page 1 detectors for fluorescence imaging klaus suhling department of physics king’s college...

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

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

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.

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)

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

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

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

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

Young’s double slit experiment


Spherical waves emanate

from slits and interfere

Thomas Young, 1801

Young’s double slit experiment


The electromagnetic spectrum

Light as electromagnetic waves - Maxwell

2eV4eV

Wave nature of light explains

interference,

diffraction,

polarization,

dipole character of emission

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

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

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"

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

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

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

Single point detectors

2 main types of detectors

• Photoelectronic devices - photomultipliers

• solid state devices - photodiodes

• both have advantages and disadvantages

Confocal Microscopy

+ optical sectioning+ easy for time-resolved detection (FLIM)- slow

detector

laser

sample

pinholedichroicbeam-splitter scanning

mirrors

Photomultipliers

Photoelectronic device - operates in vacuum

dynodes

photocathode

e-

h

anode

http://www.olympusconfocal.com/java/sideonpmt/index.html

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.“

fast responseexcellent for timing

anode

Microchannel plate (MCP)

Glass capillaries

latest technology - etched silicon

Advantages / disadvantages of photomultipliers

+ large detection area

+ high gain

+ timing independent of count rate

- saturation damages detector

- modest quantum efficiency (<50%)

- operate in vacuum

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

depletion region

h

electron-hole pair created

Avalanche Photodiodes (APDs)

reverse current varies with illumination

voltage

current

Vbias

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

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

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.

laser / lamp

Wide-field Microscopy

sample

camera

dichroicbeam-splitter

+ fast- out of focus blur

Wide-field Microscope

sample

camera

dichroicbeam-splitter

detector

laser

sample

pinholedichroicbeam-splitter scanning

mirrors

ConfocalMicroscope

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

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

Image intensifiers – photoelectronic devices (vacuum)

camera

lens

http://www.microscopyu.com/tutorials/java/digitalimaging/ccd/proximity/index.html

night vision devices

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

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

Faint Object Camera Images of Pluto

Distance from Earth 3 x 109 km

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

Microchannel plate (MCP) image intensifierMicrochannel plate (MCP) image intensifier

Glass capillaries

(latest technology - etched silicon)

Loundspeaker at resonance

frequency of glass

Image test pattern at 30 000 frames/sec

Photon counting imaging @ 30 microseconds per frame

Image this test pattern

Photon counting imaging – test pattern

sum of frames, ≈10ms centroided

Arrival time plot - cw excitation

selected region of interest – photon arrival times over 20 ms

N. Sergent et al PROC SPIE 6771, 67710X, 2007

Photon counting means timing

Use pulsed excitation source and a decaying sample

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

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

Arrival time plot - pulsed excitation

30 μs per frame

Add all photons to obtain Eu3+ POM decay

Luminescence Lifetime Image of Eu3+ POM on glass

Eu3+ POM on glass

decay time ~1.5ms

Luminescence Lifetime Image of ruby

ruby decay

around 3 ms

edge of rubyedge of intensifier

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

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

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

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

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

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

Usability Issues

• Cost

• damaged by saturation?

• Lifetime

• ease and convenience of use

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

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)

Nanowire superconducting single photon detectors for TCSPC

Fast response

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.

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

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

Detectors of the future II

• Single Photon Avalanche Diode (SPAD) arrays

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.

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

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

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.

page 1 detectors for fluorescence imaging klaus suhling department of physics king’s college...

Documents

ls slide

cork slide

elastin slide

fluorescence lifetime

encoded fluorescence

stokes fluorescence

brief history of fluorescence

wave nature of light