Bit System (4,095 Greylevels)

Intensified (OnIntensified (On--Chip Gain)Chip Gain)-- Moderate ResolutionModerate Resolution-- High Speed and SensitivityHigh Speed and Sensitivity-- 16 Bit System (65,535 Greylevels)16 Bit System (65,535 Greylevels)

Short Exposure (BT Camera)Short Exposure (BT Camera)-- High SensitivityHigh Sensitivity-- Slow, Quiet digitization of DataSlow, Quiet digitization of Data-- Various Resolution OptionsVarious Resolution Options-- 16 Bit System (65,535 Greylevels)16 Bit System (65,535 Greylevels)

Interline

EMCCD’s

Slow Scan Back

Illuminated

ResolutionResolution SpeedSpeed

IntensityIntensity

Interline

CamerasCameras

Back illuminated EMCCD

Resolution and speedResolution and speedInterline TransferInterline TransferMost versatile type of camera!Most versatile type of camera!

High Resolution / Moderate High Resolution / Moderate Sensitivity.Sensitivity.10 Frames Per Second @ 10 Frames Per Second @ 6.45um Pixel Res.6.45um Pixel Res.~60% Light Collecting Ability~60% Light Collecting AbilityBalanced SystemBalanced System

Applications

Fixed Fluorescence – Timelapse Imaging – Colocalization – Live Cell Fluorescence

Resolution and IntensityResolution and IntensityBack illuminatedBack illuminated

Various Resolution / Very Various Resolution / Very High Sensitivity.High Sensitivity.High Sensitivity SystemsHigh Sensitivity Systems

Dual or Quad viewDual or Quad view95% Light Collecting Ability95% Light Collecting AbilityVery low read noiseVery low read noiseDeeply cooledDeeply cooled

Applications

Long Term Timelapse – FRET – Co Localization – Bioluminescence

Intensified / OnIntensified / On--Chip GainChip Gain

EMCCDEMCCDModerate Resolution / Very Moderate Resolution / Very High Sensitivity.High Sensitivity.High Speed SystemHigh Speed System30 Frames Per Second @ 30 Frames Per Second @ 16um Pixel Res.16um Pixel Res.92% Light Collecting Ability92% Light Collecting Ability

Applications

Confocal Imaging – TIRF – FRET – Calcium – Motion Tracking

Do I really need an EMCCD?Do I really need an EMCCD?

`Images courtesy of Michael Davidson; FSU`Images courtesy of Michael Davidson; FSU

Is the application low light fast?Is the application low light fast?

`Images courtesy of Michael Davidson; FSU`Images courtesy of Michael Davidson; FSU

OnOn--Chip Multiplication Gain Chip Multiplication Gain TechnologyTechnology

Based on conventional CCDBased on conventional CCDFrame Transfer Frame Transfer -- fast frame rates, no shutterfast frame rates, no shutterBack illuminated Back illuminated -- highest Q.E.highest Q.E.Small pixel size Small pixel size -- high spatial resolutionhigh spatial resolutionDual Amplifier Capability Dual Amplifier Capability –– slow scan, low noise slow scan, low noise option option

Solid State detectorNo damage due to bright light

Multiplication in the solid state domainMinimum excess noiseNo need for external amplifier hardware (e.g., photocathode)Easy to vary the multiplication level

Theory of OperationTheory of Operation

OnOn--chip multiplication gain CCDchip multiplication gain CCDActivearray

Masked array

Serial register

Extended serial register

Preamplifier

Output node

ADC

Pre-amp/electronics noise is effectively overcome by multiplying the signal

ADC

Serial Register

Preamplifier


Analog GainAnalog Gain

SignalSignal--toto--Noise RatioNoise Ratio

SignalSignal--toto--Noise (SNR) is an important Noise (SNR) is an important consideration in lowconsideration in low--light level imaging light level imaging

applicationsapplications

The Key TermsThe Key Terms

Signal Signal Created when the incoming Photons (S) are Created when the incoming Photons (S) are detected as electrons. detected as electrons. DonDon’’t forget QE! (Quantum Efficiency)t forget QE! (Quantum Efficiency)

Noise (Noise (σσ)) Three main sourcesThree main sources#1: Light itself:#1: Light itself: Photon Shot Noise Photon Shot Noise √√SS

-- Law of PhysicsLaw of Physics-- Given by square root of the signalGiven by square root of the signal

#2: Detector and Electronics:#2: Detector and Electronics: Read Noise Read Noise #3: Thermal Energy:#3: Thermal Energy: Dark Noise (Dark Noise (√√D)D)

--Modern CCD detectors are cooledModern CCD detectors are cooled--not the limiting factor at high frame ratesnot the limiting factor at high frame rates

SNR: The Classic equationSNR: The Classic equation

Standard CCD SNR Equation:

SNR = S ÷ √[Sn2+Rn2+Dn2]

High read noise is the limitation for low-light detection

The Read Noise limitationThe Read Noise limitation

LowLow--light level applications were often read light level applications were often read noise limitednoise limited

i.e., signals below the read noise could not be i.e., signals below the read noise could not be detecteddetected

Read noise limited Read noise minimized

Nothing is free; Nothing is free; EMCCDEMCCD’’ss are low light fast!!are low light fast!!

SNR: The new equationSNR: The new equation

On-Chip Multiplication Gain CCD SNR:

SNR = [S*QE] ÷ √[S*QE*F2 + D*F2 + (σR/G)2]

Note: F is the excess noise factor.

For more information, refer to the Technical Note: On-chip multiplication gain

Acrobat Documen

Excess Noise FactorExcess Noise Factor

Excess noise (F) is generated in the Excess noise (F) is generated in the multiplication processmultiplication process

Increase in the pulse height distribution Increase in the pulse height distribution of the input signalof the input signal

Measured to be between 1.0 and 1.4Measured to be between 1.0 and 1.4

TwoTwo--InIn--One CameraOne Camera

Dual readout Dual readout amplifiersamplifiers

1. EM Gain amplifier for 1. EM Gain amplifier for high speed and lowhigh speed and low--light applications, light applications, operates at 10 MHz and operates at 10 MHz and 5 MHz5 MHz2. Traditional amplifier for 2. Traditional amplifier for slow scan, lowslow scan, low--noisenoiseapplications, Operates at applications, Operates at 5 MHz and 1 MHz5 MHz and 1 MHz

EMCCD ApplicationsEMCCD Applications

SingleSingle--molecule fluorescencemolecule fluorescence

HighHigh--speed motility studiesspeed motility studies

HighHigh--speed FRET/Ion imagingspeed FRET/Ion imaging

BioBio--Luminescence (NO WAY!)Luminescence (NO WAY!)

When to use an EMCCD in Life When to use an EMCCD in Life Science ApplicationsScience Applications

Less excitation light reduces Less excitation light reduces phototoxicityphototoxicity

Live cell fluorescenceLive cell fluorescenceFast bleaching dyesFast bleaching dyes

Single Molecule FluorescenceSingle Molecule FluorescenceFast kinetics studiesFast kinetics studies

MotilityMotilityRatio imaging for Calcium, pH, FRETRatio imaging for Calcium, pH, FRET

Common ApplicationsCommon Applications(a recap)(a recap)

Spinning Disk ConfocalSpinning Disk ConfocalFRETFRETLow Light TimelapseLow Light TimelapseDeconvolutionDeconvolution

Spinning Disk ConfocalSpinning Disk Confocal

High Frame Speed & Low LightHigh Frame Speed & Low LightCan be Synchronized with focusing hardwareCan be Synchronized with focusing hardwareDynamic Range helpful for decon/processing later. Dynamic Range helpful for decon/processing later. Operated at 60Operated at 60--100x Mag, large Pixel Appropriate.100x Mag, large Pixel Appropriate.

FRETFRET

Low LightLow LightLarge Dynamic Range keyLarge Dynamic Range keyCould be Fast Framerate or slow. Could be Fast Framerate or slow. Varying Magnifications usedVarying Magnifications used

Low Light TimelapseLow Light Timelapse

Low LightLow LightNo Framerate requirementNo Framerate requirementLowest camera noise possible Lowest camera noise possible Varying Magnifications usedVarying Magnifications used

DeconvolutionDeconvolution

Adequate IlluminationAdequate IlluminationVarying frame rateVarying frame rateMaximum resolutionMaximum resolution

What is the right system for your lab?What is the right system for your lab?

One Lab may have several different needs for an imaging One Lab may have several different needs for an imaging system. Can one system perform all experiments well? Is a system. Can one system perform all experiments well? Is a bundled system appropriate?bundled system appropriate?

What limitations do the microscope systems place on the What limitations do the microscope systems place on the imaging hardware? imaging hardware?

Will a dualWill a dual--view, view, piezopiezo or high speed excitation system change or high speed excitation system change your camera choice?your camera choice?

What system will provide the best performance for future What system will provide the best performance for future experiments? As experiments develop demands change.experiments? As experiments develop demands change.

Thank you!Thank you!

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