1. Page 1 Detectors for Fluorescence Imaging Klaus Suhling Department of Physics King’s College London Strand London WC2R 2LS

2. Page 2Outline What is light ? How do you detect light ? Single point detectors - photomultipliers/photodiodes Imaging detectors – cameras signal to noise considerations detectors of the future Summary & Resources

3. Page 3 BC AD 1565 1590 1665 1852 1893 1900 1926 1930 1950 1955 1990 1994 20?? fluorescence explained Stokes fluorescence lifetimes measured Gaviola fluorescence lifetime imaging Bugiel at al Wang et al fluorescence observed Monardes bioluminescence compound microscope Jansens UV fluorescence microscopy Köhler Simultaneous imaging of entire fluorescence emission contour Micrographia Hooke magnifying glasses fluorescence microscopy Haitinger et al fluorescently labelled antibodies Coons et al GFP Chalfie et al confocal microscope Minsky microscopy fluorescence Theory of microscopy Abbe A brief history of fluorescence, lifetime and imaging Adapted from: K. Suhling. “Fluorescence Lifetime Imgaging.” in Methods Express, Cell Imaging (ed D. Stephens), chapter 11, 219-245, Scion publishing, Bloxham, 2006.

4. Page 4 Optical Microscopy Micrographia, published in 1665 by Robert Hooke (1635-1703) Hooke also coined the word cell (compartments in cork)

5. Page 5 Modern Fluorescence Microscopy high contrast, exciting light eliminated (Stokes’ shift) minimally invasive & non-destructive can be performed on live cells and tissue tag specific proteins in live cells with fluorescent labels and locate them

6. Page 6 Fluorescent labels for microscopy Stain biological specimen with fluorescent dyes, nanodiamonds or quantum dots and observe stained regions Use genetically encoded fluorescence proteins, e.g. green fluorescent protein GFP Use endogenous fluorescence (“autofluorescence”), e.g. tryptophan, flavins, NaDH, collagen, elastin

7. Page 7 What is Fluorescence? krkr excited state S 1 ground state S 0 k isc T1T1 k ic radiative deactivation of the first electronically excited singlet state k ph / k ic molecular energy levels

8. Page 8 What is light? Light as a wave - Huygens principle - pinhole is centre of spherical wave Instructor's Resource CD-ROM, Physics, James S. Walker, Pearson Education 2004 Christian Huygens (1629-1695) Traite de la Lumiere, 1678

9. Page 9 Young’s double slit experiment Instructor's Resource CD-ROM, Physics, James S. Walker, Pearson Education 2004 Spherical waves emanate from slits and interfere Thomas Young, 1801

10. Page 10 Young’s double slit experiment Instructor's Resource CD-ROM, Physics, James S. Walker, Pearson Education 2004

11. Page 11 The electromagnetic spectrum Light as electromagnetic waves - Maxwell 2eV4eV

12. Page 12 Wave nature of light explains interference, diffraction, polarization, dipole character of emission

13. Page 13 However…... …some experiments cannot be explained by the wave nature of light, e.g.: blackbody spectrum (Planck, 1900), photoelectric effect (Einstein, 1905), Compton effect (inelastic scattering of photons in matter (electrons), 1920s) particle nature of light

14. Page 14 Photon Photon is smallest amount of light energy E one can have E=hν h – Planck’s constant 6.6x10 -34 J s (4.1x10 -15 eV s) ν – frequency of light = c/λ (with c speed of light and λ wavelength) massless boson, spin 1

15. Page 15 The photoelectric effect - Einstein 1905 http://en.wikipedia.org/wiki/Photoelectric_effect incoming light metal surface photoelectrons ejected E kin = hv–W W - work function, energy needed to eject photoelectron from metal Nobel Prize in Physics 1921: "for his services to Theoretical Physics, and especially for his discovery of the law of the photoelectric effect"

16. Page 16 Nobody knows what light really is Wave and particle concepts are mutually exclusive 2 complementary models explain light’s behaviour propagation of light - wave nature interaction of light with matter - particle nature detection of light

17. Page 17 Efficient collection is at least as important as using a sensitive detector! Excitation of sample Emission of fluorescence by the sample Collection of light by the objective Onward transmission to the detector Characteristics of photodetectors

18. Page 18 Types of detectors Single point detector (one pixel) Cameras Solid state detectors (diodes) Photoelectronic vacuum (photomultipliers) Solid state detectors (CCDs, CMOS) Photoelectronic vacuum (image intensifiers) Solid state and photoelectronic vacuum hybrid detectors also exist

19. Page 19 Single point detectors 2 main types of detectors Photoelectronic devices - photomultipliers solid state devices - photodiodes both have advantages and disadvantages

20. Page 20 Confocal Microscopy + optical sectioning + easy for time- resolved detection (FLIM) - slow detector laser sample pinhole dichroic beam- splitter scanning mirrors

21. Page 21 Photomultipliers Photoelectronic device - operates in vacuum dynodes photocathode e-e- h anode http://www.olympusconfocal.com/java/sideonpmt/index.html

22. Page 22 Feynman explains photomultipliers http://www.vega.org.uk/video/programme/45 after approx 37min Richard Feynman - 1965 Nobel Laureate in Physics, “for fundamental work in quantum electrodynamics, with deep-ploughing consequences for the physics of elementary particles.“

23. Page 23 fast response excellent for timing anode Microchannel plate (MCP) Glass capillaries latest technology - etched silicon

24. Page 24 Advantages / disadvantages of photomultipliers + large detection area + high gain + timing independent of count rate - saturation damages detector - modest quantum efficiency (<50%) - operate in vacuum

25. Page 25 Diodes Semiconductor device based on p-n junction - allows charge to flow in one direction, but not the other A p-n junction is formed by combining N-type (excess electrons) and P-type (excess holes) semiconductors together in very close contact voltage current forward current leakage or reverse current breakdown voltage - avalanche current

26. Page 26 depletion region h electron-hole pair created Avalanche Photodiodes (APDs) reverse current varies with illumination voltage current V bias

27. Page 27 Single Photon Avalanche Diodes (SPADs) voltage current operated with much higher reverse bias - above breakdown voltage. This allows each photoelectron to be multiplied by avalanche breakdown, resulting in internal gain within the photodiode. Allows photon counting. Circuit needs to be quenched. V bias

28. Page 28 Advantages / disadvantages of APDs / SPADs compared to photomultipliers + high quantum efficiency, typically 80% + no high voltage or vacuum required + Low cost + Compact and light weight + Long lifetime - small active area - noise increases with area - small gain (1, or 10 2 –10 3 for avalanche photodiodes) - slow response time, can be count rate dependent

29. Page 29 Photon timing is easy with both diodes and PMs TCSPC allows photon arrival times picoseconds after excitation laser pulse to be measured X. Michalet et al, J Mod Opt 54 (2-3), 239-281, 2007.

30. Page 30 laser / lamp Wide-field Microscopy sample camera dichroic beam- splitter + fast - out of focus blur

31. Page 31 Wide-field Microscope sample camera dichroic beam- splitter detector laser sample pinhole dichroic beam- splitter scanning mirrors Confocal Microscope

32. Page 32 Imaging detectors CCD - Charge coupled device – solid state device (silicon) Nobel Prize in Physics 2009 The prize is being awarded with one half to: CHARLES K. KAO for groundbreaking achievements concerning the transmission of light in fibers for optical communication and the other half jointly to: WILLARD S. BOYLE and GEORGE E. SMITH for the invention of an imaging semiconductor circuit - the CCD sensor. CCDs invented at Bell Labs in 1969 G.E. Smith, The invention and early history of the CCD, Nuclear Instr Meth Phys Res A 607 (2009) 1–6

33. Page 33 Imaging detectors CCDs - Charge coupled device – solid state devices (silicon) latest development: electron-multiplying CCDs - EMCCDs with gain in read-out process (impact ionisation) http://www.microscopyu.com/tutorials/java/digitalimaging/ccd/fullframe/index.html

34. Page 34 Image intensifiers – photoelectronic devices (vacuum) camera lens http://www.microscopyu.com/tutorials/java/digitalimaging/ccd/proximity/index.html night vision devices

35. Page 35 Electron Bombarded CCD (EB CCD) Hybrid detector - photocathode and CCD (vacuum & solid state) no microchannel plate Low noise amplification of electrons 100 % open area ratio - no loss of photoelectrons no lag, no distortion Real time camera using frame transfer CCD chip Ultra low light camera using Full frame cooled slow scan CCD Chip each 3.6eV creates electron/hole pair Ceramic bulb -8kV p Back thinned CCD Structure Photocathode e eeeee also single point hybrid detectors – no afterpulses, useful for FCS

36. Page 36 Detection of Light Astronomers cannot do experiments - the only way they can find out about stars and the universe is to watch Astronomers have very powerful telescopes with very sensitive cameras to observe the universe Hubble’s photon counting imaging Faint Object Camera (FOC) The most sensitive imaging method Hubble Space telescope

37. Page 37 Faint Object Camera Images of Pluto Distance from Earth 3 x 10 9 km

38. Page 38 Photon counting imaging Single frameIntegrated image + large dynamic range + zero read out noise + photon timing - photocathodes: low QE - slow, acquisition speed limited by frame rate of camera K. Suhling et al. Nucl Instrum and Methods A 437: 393-418, 1999 & Rev Sci Instrum 73: 2917-2922, 2002

39. Page 39 Microchannel plate (MCP) image intensifier Glass capillaries (latest technology - etched silicon)

40. Page 40 Loundspeaker at resonance frequency of glass

41. Page 41 Image test pattern at 30 000 frames/sec Photon counting imaging @ 30 microseconds per frame Image this test pattern

42. Page 42 Photon counting imaging – test pattern sum of frames, ≈10ms centroided

43. Page 43 Arrival time plot - cw excitation selected region of interest – photon arrival times over 20 ms N. Sergent et al PROC SPIE 6771, 67710X, 2007

44. Page 44 Photon counting means timing Use pulsed excitation source and a decaying sample

45. Page 45 Polyoxometalate (POM) nanoparticles with Europium POMs placed on glass slide excite with pulsed diode laser at 470nm @ around 10 Hz repetition rate Emission monitored >550nm take 1000 images after each excitation pulse

46. Page 46 Eu 3+ POM excitation and emission spectra 5D0  7F45D0  7F4 5D0  7F25D0  7F2 5D0  7F15D0  7F1 7F0  5D27F0  5D2 7F0  5L67F0  5L6 Charge transfer band millisecond luminescence decay time

47. Page 47 Arrival time plot - pulsed excitation 30 μs per frame

48. Page 48 Add all photons to obtain Eu 3+ POM decay

49. Page 49 Luminescence Lifetime Image of Eu 3+ POM on glass Eu 3+ POM on glass decay time ~1.5ms

50. Page 50 Luminescence Lifetime Image of ruby ruby decay around 3 ms edge of ruby edge of intensifier

51. Page 51 Fast timing with imaging detectors is difficult X. Michalet et al, J Mod Opt 54 (2-3), 239-281, 2007. Quadrant anodes or wedge and strip anode allow picosecond timing

52. Page 52 Quantum efficiency Dotted lines show how response can be extended into the UV if the device has a quartz instead of glass window. Number of photoelectrons produced per incident photon QE = pe - / hν

53. Page 53 Signal to Noise Ratio - the Key to Sensitivity S N SNR: Signal to Noise Ratio S (electron): Signal detected by the detector N (electron): Total noise SNR  IQE  T  S  S (electron): Signal I (photon/sec): Input light level QE (electron/photon):Quantum Efficiency T (sec): Integration time Signal to Noise Ratio determines the sensitivity

54. Page 54 Noise NS Shot  N Shot (electron): Signal Shot Noise S (electron): Signal Signal Shot Noise NDT Dark  N Dark (electron):Dark Noise D (electron/sec):Dark current T (sec):Integration time Camera Dark Noise Camera 2 Read 2 Dark NNN  N Camera (electron): Camera Noise N Read (electron):Read Noise N Dark (electron):Dark Noise Total Camera Noise N NN 2 Shot 2 Camera  N(electron):Total Noise N Shot (electron):Signal Shot Noise N Camera (electron):Camera Noise Total Noise

55. Page 55 http://www.microscopyu.com/tutorials/java/digitalimaging/signaltonoise/index.html

56. Page 56 Measure readout noise experimentally K.A. Lidke et al, IEEE Trans Image Proc 14(9), 1237-1245, 2005. Take series of images, subtract background, normalise each image by integrated intensity, plot variance vs mean intensity

57. Page 57 Usability Issues Cost damaged by saturation? Lifetime ease and convenience of use

58. Page 58 Fluorescence can be characterised by: position intensity wavelength lifetime polarization -> obtain all these parameters in a single measurement for maximum information content (with maximum resolution, maximum sensitivity and minimum acquisition time) Instrumentation challenge

59. Page 59 Detectors of the future I Superconducting single photon detectors Superconducting tunnel junction detectors Transition edge sensors (calorimeter) Have an intrinsic wavelength resolution Work with superconductivity, i.e. no resistance when current flows at low temperatures (liquid helium temperatures, - 270 o C)

60. Page 60 Nanowire superconducting single photon detectors for TCSPC Fast response

61. Page 61 Nanowire superconducting single photon detectors for TCSPC FIG. 2. Instrument response functions of three detectors: SSPD red open circles, conventional Si APD dashed green curve, and fast Si APD dotted blue curve. The solid red curve is a Gaussian fit to the measured SSPD response function. FIG. 3. SSPD lifetime measurements: IRF open green squares, measured decay closed blue circles and fit solid red curve for a quantum well at 935 nm.

62. Page 62 Superconducting tunnel junction detectors h superconducting cathode (Cooper Pairs, milli-electronvolt binding energy) tunnel junction amplification high quantum yield, signal proportional to photon energy, ie intrinsic resolution

63. Page 63 STJs have also been applied to measure fluorescently labelled DNA Fraser et al, Nucl Instrum Meth A 559, 782–784, 2006. Fraser et al, Rev Sci Instrum 74, 4140-4144, 2003. Slow response time, Pixellated devices, have been used on telescopes, in IR and X-ray, UV etc

64. Page 64 Detectors of the future II Single Photon Avalanche Diode (SPAD) arrays

65. Page 65 SPAD array Fig. 7. Photomicrograph of the TCSPC image sensor with a pixel detail in the inset. The integrated circuit, fabricated in a 0.35 µm CMOS technology, has a surface of 8x5mm 2. The pixel pitch is 25 µm, which leads to an active area fill factor of 6.16%. Fig. 10. Time jitter measurement of the SPAD detector and overall circuitry using the integrated TDCs. In the inset, a logarithmic plot is shown.

66. Page 66 SPAD array for Laser Range finding and detection (LIDAR) Fig. 12. Experimental 3-D image with model picture in inset. Measurement based on a target distance of 1 m. FLIM also possible, but low fill factor problematic - need microlens array

67. Page 67 Conclusion basically 2 types of detectors - photoelectronic and solid state devices (hybrid detectors exist) single point and imaging detectors which to choose depends on the requirements of the application (eg timing required?) there is no “ideal” choice yet to fit all applications future detectors (long term) will have an intrinsic wavelength resolution

68. Page 68 Resources http://www.microscopyu.com/ Instruments for fluorescence imaging, W.B. Amos, in Protein Localization by Fluorescence Microscopy, ed V.J. Allen, Oxford University Press 2000. Ultraviolet and visible detectors for future space astrophysics missions, ed J Chris Blades, Space Telescope Science Institute, 2000. Detectors for single-molecule fluorescence imaging and spectroscopy X. Michalet, O.H.W. Siegmund, J.V. Vallerga, P. Jelinsky, J.E. Millaud and S. Weiss. J Mod Opt 54(2-3), 239-281, 2007. The Role of Photon Statistics in Fluorescence Anisotropy Imaging. K.A. Lidke, B. Rieger, D.S. Lidke and T.M. Jovin. IEEE Trans Image Proc 14(9), 1237-1245, 2005.