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Transcript of Page 1 Detectors for Fluorescence Imaging Klaus Suhling Department of Physics King’s College...
Page 1
Detectors for Fluorescence ImagingDetectors for Fluorescence Imaging
Klaus SuhlingKlaus Suhling
DeDepartment of Physicspartment of Physics King’s College King’s College LondonLondon
StrandStrandLondon WC2R 2LSLondon WC2R 2LS
Page 2
OutlineOutline
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
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 imagingA 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.
Page 4
Optical Microscopy
MicrographiaMicrographia, published in 1665 , published in 1665 by by
Robert Hooke (1635-1703)Robert Hooke (1635-1703)
Hooke also coined the word cell (compartments in cork)
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
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
Page 7
What is Fluorescence?What is Fluorescence?
kr
excitedstate S1
groundstate S0
kisc
T1kic
radiative deactivation of the first
electronically excited singlet state
kph / kic
molecular energy levels
Page 8
What is light?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
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
Page 10
Young’s double slit experiment
Instructor's Resource CD-ROM, Physics, James S. Walker, Pearson Education 2004
Page 11
The electromagnetic spectrum
Light as electromagnetic waves - Maxwell
2eV4eV
Page 12
Wave nature of light explains
interference,
diffraction,
polarization,
dipole character of emission
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
Page 14
PhotonPhoton is smallest amount of light energy E one
can have
E=hνh – 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
Page 15
The photoelectric effect - Einstein 1905
http://en.wikipedia.org/wiki/Photoelectric_effect
incoming light
metal surface
photoelectrons ejected
Ekin= 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"
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
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
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
Page 19
Single point detectors
2 main types of detectors
• Photoelectronic devices - photomultipliers
• solid state devices - photodiodes
• both have advantages and disadvantages
Page 20
Confocal Microscopy
+ optical sectioning+ easy for time-resolved detection (FLIM)- slow
detector
laser
sample
pinholedichroicbeam-splitter scanning
mirrors
Page 21
Photomultipliers
Photoelectronic device - operates in vacuum
dynodes
photocathode
e-
h
anode
http://www.olympusconfocal.com/java/sideonpmt/index.html
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.“
Page 23
fast responseexcellent for timing
anode
Microchannel plate (MCP)
Glass capillaries
latest technology - etched silicon
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
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
currentleakage
or reverse
current
breakdown
voltage -
avalanche
current
Page 26
depletion region
h
electron-hole pair created
Avalanche Photodiodes (APDs)
reverse current varies with illumination
voltage
current
Vbias
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.
Vbias
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 102–103 for avalanche photodiodes)
- slow response time, can be count rate dependent
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.
Page 30
laser / lamp
Wide-field Microscopy
sample
camera
dichroicbeam-splitter
+ fast- out of focus blur
Page 31
Wide-field Microscope
sample
camera
dichroicbeam-splitter
detector
laser
sample
pinholedichroicbeam-splitter scanning
mirrors
ConfocalMicroscope
Page 32
Imaging detectors• CCD - Charge coupled device – solid state device (silicon)
Nobel Prize in Physics 2009The 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
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
Page 34
Image intensifiers – photoelectronic devices (vacuum)
camera
lens
http://www.microscopyu.com/tutorials/java/digitalimaging/ccd/proximity/index.html
night vision devices
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
Page 36
Detection of LightAstronomers 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
methodHubble Space telescope
Page 37
Faint Object Camera Images of Pluto
Distance from Earth 3 x 109 km
Page 38
Photon counting imagingSingle frame Integrated 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
Page 39
Microchannel plate (MCP) image intensifierMicrochannel plate (MCP) image intensifier
Glass capillaries
(latest technology - etched silicon)
Page 40
Loundspeaker at resonance
frequency of glass
Page 41
Image test pattern at 30 000 frames/sec
Photon counting imaging @ 30 microseconds per frame
Image this test pattern
Page 42
Photon counting imaging – test pattern
sum of frames, ≈10ms centroided
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
Page 44
Photon counting means timing
Use pulsed excitation source and a decaying sample
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
Page 46
Eu3+ POM excitation and emission spectra
400 500 600 7000
20000
40000
60000
180000
190000
200000in
ten
sity
/co
un
ts
wavelength /nm
5D0 7F4
5D0 7F2
5D0 7F1
7F0 5D2
7F0 5L6
Charge transfer band
millisecond luminescence decay time
Page 47
Arrival time plot - pulsed excitation
30 μs per frame
Page 48
Add all photons to obtain Eu3+ POM decay
Page 49
Luminescence Lifetime Image of Eu3+ POM on glass
Eu3+ POM on glass
decay time ~1.5ms
Page 50
Luminescence Lifetime Image of ruby
ruby decay
around 3 ms
edge of rubyedge of intensifier
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
Page 52
Quantum efficiency
Dotted lines showhow response can beextended into the UVif the device has a quartz instead ofglass window.
Number of photoelectrons produced per incident photon
QE = pe- / hν
Page 53
Signal to Noise Ratio - the Key to Sensitivity
S
NSNR: Signal to Noise RatioS (electron): Signal detected by the detectorN (electron): Total noise
SNR
I QE TS S (electron): SignalI (photon/sec): Input light levelQE (electron/photon):Quantum Efficiency
T (sec): Integration time
Signal to Noise Ratio determines the sensitivity
Page 54
Noise
N SShot NShot (electron): Signal Shot NoiseS (electron): Signal
Signal Shot Noise
N D TDark NDark (electron): Dark NoiseD (electron/sec): Dark currentT (sec): Integration time
Camera Dark Noise
Camera2
Read2
DarkN N N NCamera (electron): Camera NoiseNRead (electron): Read NoiseNDark (electron): Dark Noise
Total Camera Noise
N N N2Shot
2Camera
N(electron): Total NoiseNShot (electron): Signal Shot NoiseNCamera (electron): Camera Noise
Total Noise
Page 55
•http://www.microscopyu.com/tutorials/java/digitalimaging/signaltonoise/index.html
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
Page 57
Usability Issues
• Cost
• damaged by saturation?
• Lifetime
• ease and convenience of use
Page 58
Fluorescence can be characterised by:
• position• intensity
• wavelength• lifetime
• polarization
-> obtain all these parameters in a single measurementfor maximum information content (with maximum resolution, maximum sensitivity and minimum acquisition time)
Instrumentation challengeInstrumentation challenge
Page 59
Detectors of the future ISuperconducting single photon detectors
Superconducting tunnel junction detectorsTransition edge sensors (calorimeter)Have an intrinsic wavelength resolution
Work with superconductivity, i.e. no resistance when current flows at low temperatures (liquid helium temperatures, -270oC)
Page 60
Nanowire superconducting single photon detectors for TCSPC
Fast response
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.
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, ieintrinsic resolution
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
Page 64
Detectors of the future II
• Single Photon Avalanche Diode (SPAD) arrays
Page 65
SPAD array
Fig. 7. Photomicrograph of the TCSPC image sensor with a pixel detail in theinset. The integrated circuit, fabricated in a 0.35 µm CMOS technology, has a surface of 8x5mm2. 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.
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
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
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.