photodetection in flow cytometry - hamamatsu...
TRANSCRIPT
Slide # 1
Photodetection in Flow Cytometry
Slawomir PiatekNJIT/HamamatsuCopyright 2019 Hamamatsu
Slide # 2
Outline
9/24/2019 Photodetection in Flow Cytometry
1. Introduction to flow cytometry
b. Modes of light-cell interactions and signal formationa. Basic setup
c. Forward- and side-scatter configurationsc. Data acquisition and data display
2. Introduction to photodetectorsa. Structure and operationb. Opto-electronic characteristics
Slide # 3
Outline
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3. Photodetection
a. Noise and signal-to-noise ratiob. Linearity and dynamic range
4. Summary and conclusion
c. Impact on scatter plots
Slide # 4
1. Introduction to flow cytometry
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Slide # 5
Flow Cytometer consists of three components:
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Fluidics
Optics
ElectronicsPhotodetectors bridge these two components
Photodetection in Flow Cytometry
Slide # 6
Fluidics β sample injection
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Interrogation oranalysis point
~100 ΞΌm~20 ΞΌm
β’ Hydrodynamic focusing creates a narrow flow, known as the core, where the cells arrange in a one-by-one file.
β’ The diameter of the core depends on the pressure difference between the sheath fluid and the core fluid.
β’ Only one cell at the time should be passing through the interrogation point.
β’ Sampling rate ~ 1,000 β 100,000 s-1
Photodetection in Flow Cytometry
Slide # 7
Optics
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FD
SCD
FSD
Laser 1
Lase
r 2Data collection & analysis
Beam mixer
DM
DM
Flow
Legend:FSD β forward-scatter detectorSCD β side-scatter detector
FD β fluorescence detectorDM β dichroic mirror
Fluorescence-tagged cell
Photodetection in Flow Cytometry
Slide # 8
Interrogation point
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Beam profile (typically Gaussian)before cylindrical lens (not to scale)
Beam profile at the interrogationpoint (not to scale). The long axis is perpendicular to the flow.
~100 ΞΌm~20 ΞΌm
interrogation point
cylindrical lens
Photodetection in Flow Cytometry
Slide # 9
Light sources
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β’ Solid-state lasers are the most common illuminators in modern flow cytometers.
β’ They are compact, light weights, and can provide up to 150 mW of output power.
β’ Typical wavelengths [in nm]: 488, 505, 514, 532, 552, 561, 594
Photodetection in Flow Cytometry
Slide # 10
Interaction of light with the cell: light signal generation
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reflection
refraction
Diffraction/scattering
fluorescence
absorption
cell
Photodetection in Flow Cytometry
Slide # 11
Spurious light
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ππ1 ππ2
Spurious light can be due to:
Impurities
Index of refraction differences
Flow turbulence
Slide # 12
Light signal formation
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20 ΞΌm
100 ΞΌm
10-ΞΌm cell
β’ For reasonable assumption, a 10 β ππm cell will scatter about 5Γ1012 photons at the interrogation rate of 1,000 cells per second.
Photodetection in Flow Cytometry
Slide # 13
Anatomy of a light signal
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ππ
Pulse of light lens
ππPo
wer
time
Pulse duration
Peak power
Area of the pulse = total energy in the pulse
Photodetection in Flow Cytometry
Slide # 14
Anatomy of a light signal
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Approximating the pulse with a triangle, the peak power is related to the number of photon in the pulse as by:
ππππππππ =2πππππππππ
For ππ = 10 πππ π and ππ = 488 nm
ππ ππππππππ [W]
10
100
1,000
10,000
100,000
7.7 οΏ½ 10β13
7.7 οΏ½ 10β12
7.7 οΏ½ 10β11
7.7 οΏ½ 10β10
7.7 οΏ½ 10β9
Slide # 15
Forward scatter
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Iris
Lens
Beam blocker
Flow cell
Photodetectortypically a photodiode
FilterΞ»center = Ξ»laser
1. The forward scatter signal is primarily determined by the size of the interrogated cell.
2. The peak forward scatter power at Ξ» = 488 nm using 2-ΞΌm microspheres and 20-mW laser is about 4 ΞΌW.
side view
Photodetection in Flow Cytometry
Slide # 16
Side scatter
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1. Light is a mixture of scattered laser light and fluorescence
2. Light level much lower than in the forward scatter
4. Need to use photodetectors with with intrinsic gain
3. Multiple fluorescence wavelengths can be present
Photodetection in Flow Cytometry
Slide # 17
Side scatter optical setup
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APD/PMT/SiPM
Data processing
illuminating beam flow cell/interrogation point
collector and collimator
dich
roic
mirr
ors
Photodetection in Flow Cytometry
Slide # 18
The role of the photodetector
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time
light
leve
l
time
photodetector electronics
voltage
β’ A photodetector + electronics convert the input light signal (a pulse) into electrical pulse (voltage as a function of time).
to A/D converter
Photodetection in Flow Cytometry
Slide # 19
Current-to-voltage conversion
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PD R
i
Resistive termination
PD R
iβ
+
RF
Transimpedance amplifier (pre-amplifier)
+ Simple circuit and inexpensive
+ Well-behaved frequency response
+ Well-understood noise (Johnson)
β Loads the PD, can lead to non-linearity
+ Virtual ground, no loading of the PDβ Complex noise and frequency responseβ Needs biasing
Photodetection in Flow Cytometry
ππππππππ = ππππππππππππ = βπππππΉπΉ
virtual ground!
Slide # 20
Detection and data processing
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-
+
Vo
R
PD
Pre-amplifier
electronicsto measurepeak, area,or FWHM
A/D
Photodetection in Flow Cytometry
Slide # 21
Flow Cytometer: what is measured?
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time
FWHM
Area
Peak
Out
put v
olta
ge fr
om p
ream
plifi
er
β’ The front end electronics can be set up to measure the peak value of the pulse, its FWHM, and/or area under the curve. These different measurements provide specific information about the cell.
Photodetection in Flow Cytometry
Slide # 22
Scatter plots
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Forward-scatter signal
Side
-sca
tter s
igna
l
Fluorescence ch. 1Fl
uore
scen
ce c
h. 2
Photodetection in Flow Cytometry
Scatter plots are ubiquitous to flow cytometry
Slide # 23
Histograms
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No.
of c
ells
(eve
nts)
Signal, e.g. side scatter
Photodetection in Flow Cytometry
Histograms of a given measured quantity are also common
Slide # 24
2. Introduction to photodetectors
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photomultiplier tube
photodiode
avalanche photodiode
silicon photomultiplier
Photodetection in Flow Cytometry
Slide # 25
Photomultiplier tube
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R1 R2 R3 R4 R5 R6 R7
C1 C2 C3
K P
D1 D2 D3 D4 D5 D6
V ~ 1000 VTypical voltage divider
Lighteβ
Rf
VoutIPIK
Intrinsic gain of a PMT is about 105 - 107
Photodetection in Flow Cytometry
Slide # 26
Solid-state photodetectors
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Si PIN photodiode Si APD SiPM
p
i
n
πππ΅π΅π΅π΅π΅π΅π΅π΅
ππ ππππππππ
hΞ½
p
n
ππ ππππππππ
hΞ½
Gain =1 Gain up to ~100
ππ ππππππππ
hΞ½ hΞ½
Gain ~106
Photodetection in Flow Cytometry
πππ΅π΅π΅π΅π΅π΅π΅π΅ πππ΅π΅π΅π΅π΅π΅π΅π΅
Slide # 27
Desired characteristics of photodetectors: photosensitivity
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Can we detect the βredβ pulse?
PD E
β’ A photodetectorβs photosensitivity depends on its quantum efficiency (function of wavelength) and intrinsic gain.
Photodetection in Flow Cytometry
light
Slide # 28
Important points
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1. Whether a light pulse is detected (that is, it has provided usefulscientific information), depends not only on the characteristics of thephotodetector but also on the characteristics of the front-endelectronics.
2. Without the front-end electronics, the photodetector is useless.
Slide # 29
Photosensitivity
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wavelength
Qua
ntum
effi
cien
cy
wavelength
Spec
tral s
ensi
tivity
For a given wavelength, quantum efficiency, ππ, and spectral sensitivity, ππππ, are related.
ππ =1240 οΏ½ ππππππ ππππ
Slide # 30
Photosensitivity: output current
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πΌπΌππππππ = ππππππππππππ ππππ
1240ππ = ππππππππππππππ
PD
πππππππππΌπΌππππππ
ππππππππ - Peak input light power
πΌπΌππππππ - Peak output current
ππ - Quantum efficiency
light current
ππ - Wavelength (in nm)
ππ β Gain of the photodetector
ππππ β Spectral sensitivity
Photodetection in Flow Cytometry
Slide # 31
Closer look at the intrinsic gain
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PDOne photon
ππ electrons
R
Vout
PDOne photon
Oneelectron
R
Voutππ
Photodetector with gain ππ
=v/v
ππππ
ππππ
Photodetection in Flow Cytometry
Slide # 32
Dark current
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1. Photodetectorβs dark current results in the output signal offset from zero.
PD E
2. The variation around the mean is noise.
3. The magnitude of dark current depends on the photodetectorβs bias voltage and temperature.
Photodetection in Flow Cytometry
Slide # 33
Bandwidth
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Is there output signal pileup?Is a pulse structure resolved?
PD E
1. Insufficient bandwidth degrades signal fidelity. At high counting rates this may lead to signal pileup.
2. Detection bandwidth is determined by the photodetector and front-end electronics.
Photodetection in Flow Cytometry
Slide # 34
Fourier representation and detection bandwidth
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time frequency
ππ ππ
time
Arb.
frequency
ππ ππ
β
β
timefrequency
ππ ππβ
Arb.
Arb. βwhiteβ noise
Photodetection in Flow Cytometry
Slide # 35
Detection bandwidth
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PD
RIntensity-modulated light input; ππ modulation frequency
ππππ
ππ 0
ππ
12
Bandwidth
ππππ
β’ Both the photodetector and front-end electronics determine detection bandwidth.
Photodetection in Flow Cytometry
Slide # 36
How much bandwidth do I need?
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time
|f(t)|2
|fmax|2
Β½|fmax|2 Ξ΄t
|f(Ο)|2
|fmax|2
Β½|fmax|2Ξ΄Ο
0
~
~
~
Fourier transform
β’ The spectral composition depends on the pulse shape and its duration.
Ο
For Gaussian pulse:
Ξ΄t.Ξ΄Ο = 4.log(2) β 2.7726 (time-bandwidth product)
Photodetection in Flow Cytometry
Slide # 37
Bandwidth
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β’ Too much bandwidth adds noise (indicated by thicker line)
PD E
Photodetection in Flow Cytometry
Slide # 38
Bandwidth
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100 kHz bandwidth 10 MHz bandwidth
Slide # 39
3. Photodetection
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Slide # 40
Detection signal-to-noise ratio
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R
light
PD
To signal processing
ππππ
Photodetection in Flow Cytometry
Slide # 41
Sources of noise: gain variation
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οΏ½ππππ ππππ
οΏ½ππππ ππππππ
Gain section of a photodetector
F=οΏ½ππππ ππππ
οΏ½ππππ ππππππ
Excess noise factor
Photodetection in Flow Cytometry
Slide # 42
Sources of noise: Dark current shot noise
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PD
Gain ππ
πΌπΌπ·π·
π‘π‘
ππ2 = 2πππππππππΌπΌπ·π·Dark current shot noise variance
πΌπΌπ·π·
Photodetection in Flow Cytometry
Slide # 43
Sources of noise: Photon shot noise
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PD
Gain ππ
πΌπΌ = ππππππππ
π‘π‘
ππ2 = 2πππππππππΌπΌ = 2ππππππππ2ππππππ
Photon shot noise variance
πΌπΌ
Photodetection in Flow Cytometry
Slide # 44
Sources of noise: Johnson thermal noise
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ππ
πΌπΌ
ππ2 =4ππππππππ
Johnson noise variance
Photodetection in Flow Cytometry
Slide # 45
Detection signal-to-noise ratio (resistive termination)
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SΞ» β Detectorβs sensitivity
ππ β Detectorβs intrinsic gain
ID β Detectorβs dark current
F β Detectorβs excess noise factor
B β Detection bandwidth
PB β Background light optical power
e β elementary charge; k β Boltzmann constant; T β temperature
ππππ
=ππ οΏ½ ππππ οΏ½ ππ
2ππππ ππ + πππ΅π΅ ππππ + πΌπΌπ·π· ππππ2 + 4ππππππππ
P β Instantaneous optical power
Photodetection in Flow Cytometry
Slide # 46
Detection signal-to-noise ratio
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ππππ
=ππ οΏ½ ππππ οΏ½ ππ
2ππππ ππ + πππ΅π΅ ππππ + πΌπΌπ·π· ππππ2 + 4ππππππππ
ππππ
=ππ οΏ½ ππππ
2ππππ ππ + πππ΅π΅ ππππ + πΌπΌπ·π· ππ
If the photodetector has a large intrinsic gain ππ, then the above equation for SN
reduces to:
Photodetection in Flow Cytometry
(βlargeβ intrinsic gain)
The effect of the intrinsic gain is to eliminate noise contribution from the front-end electronics.
Slide # 47
Closer look at the impact of photodetector gain
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ππππ
=ππ οΏ½ ππππ
2ππππ ππ + πππ΅π΅ ππππ + πΌπΌπ·π· ππ + 4ππππππππππ2
1. The purpose of the photodetectorβs gain is to make the term 4πππππ΅π΅π π ππ2
negligible compared to the other noise terms.
2. For a given gain, the term 4πππππ΅π΅π π ππ2
can also be made smaller by increasing ππ.
3. (!!!) However, increasing ππ may reduce detection bandwidth, reduce photodetectorβs linearity, and cause electronics ringing (due to impedance mismatch).
Photodetection in Flow Cytometry
Slide # 48
Closer look at the impact of photodetector gain
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The gain comes with excess noise factor, ππ.
PMT
APD
SiPM
105 β 107
105 β 106
1β100
ππππ
~1.2
~3 β 4
~1.1
ππ βπΏπΏ
πΏπΏ β 1
ππ β ππ0.3
ππ β 1 + ππππππ
Photodetection in Flow Cytometry
ππππ
=ππ οΏ½ ππππ
2ππππ ππ + πππ΅π΅ ππππ + πΌπΌπ·π· ππ + 4ππππππππππ2
Slide # 49
S/N comparison between photodetectors
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1. In what follows, we compare the performance of four photodetectors given the same light input and front-end electronics.
2. Although not revealed, the photodetectors are those used in flow cytometry.
Slide # 50
Signal-to-Noise ratio: H10770A-40 PMT
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PMT (H10770A-40)
R = 10 kΞ©
Ξ» = 520 nm
Specifications
Photocathode spectral sensitivity SK at 520 nm = 175 mA/W
Gain ΞΌ = 5Γ105
Anode dark current ID = 3.3Γ10-9 A = 3.3 nA
Excess noise figure F β 1.2
Maximum average anode current = 2 ππA
Temperature T = 20 β = 293 K
Photodetection in Flow Cytometry
Slide # 51
Signal-to-Noise ratio: S8664-30K APD
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APD (S8664-30K)
R = 10 kΞ©
Ξ» = 520 nm
Specifications
Spectral sensitivity Ο at 520 nm = 0.34 A/W
Gain ΞΌ = 50
Dark current ID = 400 pA
Excess noise figure F β 500.2 = 2.2
Temperature T = 20 β = 293 K
Photodetection in Flow Cytometry
Slide # 52
Signal-to-Noise ratio: S3072 PIN PD
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PD (S3072)
R = 10 kΞ©
Ξ» = 520 nm
Specifications
Spectral sensitivity Ο at 4520 nm = 0.35 A/W
Dark current ID = 0.3 nA (VR = 10 V)
Temperature T = 20 β = 293 K
Photodetection in Flow Cytometry
Slide # 53
Signal-to-Noise ratio: S13360-3025CS SiPM
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SiPM (S13360-3025CS)
R = 10 kΞ©
Ξ» = 520 nm
Specifications
Photodetection efficiency at 520 nm Ξ· = 24%
Dark current ID = 44.8 nA
Temperature T = 20 β = 293 K
Gain ΞΌ = 7Γ105
Excess noise figure F β 1.01
Number of microcells = 14,400
Photodetection in Flow Cytometry
Slide # 54
Signal-to-noise ratio comparison
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ππ = 10 ππΞ©
PMT
SiPM
APD
PD
ππ = 520 ππππ
log10 ππ (Incident Power) [W]
log 1
0ππ ππ
ππ
Slide # 55
Signal-to-noise ratio comparison
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ππ = 10 ππΞ©ππ = 1 ππππ
PMT
SiPM
APD
PD
This PMT has almost no sensitivity above 750 nm. However, there are other PMTs that have IR sensitivity.
Wavelength [nm]
log 1
0ππ ππ
ππ
Slide # 56
Importance of picking the right photocathode (PMT)
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For a family of PMT photocathodes, the spectral response ranges from UV to IR.
Slide # 57
Signal-to-noise ratio comparison
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β’ As radiant incident power increases, π΅π΅ππ
for each photodetector increases and the intrinsic gain plays a less significant role.
ππ = 10 ππΞ©
ππ = 1 ππππ
PMT
SiPM
APD
PD
Wavelength [nm]
log 1
0ππ ππ
ππ
Slide # 58
Signal shot noise limited case
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ππππ
=ππ οΏ½ ππππ οΏ½ ππ
2ππππ ππ + πππ΅π΅ ππππ + πΌπΌπ·π· ππππ2 + 4ππππππππ
If ππ is large so that the photon shot noise is dominant, the equation for π΅π΅ππ
becomes:
ππππ
=ππππππ2ππππππ (signal shot-noise limited case)
Slide # 59
Comments on the S/N comparison
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1. The above side-by-side comparison is not optimal. The performance of the detection system is not only a function of the photodetector but also of the front-end electronics: the two interact in a unique way. An optimal comparison would use font-end electronics for each photodetector that maximizes π΅π΅
ππ.
2. At a low incident power level, the PMT yields a higher π΅π΅ππ
than does the APD because of the PMTβs higher gain and lower dark current.
Slide # 60
Comments on the S/N comparison
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3. At a higher incident power level, the performance of the APD, as measured by π΅π΅ππ
approaches that of the PMT.
4. The performance of the APD exceeds that of the PMT for wavelengths greater than about 750 nm. The PMT used in the above example has practically no sensitivity for wavelengths greater than about 750 nm.
Slide # 61
Desired characteristics of photodetectors: linearity and dynamic range
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Is the output proportional to the input?
PD E
β’ Linearity and dynamic range depend on the properties of the photodetector and detection electronics.
saturation
Photodetection in Flow Cytometry
Slide # 62
Linearity and dynamic range
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Input [Arb.]
Out
put [
Arb
.]
Range of measurable light levels (with dark
correction)
Range of outputs
Saturation
Ideal response
Range of measured outputs with S/N > 1
up to saturation
Slide # 63
Closer look at dynamic range for a PMT
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Output current [mA]
Dev
iatio
n [%
]
H10720 10% nonlinearity: 75 mA, TP = 0.5 ππs
Gain = 2Γ106, QE = 28%
No. of incident photons at 450 nm per pulse:
4.2Γ105
β’ Dynamic range and linearity of a PMT can be controlled by the voltage divider.
Slide # 64
Closer look at dynamic range for a SiPM
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πππΎπΎ οΏ½ ππππππ = 1.1 Γ 105 (ideal response for 4.2 Γ 105 photons )
οΏ½ππππππππππππ = 0.75 Γ 105 or 32% below an ideal response
ππππ β Pulse duration
π‘π‘ππ β SiPM recovery time
οΏ½ππππππππππππ βAverage number of activated microcells
ππππππππ β Total number of microcells
πππΎπΎ β Number of photons in a pulse PDE β Photon detection efficiency
β’ Dynamic range and linearity of an SiPM is limited by the number of microcells.
Slide # 65
Dynamic range and linearity of a PD and APD
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R
light
Photodiode or APD
ππππ
1. The lower bound of dynamic range is limited by the photodetectorβs dark current shot noise and noise from the front-end electronics (resistor or TIA).
2. The upper bound of the dynamic range depends on the voltage bias (especially for photodiode), value of the load resistor, or dynamic range of the TIA.
3. The upper bound of the dynamic range of a photodiode and APD is generally larger than that for a PMT and SiPM.
Slide # 669/24/2019 Photodetection in Flow Cytometry
Forward-scatter signal
Forward-scatter signal Forward-scatter signal
Forward-scatter signal
Forward-scatter signal
Forward-scatter signal
Side
-sca
tter s
igna
l
Side
-sca
tter s
igna
l
Side
-sca
tter s
igna
l
Side
-sca
tter s
igna
l
Side
-sca
tter s
igna
l
Side
-sca
tter s
igna
l
Ideal Sample & Optics Variation Photon shot noise
QE conversion noise Gain variation noise Random electronic noise
Slide # 67
4. Summary and conclusions
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1. Flow cytometry is a versatile technique to study cells and microparticles.
2. Photodetector is an indispensable component of every flow cytometer.
3. The choice of the photodetector should be based on the best π΅π΅ππ
performance of the detection system.
Slide # 68
4. Summary and conclusions
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4. Limitations of the detection system will affect scatter plots and histograms masking or distorting science.
5. Idiosyncrasies of photodetectors prevent a simple swap of one detector for the other without altering the rest of the detection system.
Slide # 69Slide # 69
Thank You Fluorescence Microscopy Images
9/24/2019
Contact InformationSlawomir [email protected]
Photodetection in Flow Cytometry