From CCD to EMCCDScientific imaging for today’s microscopy

From CCD to EMCCD

Is this a right topic?

Who should care what?

The CCD manufactories: Sony, Kodak, Texas Instruments, e2V ….

2. Cameras: Read noise, cooling, interface, speed, sensitivities …..

MAG !!

3. Imaging Systems:

1. CCD, CMOS, EMCCD, Interline, Color, Frame Transfer……

System Integrators, LIN Trading !!

From CCD to EMCCD

Is this a right topic?

What actually do you/researchers care about?

Beautiful image!!!

Publication and

Quantization !!!

From CCD to EMCCD

The right topic

What make a good image?

• better resolution

• higher Signal-Noise Ratio

• Good Contrast (Dynamic Range)

• Resolution (Digital Resolution, Spatial Resolution)

• Signal-Noise Ratio

• Contrast / Dynamic Range

From CCD to EMCCD

Revised topic: important characters of digital image

electronics

CCD / CMOS

image sensor

Camera (CCD?)

• Resolution (Digital Resolution, Spatial Resolution)

• Signal-Noise Ratio

• Contrast (Dynamic Range)

From CCD to EMCCD

Revised topic: important characters of digital image

From CCD to EMCCD

Digital Resolution

Approach:

1. Bigger Chip;

High cost for high grade chips

2. Smaller Pixels;

Lower sensitivity (Signal/Noise Ratio)

3. Micro Scanning

Good Balance of the above, slow speed.

From CCD to EMCCD

Digital Resolution

Useful resolution for microscopy

Camera Resolving Power > Optical Resolving Power

1. Specimen details resolved by the objectives, need to be acquired by the camera ;2. Avoid “empty resolution“, empty resolution only create unnecessary large files.

Tip to be remembered:Camera resolution should match optical resolution;Low magnifications normally require higher camera resolutions.

From CCD to EMCCD

Digital Resolution : The higher, the better ?

25 mm

2/3 “ Chip

Adaptation 1,0 x 18, 3% of view area 20

Adaptation 0,5 x , 58% of view area 20

sensor size 8,8 mm x 6,6 mm

8.8mm6

.6m

m

1.22 x N.A.Objective + N.A.Condenser

d0 =

Field of View = Field Number/mag.

Requested resolution = 2* FOV / d0

Requested Resolution

The higher, the better?

1435 x 108184714 x 53842 1,4100

921 x 69354459 x 34627 0,9100

2270 x 17091341139 x 85867 1,463

3320 x 24991951666 x 1254981,340

1906 x 1435112952 x 717560,7540

3255 x 24511911632 x 1229960,8025

3830 x 28862251921 x 14461130,7520

2548 x 19181501275 x 960 750,520

5097 x 38373002550 x 19201500,510

2548 x 19181501275 x 960750,2510

3063 x 23051801530 x 1152900,155

4905 x 36932882448 x 18431440,122,5

3210 x 24511921632 x 1229960,041,25

Necessary camera

resolution

Lines/mm(TV- 0,5 x)

Necessary camera

resolution

Lines/mm(TV-1.0 x)

N.A.Magnificati

on

Nyquist Theorem: Sampling frequency should be double the frequency of the signal.

From CCD to EMCCD

Digital Resolution: the higher, the better?

1. GREEN resolution/QE ->50％；2. RED resolution/QE -> 25％； 3. BLUE resolution/QE -> 25％； 4. The color interpolation decreases camera resolution;

4.2 Pixels

From CCD to EMCCD

Digital Resolution: Color or Mono?

1 Pixels

Exposure Time: Color 3.34 ms VS. Mono 0.9 ms

From CCD to EMCCD

Digital Resolution: Color or Mono?

• Resolution (Digital Resolution, Spatial Resolution)

• Signal-Noise Ratio

• Contrast (Dynamic Range)

From CCD to EMCCD

Revised topic: important characters of digital image

Important Camera Specs affect Signal-Noise Ratio

1. Quantum Efficiency: higher signal

2. Noise: Photon noise, readout noise, dark current

3. Signal-Noise Ratio, Camera sensitivity

From CCD to EMCCD

Signal-Noise Ratio: Low light considerations

Quantum Efficiency : The Spectral Response / Photon to Electron converting efficiency

From CCD to EMCCD

Signal-Noise Ratio: Quantum Efficiency

Front vs Backside Illuminated CCD

From CCD to EMCCD

Signal-Noise Ratio: Quantum Efficiency

– Photon-induced shot noise– Readout noise– Dark current noise

• Total System Noise = all noise sources added in quadrature

Main Noise Sources in CCDs

From CCD to EMCCD

Signal-Noise Ratio: Noise

Photon Noise (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

Photon noise = √Signal electrons

- Poisson distribution- When photon noise exceeds system noise, image data is photon (shot) noise limited

From CCD to EMCCD

Signal-Noise Ratio: Noise

Serial Register

Preamplifier

Output NodeActive Array

ADC

From CCD to EMCCD

Signal-Noise Ratio: Noise

CCD Readout

Read Noise (preamplifier noise)

- Higher readout speed leads to higher Read Noise; example: Readout speed = 1 MHZ, Readout Noise = 3 e; -> 0.5 frame/second Readout speed = 20 MHZ, Readout Noise = 8 e; -> 10 frames/second

- 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

From CCD to EMCCD

Signal-Noise Ratio: Noise

Dark current: • Electrons created by thermal emission;• Increases with time and temperature;• Cooling CCD reduces Dark Current;

Dark current is cut in half as the CCD temperature drops approximately every 6.7° C• Reduced by utilizing multi-pinned-phase (MPP) technology

Rule: 6~7 degree doubling

From CCD to EMCCD

Signal-Noise Ratio: Noise

Tip: Readout Noise is the major equipmental noise contributor for a cooled camera!

Total equipment noise=√readout noise2+dark noise2

We use a typical readout noise = 8e, Dark noise =√total dark current =√dark current x exposure time

camera A cooled 25°C lower than ambient, dark current = 0.15e/p/scamera B cooled to -25°C, dark current = 0.015e/p/s

With exposure 30s, Total noise of camera A = 8.27e Total noise of camera B = 8.02e

With exposure 1mins, Total noise of camera A = 8.54e Total noise of camera A = 8.06e

From CCD to EMCCD

Signal-Noise Ratio: the cooler, the better?

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

– Photon Noise - A law of physics!

– Readout Noise - Reduced by careful electronics design

– Dark Current Noise - Reduced by cooling and MPP

Noise Reduction in CCD

From CCD to EMCCD

Signal-Noise Ratio

From CCD to EMCCD

Signal-Noise Ratio: The final Equation

Signal-to-Noise Ratio of an Image = Total Photon collected / Noise1. Total Photon Collected where P=total incident photons,

DQE = QE at specific wavelength

2. Shot Noise

From CCD to EMCCD

Signal-Noise Ratio: The final Equation

Sensitivity of a camera: the lowest signal can be differentiated from background noise by the camera

Read Noise limited region

Photon Noise limited region

• Resolution (Digital Resolution, Spatial Resolution)

• Signal-Noise Ratio

• Dynamic Range (Contrast)

From CCD to EMCCD

Revised topic: important characters of digital image

Well capacity (Well depth):Number of electrons can be hold by a pixel before saturation

Well Capacity will be higher when pixel size is bigger:

• Same resolution, larger chip size;

• Same chip size, lower resolution;

• Binning

Note: If the charge capacity is exceeded,the excess charge will overflow into

adjacent pixels and produce artifacts known as blooming and smear.

NoiseOverflowing and Blooming

Charging

From CCD to EMCCD

Revised topic: important characters of digital image

Dynamic Range

Dynamic Range = Well capacity / Read noiseDynamic Range (dB) = 20 x Log10 (Well capacity /Read noise)

Tip: if your sample contains both very dark and very bright signals, a higher dynamic range camera is needed to imaging them in one shot!

From CCD to EMCCD

Revised topic: Dynamic Range

Dynamic Range of CCD should be matched to A/D Converter. 12, 14, 16 bit

Binning

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

From CCD to EMCCD

Revised topic: Dynamic Range

Slower readout -> Lower noise

From CCD to EMCCD Low light – slower readout or longer exposure

Longer exposure -> Stronger

Signal

same exposure same readout speed

short exposure less photon collected

high readout high noise

From CCD to EMCCD

Low light & High Speed -> Short exposure + High Readout ??!!

Signal-to-Noise Ratio (SNR) = Total Photon collected / Noise

• EMCCD: Electron Multiplying Charge Coupled Device

• Operates by applying high voltage during readout before the preamp stage of the CCD. Occurs through a probabilistic phenomenon where the gain is determined by:

Gain = (1 + g)N

where g is the probability of creating a second electron (typically in the vicinity of 0.01 – 0.016) and N is the number of elements (usually 500+)

From CCD to EMCCD Low light & High Speed -> The EMCCD Technology !

Signal-to-Noise Ratio (SNR) = Total Photon collected / Noise

Frame Transfer CCD

Serial Register

Preamplifier

Output NodeActive Array

Frame Transfer CCD

Serial Register

Preamplifier

Output NodeActive Array

Frame Transfer CCD

Serial Register

Preamplifier

Output NodeActive Array

Frame Transfer CCD

Serial Register

Preamplifier

Output NodeActive Array

Frame Transfer CCD

Serial Register

Preamplifier

Output NodeActive Array

ADC

ReadoutSignal = 1

Note: if read noise is 1 then S/N = 1/1

Frame Transfer EMCCD

Active Array

Output Node

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register Preamplifier

Output Node

Active Array

EM Register

Frame Transfer EMCCD

Serial Register

Active Array

Preamplifier

Output Node

EM Register

Frame Transfer EMCCD

Serial Register Preamplifier

Output Node

Active Array

ADC

ReadoutSignal = 5

Note: if read noise is 1 then S/N = 5/1! Vast improvement

EM Register

On-Chip Multiplication Gain CCD SNR:

SNR=[S*QE]÷√[S*QE*F2 + D*F2 +(σR/G)2]

Note: F is the excess noise factor.

From CCD to EMCCD SNR: The new equation

From CCD to EMCCD Types of Noise in EM Cameras

• Dark Current– Dependent on exposure time– Increases when gain is increased -> cooling important

• Read Noise– Changes with readout speed

• Spurious Noise (aka clock induced charge)– Not dependent on exposure time– Lower cooling increases chance of spurious charge– Occurs during high pulse clocking of CCD and generates a

secondary electron, even though no primary is present– Usually combined with the overall dark charge

• Excess Noise Factor– Based on deviation or uncertainty in on-chip multiplication gain

EM camera Applications

• Total internal reflection fluorescence (TIRF) microscopy

• Spinning-disk confocal microscopy

• Dynamic ratio imaging (e.g., pH and low-concentration flux)

• Fluorescence recovery after photo bleaching (FRAP)

• Live-cell fluorescent protein imaging

Very high sensitivity up to single molecular detection!

From CCD to EMCCD When to use EMCCD?

Signal-to-Noise ratio curve

From CCD to EMCCD

Dual Amplifier EMCCD: Traditional Amplifier for Wide-dynamic range operation

From CCD to EMCCDScientific imaging for today’s microscopy

Thank you for your attention!