1. From CCD to EMCCD
Scientific imaging for today’s microscopy

2. Is this a right topic? From CCD to EMCCD Who should care what?
1. CCD, CMOS, EMCCD, Interline, Color, Frame Transfer……
The CCD manufactories: Sony, Kodak, Texas Instruments, e2V ….
2. Cameras: Read noise, cooling, interface, speed, sensitivities …..
MAG !!
3. Imaging Systems:
System Integrators, LIN Trading !!

3. Is this a right topic? From CCD to EMCCD
What actually do you/researchers care about?
Beautiful image!!!
Publication and
Quantization !!!

4. From CCD to EMCCD The right topic What make a good image?
higher Signal-Noise Ratio
better resolution
Good Contrast (Dynamic Range)

5. (Digital Resolution, Spatial Resolution) Signal-Noise Ratio
From CCD to EMCCD
Revised topic: important characters of digital image
Resolution
(Digital Resolution, Spatial Resolution)
Signal-Noise Ratio
Contrast / Dynamic Range

6. Camera (CCD?)
electronics
CCD / CMOS
image sensor

7. (Digital Resolution, Spatial Resolution) Signal-Noise Ratio Contrast
From CCD to EMCCD
Revised topic: important characters of digital image
Resolution
(Digital Resolution, Spatial Resolution)
Signal-Noise Ratio
Contrast
(Dynamic Range)

8. From CCD to EMCCD
Digital Resolution

9. From CCD to EMCCD Digital Resolution Approach: Bigger Chip;
High cost for high grade chips
Smaller Pixels;
Lower sensitivity (Signal/Noise Ratio)
3. Micro Scanning
Good Balance of the above, slow speed.

10. From CCD to EMCCD Digital Resolution : The higher, the better ?
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.

11. 1.22 x l N.A.Objective + N.A.Condenser
Requested Resolution
The higher, the better?
1.22 x l N.A.Objective + N.A.Condenser
d0 =
Field of View = Field Number/mag.
Requested resolution = 2* FOV / d0
25 mm
Adaptation 1,0 x  18, 3% of view area 20
8.8mm
6.6mm
Adaptation 0,5 x , 58% of view area 20
2/3 “ Chip
sensor size 8,8 mm x 6,6 mm

12. Necessary camera resolution
From CCD to EMCCD
Digital Resolution: the higher, the better?
1435 x 1081 84
714 x 538 42
1,4
100
921 x 693 54
459 x 346 27
0,9
2270 x 1709
134
1139 x 858 67 63
3320 x 2499
195
1666 x 1254 98
1,3 40
1906 x 1435
112
952 x 717 56
0,75
3255 x 2451
191
1632 x 1229 96
0,80 25
3830 x 2886
225
1921 x 1446
113 20
2548 x 1918
150
1275 x 960 75
0,5
5097 x 3837
300
2550 x 1920 10
0,25
3063 x 2305
180
1530 x 1152 90
0,15 5
4905 x 3693
288
2448 x 1843
144
0,12
2,5
3210 x 2451
192
0,04
1,25
Necessary camera resolution
Lines/mm (TV- 0,5 x)
Lines/mm (TV-1.0 x)
N.A.
Magnification
Nyquist Theorem: Sampling frequency should be double the frequency of the signal.

13. From CCD to EMCCD Digital Resolution: Color or Mono?
1. GREEN resolution/QE ->50％；
2. RED resolution/QE -> 25％；
3. BLUE resolution/QE -> 25％；
4. The color interpolation decreases camera resolution;
1 Pixels
4.2 Pixels

14. Exposure Time: Color 3.34 ms VS. Mono 0.9 ms
From CCD to EMCCD
Digital Resolution: Color or Mono?
Exposure Time: Color 3.34 ms VS. Mono 0.9 ms

15. (Digital Resolution, Spatial Resolution) Signal-Noise Ratio Contrast
From CCD to EMCCD
Revised topic: important characters of digital image
Resolution
(Digital Resolution, Spatial Resolution)
Signal-Noise Ratio
Contrast
(Dynamic Range)

16. From CCD to EMCCD Signal-Noise Ratio: Low light considerations
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

17. From CCD to EMCCD Signal-Noise Ratio: Quantum Efficiency
The Spectral Response / Photon to Electron converting efficiency

18. From CCD to EMCCD Signal-Noise Ratio: Quantum Efficiency
Front vs Backside Illuminated CCD

19. Main Noise Sources in CCDs
From CCD to EMCCD
Signal-Noise Ratio: Noise
Main Noise Sources in CCDs
Photon-induced shot noise
Readout noise
Dark current noise
Total System Noise =
all noise sources added in quadrature

20. Photon Noise (Shot Noise)
From CCD to EMCCD
Signal-Noise Ratio: Noise
Photon Noise (Shot Noise)
- 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
- 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

21. From CCD to EMCCD CCD Readout ADC Signal-Noise Ratio: Noise
Preamplifier
Serial Register
ADC
Output
Node
Active Array

22. Read Noise (preamplifier noise)
From CCD to EMCCD
Signal-Noise Ratio: Noise
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

23. 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

24. From CCD to EMCCD Signal-Noise Ratio: the cooler, the better?
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/s
camera 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

25. From CCD to EMCCD Noise Reduction in CCD Signal-Noise Ratio
Photon Noise - A law of physics!
Readout Noise - Reduced by careful electronics design
Dark Current Noise - Reduced by cooling and MPP
Ultimately, a High-Performance CCD camera is limited only by Readout Noise and Photon Noise.

26. From CCD to EMCCD Signal-Noise Ratio: The final Equation
Signal-to-Noise Ratio of an Image = Total Photon collected / Noise
1. Total Photon Collected where P=total incident photons,
DQE = QE at specific wavelength
2. Shot Noise

27. Read Noise limited region Photon Noise limited region
From CCD to EMCCD
Signal-Noise Ratio: The final Equation
Read Noise limited region
Photon Noise limited region
Sensitivity of a camera: the lowest signal can be differentiated from background noise by the camera

28. (Digital Resolution, Spatial Resolution) Signal-Noise Ratio
From CCD to EMCCD
Revised topic: important characters of digital image
Resolution
(Digital Resolution, Spatial Resolution)
Signal-Noise Ratio
Dynamic Range (Contrast)

29. 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.
Charging
Overflowing and Blooming
Noise

30. From CCD to EMCCD Dynamic Range Revised topic: Dynamic Range
Dynamic Range = Well capacity / Read noise
Dynamic Range (dB) = 20 x Log10 (Well capacity /Read noise)
Dynamic Range of CCD should be matched to A/D Converter. 12, 14, 16 bit
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!

31. Binning From CCD to EMCCD Revised topic: Dynamic Range
- Higher Dynamic Range - Higher Signal-to-Noise Ratio
- Faster Readout
- Dynamically Change Pixel Size/Aspect Ratio

32. From CCD to EMCCD Low light – slower readout or longer exposure
Slower readout -> Lower noise
Longer exposure -> Stronger Signal
same exposure
same readout speed

33. From CCD to EMCCD Low light & High Speed -> Short exposure + High Readout ??!!
Signal-to-Noise Ratio (SNR) = Total Photon collected / Noise
short exposure  less photon collected
high readout  high noise

34. From CCD to EMCCD Low light & High Speed -> The EMCCD Technology !
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+)
Signal-to-Noise Ratio (SNR) = Total Photon collected / Noise

35. Frame Transfer CCD Preamplifier Serial Register Output Node
Active Array

36. Frame Transfer CCD Preamplifier Serial Register Output Node
Active Array

37. Frame Transfer CCD Preamplifier Serial Register Output Node
Active Array

38. Frame Transfer CCD Preamplifier Serial Register Output Node
Active Array

39. Frame Transfer CCD Note: if read noise is 1 then S/N = 1/1
Preamplifier
Serial Register
ADC
Output
Node
Active Array
Readout
Signal = 1

40. Frame Transfer EMCCD
Output
Node
Active Array

41. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

42. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

43. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

44. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

45. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

46. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

47. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

48. Frame Transfer EMCCD Serial Register EM Register Preamplifier Output
Node
Active Array

49. Frame Transfer EMCCD Serial Register EM Register Preamplifier ADC
Output
Node
Readout
Signal = 5
Note: if read noise is 1 then S/N = 5/1!
Vast improvement
Active Array

50. From CCD to EMCCD SNR: 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.

51. 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
Excess noise factor is based on the fact that the gain is a probabilistic phenomenon and there is deviation
Regarding quantization: EMCCDs have a reputation for not being quantitative and to some level this is true. However, CCDs can also be considered non-quantitative too, it is just that the error bars are much smaller than an EMCCD and therefore not as pronounced.
SNR = (Signal*QE)/Total System Noise
Total System Noise = sq root (Signal*QE*F2 + Dark Noise * F2 + Read Noise * F2)

52. EM camera Applications
Very high sensitivity up to single molecular detection!
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

53. From CCD to EMCCD When to use EMCCD?
Signal-to-Noise ratio curve

54. From CCD to EMCCD Dual Amplifier EMCCD:
Traditional Amplifier for Wide-dynamic range operation

55. Thank you for your attention!
From CCD to EMCCD
Scientific imaging for today’s microscopy
Thank you for your attention!