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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
9/24/2019 Photodetection in Flow Cytometry
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
9/24/2019 Photodetection in Flow Cytometry
Slide # 5
Flow Cytometer consists of three components:
9/24/2019
Fluidics
Optics
ElectronicsPhotodetectors bridge these two components
Photodetection in Flow Cytometry
Slide # 6
Fluidics – sample injection
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Interrogation oranalysis point
~100 μm~20 μm
• Hydrodynamic focusing creates a narrow flow, known as the core, where the cells arrange in a one-by-one file.
• The diameter of the core depends on the pressure difference between the sheath fluid and the core fluid.
• Only one cell at the time should be passing through the interrogation point.
• Sampling rate ~ 1,000 – 100,000 s-1
Photodetection in Flow Cytometry
Slide # 7
Optics
9/24/2019
FD
SCD
FSD
Laser 1
Lase
r 2Data collection & analysis
Beam mixer
DM
DM
Flow
Legend:FSD – forward-scatter detectorSCD – side-scatter detector
FD – fluorescence detectorDM – 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 μm~20 μm
interrogation point
cylindrical lens
Photodetection in Flow Cytometry
Slide # 9
Light sources
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• Solid-state lasers are the most common illuminators in modern flow cytometers.
• They are compact, light weights, and can provide up to 150 mW of output power.
• 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
9/24/2019 Photodetection in Flow Cytometry
𝑛𝑛1 𝑛𝑛2
Spurious light can be due to:
Impurities
Index of refraction differences
Flow turbulence
Slide # 12
Light signal formation
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20 μm
100 μm
10-μm cell
• For reasonable assumption, a 10 − 𝜇𝜇m cell will scatter about 5×1012 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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𝜏𝜏
Pulse of light lens
𝜏𝜏Po
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
9/24/2019 Photodetection in Flow Cytometry
Approximating the pulse with a triangle, the peak power is related to the number of photon in the pulse as by:
𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚 =2𝑁𝑁𝑁𝑁𝑁𝜏𝜏𝜏𝜏
For 𝜏𝜏 = 10 𝜇𝜇𝑠𝑠 and 𝜏𝜏 = 488 nm
𝑁𝑁 𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚 [W]
10
100
1,000
10,000
100,000
7.7 � 10−13
7.7 � 10−12
7.7 � 10−11
7.7 � 10−10
7.7 � 10−9
Slide # 15
Forward scatter
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Iris
Lens
Beam blocker
Flow cell
Photodetectortypically a photodiode
Filterλcenter = λlaser
1. The forward scatter signal is primarily determined by the size of the interrogated cell.
2. The peak forward scatter power at λ = 488 nm using 2-μm microspheres and 20-mW laser is about 4 μW.
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
• 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
i−
+
RF
Transimpedance amplifier (pre-amplifier)
+ Simple circuit and inexpensive
+ Well-behaved frequency response
+ Well-understood noise (Johnson)
− Loads the PD, can lead to non-linearity
+ Virtual ground, no loading of the PD− Complex noise and frequency response− Needs biasing
Photodetection in Flow Cytometry
𝑉𝑉𝑜𝑜𝑜𝑜𝑜𝑜 = 𝑖𝑖𝑖𝑖𝑉𝑉𝑜𝑜𝑜𝑜𝑜𝑜 = −𝑖𝑖𝑖𝑖𝐹𝐹
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
• 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
Lighte−
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
𝑉𝑉𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵
𝑖𝑖 𝑉𝑉𝑜𝑜𝑜𝑜𝑜𝑜
hν
p
n
𝑖𝑖 𝑉𝑉𝑜𝑜𝑜𝑜𝑜𝑜
hν
Gain =1 Gain up to ~100
𝑖𝑖 𝑉𝑉𝑜𝑜𝑜𝑜𝑜𝑜
hν hν
Gain ~106
Photodetection in Flow Cytometry
𝑉𝑉𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑉𝑉𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵
Slide # 27
Desired characteristics of photodetectors: photosensitivity
9/24/2019
Can we detect the “red” pulse?
PD E
• A photodetector’s photosensitivity depends on its quantum efficiency (function of wavelength) and intrinsic gain.
Photodetection in Flow Cytometry
light
Slide # 28
Important points
9/24/2019 Photodetection in Flow Cytometry
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
9/24/2019 Photodetection in Flow Cytometry
wavelength
Qua
ntum
effi
cien
cy
wavelength
Spec
tral s
ensi
tivity
For a given wavelength, quantum efficiency, 𝜂𝜂, and spectral sensitivity, 𝑆𝑆𝜆𝜆, are related.
𝜂𝜂 =1240 � 𝑆𝑆𝜆𝜆𝜏𝜏 𝑛𝑛𝑛𝑛
Slide # 30
Photosensitivity: output current
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𝐼𝐼𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚𝜂𝜂𝜏𝜏 𝑛𝑛𝑛𝑛
1240𝜇𝜇 = 𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚𝑆𝑆𝜆𝜆𝜇𝜇
PD
𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚𝐼𝐼𝑚𝑚𝑚𝑚𝑚𝑚
𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚 - Peak input light power
𝐼𝐼𝑚𝑚𝑚𝑚𝑚𝑚 - Peak output current
𝜂𝜂 - Quantum efficiency
light current
𝜏𝜏 - Wavelength (in nm)
𝜇𝜇 – Gain of the photodetector
𝑆𝑆𝜆𝜆 – Spectral sensitivity
Photodetection in Flow Cytometry
Slide # 31
Closer look at the intrinsic gain
9/24/2019
PDOne photon
𝜇𝜇 electrons
R
Vout
PDOne photon
Oneelectron
R
Vout𝜇𝜇
Photodetector with gain 𝜇𝜇
=v/v
𝑆𝑆𝑁𝑁
𝑆𝑆𝑁𝑁
Photodetection in Flow Cytometry
Slide # 32
Dark current
9/24/2019
1. Photodetector’s 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 photodetector’s 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
𝑃𝑃 𝜔𝜔
time
Arb.
frequency
𝑃𝑃 𝜔𝜔
↔
↔
timefrequency
𝑃𝑃 𝜔𝜔↔
Arb.
Arb. ”white” noise
Photodetection in Flow Cytometry
Slide # 35
Detection bandwidth
9/24/2019
PD
RIntensity-modulated light input; 𝜔𝜔 modulation frequency
𝑉𝑉𝜔𝜔
𝑉𝑉 0
𝜔𝜔
12
Bandwidth
𝑉𝑉𝜔𝜔
• Both the photodetector and front-end electronics determine detection bandwidth.
Photodetection in Flow Cytometry
Slide # 36
How much bandwidth do I need?
9/24/2019
time
|f(t)|2
|fmax|2
½|fmax|2 δt
|f(ω)|2
|fmax|2
½|fmax|2δω
0
~
~
~
Fourier transform
• The spectral composition depends on the pulse shape and its duration.
ω
For Gaussian pulse:
δt.δω = 4.log(2) ≈ 2.7726 (time-bandwidth product)
Photodetection in Flow Cytometry
Slide # 37
Bandwidth
9/24/2019
• Too much bandwidth adds noise (indicated by thicker line)
PD E
Photodetection in Flow Cytometry
Slide # 38
Bandwidth
9/24/2019 Photodetection in Flow Cytometry
100 kHz bandwidth 10 MHz bandwidth
Slide # 39
3. Photodetection
9/24/2019 Photodetection in Flow Cytometry
Slide # 40
Detection signal-to-noise ratio
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R
light
PD
To signal processing
𝑆𝑆𝑁𝑁
Photodetection in Flow Cytometry
Slide # 41
Sources of noise: gain variation
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�𝑆𝑆𝑁𝑁 𝑖𝑖𝑖𝑖
�𝑆𝑆𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜
Gain section of a photodetector
F=�𝑆𝑆𝑁𝑁 𝑖𝑖𝑖𝑖
�𝑆𝑆𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜
Excess noise factor
Photodetection in Flow Cytometry
Slide # 42
Sources of noise: Dark current shot noise
9/24/2019
PD
Gain 𝜇𝜇
𝐼𝐼𝐷𝐷
𝑡𝑡
𝑖𝑖2 = 2𝑒𝑒𝑒𝑒𝑒𝑒𝜇𝜇𝐼𝐼𝐷𝐷Dark current shot noise variance
𝐼𝐼𝐷𝐷
Photodetection in Flow Cytometry
Slide # 43
Sources of noise: Photon shot noise
9/24/2019
PD
Gain 𝜇𝜇
𝐼𝐼 = 𝜇𝜇𝑃𝑃𝑆𝑆𝜆𝜆
𝑡𝑡
𝑖𝑖2 = 2𝑒𝑒𝑒𝑒𝑒𝑒𝜇𝜇𝐼𝐼 = 2𝑒𝑒𝑒𝑒𝑒𝑒𝜇𝜇2𝑃𝑃𝑆𝑆𝜆𝜆
Photon shot noise variance
𝐼𝐼
Photodetection in Flow Cytometry
Slide # 44
Sources of noise: Johnson thermal noise
9/24/2019
𝑖𝑖
𝐼𝐼
𝑖𝑖2 =4𝑘𝑘𝑘𝑘𝑒𝑒𝑖𝑖
Johnson noise variance
Photodetection in Flow Cytometry
Slide # 45
Detection signal-to-noise ratio (resistive termination)
9/24/2019
Sλ – Detector’s sensitivity
𝜇𝜇 – Detector’s intrinsic gain
ID – Detector’s dark current
F – Detector’s excess noise factor
B – Detection bandwidth
PB – Background light optical power
e – elementary charge; k – Boltzmann constant; T – temperature
𝑆𝑆𝑁𝑁
=𝑃𝑃 � 𝑆𝑆𝜆𝜆 � 𝜇𝜇
2𝑒𝑒𝑒𝑒 𝑃𝑃 + 𝑃𝑃𝐵𝐵 𝑆𝑆𝜆𝜆 + 𝐼𝐼𝐷𝐷 𝑒𝑒𝜇𝜇2 + 4𝑘𝑘𝑘𝑘𝑒𝑒𝑖𝑖
P – Instantaneous optical power
Photodetection in Flow Cytometry
Slide # 46
Detection signal-to-noise ratio
9/24/2019
𝑆𝑆𝑁𝑁
=𝑃𝑃 � 𝑆𝑆𝜆𝜆 � 𝜇𝜇
2𝑒𝑒𝑒𝑒 𝑃𝑃 + 𝑃𝑃𝐵𝐵 𝑆𝑆𝜆𝜆 + 𝐼𝐼𝐷𝐷 𝑒𝑒𝜇𝜇2 + 4𝑘𝑘𝑘𝑘𝑒𝑒𝑖𝑖
𝑆𝑆𝑁𝑁
=𝑃𝑃 � 𝑆𝑆𝜆𝜆
2𝑒𝑒𝑒𝑒 𝑃𝑃 + 𝑃𝑃𝐵𝐵 𝑆𝑆𝜆𝜆 + 𝐼𝐼𝐷𝐷 𝑒𝑒
If the photodetector has a large intrinsic gain 𝜇𝜇, then the above equation for SN
reduces to:
Photodetection in Flow Cytometry
(“large” 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
9/24/2019
𝑆𝑆𝑁𝑁
=𝑃𝑃 � 𝑆𝑆𝜆𝜆
2𝑒𝑒𝑒𝑒 𝑃𝑃 + 𝑃𝑃𝐵𝐵 𝑆𝑆𝜆𝜆 + 𝐼𝐼𝐷𝐷 𝑒𝑒 + 4𝑘𝑘𝑘𝑘𝑒𝑒𝑖𝑖𝜇𝜇2
1. The purpose of the photodetector’s gain is to make the term 4𝑘𝑘𝑘𝑘𝐵𝐵𝑅𝑅𝜇𝜇2
negligible compared to the other noise terms.
2. For a given gain, the term 4𝑘𝑘𝑘𝑘𝐵𝐵𝑅𝑅𝜇𝜇2
can also be made smaller by increasing 𝑖𝑖.
3. (!!!) However, increasing 𝑖𝑖 may reduce detection bandwidth, reduce photodetector’s linearity, and cause electronics ringing (due to impedance mismatch).
Photodetection in Flow Cytometry
Slide # 48
Closer look at the impact of photodetector gain
9/24/2019
The gain comes with excess noise factor, 𝑒𝑒.
PMT
APD
SiPM
105 − 107
105 − 106
1−100
𝑒𝑒𝜇𝜇
~1.2
~3 − 4
~1.1
𝑒𝑒 ≈𝛿𝛿
𝛿𝛿 − 1
𝑒𝑒 ≈ 𝜇𝜇0.3
𝑒𝑒 ≈ 1 + 𝑃𝑃𝑐𝑐𝑜𝑜
Photodetection in Flow Cytometry
𝑆𝑆𝑁𝑁
=𝑃𝑃 � 𝑆𝑆𝜆𝜆
2𝑒𝑒𝑒𝑒 𝑃𝑃 + 𝑃𝑃𝐵𝐵 𝑆𝑆𝜆𝜆 + 𝐼𝐼𝐷𝐷 𝑒𝑒 + 4𝑘𝑘𝑘𝑘𝑒𝑒𝑖𝑖𝜇𝜇2
Slide # 49
S/N comparison between photodetectors
9/24/2019 Photodetection in Flow Cytometry
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
9/24/2019
PMT (H10770A-40)
R = 10 kΩ
λ = 520 nm
Specifications
Photocathode spectral sensitivity SK at 520 nm = 175 mA/W
Gain μ = 5×105
Anode dark current ID = 3.3×10-9 A = 3.3 nA
Excess noise figure F ≈ 1.2
Maximum average anode current = 2 𝜇𝜇A
Temperature T = 20 ℃ = 293 K
Photodetection in Flow Cytometry
Slide # 51
Signal-to-Noise ratio: S8664-30K APD
9/24/2019
APD (S8664-30K)
R = 10 kΩ
λ = 520 nm
Specifications
Spectral sensitivity σ at 520 nm = 0.34 A/W
Gain μ = 50
Dark current ID = 400 pA
Excess noise figure F ≈ 500.2 = 2.2
Temperature T = 20 ℃ = 293 K
Photodetection in Flow Cytometry
Slide # 52
Signal-to-Noise ratio: S3072 PIN PD
9/24/2019
PD (S3072)
R = 10 kΩ
λ = 520 nm
Specifications
Spectral sensitivity σ at 4520 nm = 0.35 A/W
Dark current ID = 0.3 nA (VR = 10 V)
Temperature T = 20 ℃ = 293 K
Photodetection in Flow Cytometry
Slide # 53
Signal-to-Noise ratio: S13360-3025CS SiPM
9/24/2019
SiPM (S13360-3025CS)
R = 10 kΩ
λ = 520 nm
Specifications
Photodetection efficiency at 520 nm η = 24%
Dark current ID = 44.8 nA
Temperature T = 20 ℃ = 293 K
Gain μ = 7×105
Excess noise figure F ≈ 1.01
Number of microcells = 14,400
Photodetection in Flow Cytometry
Slide # 54
Signal-to-noise ratio comparison
9/24/2019 Photodetection in Flow Cytometry
𝑖𝑖 = 10 𝑘𝑘Ω
PMT
SiPM
APD
PD
𝜏𝜏 = 520 𝑛𝑛𝑛𝑛
log10 𝑃𝑃 (Incident Power) [W]
log 1
0𝑆𝑆 𝑁𝑁
𝑒𝑒
Slide # 55
Signal-to-noise ratio comparison
9/24/2019 Photodetection in Flow Cytometry
𝑖𝑖 = 10 𝑘𝑘Ω𝑃𝑃 = 1 𝑝𝑝𝑝𝑝
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
0𝑆𝑆 𝑁𝑁
𝑒𝑒
Slide # 56
Importance of picking the right photocathode (PMT)
9/24/2019 Photodetection in Flow Cytometry
For a family of PMT photocathodes, the spectral response ranges from UV to IR.
Slide # 57
Signal-to-noise ratio comparison
9/24/2019 Photodetection in Flow Cytometry
• As radiant incident power increases, 𝐵𝐵𝑁𝑁
for each photodetector increases and the intrinsic gain plays a less significant role.
𝑖𝑖 = 10 𝑘𝑘Ω
𝑃𝑃 = 1 𝑛𝑛𝑝𝑝
PMT
SiPM
APD
PD
Wavelength [nm]
log 1
0𝑆𝑆 𝑁𝑁
𝑒𝑒
Slide # 58
Signal shot noise limited case
9/24/2019 Photodetection in Flow Cytometry
𝑆𝑆𝑁𝑁
=𝑃𝑃 � 𝑆𝑆𝜆𝜆 � 𝜇𝜇
2𝑒𝑒𝑒𝑒 𝑃𝑃 + 𝑃𝑃𝐵𝐵 𝑆𝑆𝜆𝜆 + 𝐼𝐼𝐷𝐷 𝑒𝑒𝜇𝜇2 + 4𝑘𝑘𝑘𝑘𝑒𝑒𝑖𝑖
If 𝑃𝑃 is large so that the photon shot noise is dominant, the equation for 𝐵𝐵𝑁𝑁
becomes:
𝑆𝑆𝑁𝑁
=𝑃𝑃𝑆𝑆𝜆𝜆2𝑒𝑒𝑒𝑒𝑒𝑒 (signal shot-noise limited case)
Slide # 59
Comments on the S/N comparison
9/24/2019 Photodetection in Flow Cytometry
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 𝐵𝐵
𝑁𝑁.
2. At a low incident power level, the PMT yields a higher 𝐵𝐵𝑁𝑁
than does the APD because of the PMT’s higher gain and lower dark current.
Slide # 60
Comments on the S/N comparison
9/24/2019 Photodetection in Flow Cytometry
3. At a higher incident power level, the performance of the APD, as measured by 𝐵𝐵𝑁𝑁
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
9/24/2019
Is the output proportional to the input?
PD E
• 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
9/24/2019 Photodetection in Flow Cytometry
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
9/24/2019 Photodetection in Flow Cytometry
Output current [mA]
Dev
iatio
n [%
]
H10720 10% nonlinearity: 75 mA, TP = 0.5 𝜇𝜇s
Gain = 2×106, QE = 28%
No. of incident photons at 450 nm per pulse:
4.2×105
• Dynamic range and linearity of a PMT can be controlled by the voltage divider.
Slide # 64
Closer look at dynamic range for a SiPM
9/24/2019 Photodetection in Flow Cytometry
𝑁𝑁𝛾𝛾 � 𝑃𝑃𝑃𝑃𝑃𝑃 = 1.1 × 105 (ideal response for 4.2 × 105 photons )
�𝑁𝑁𝑓𝑓𝑖𝑖𝑓𝑓𝑓𝑓𝑓𝑓 = 0.75 × 105 or 32% below an ideal response
𝑘𝑘𝑃𝑃 − Pulse duration
𝑡𝑡𝑓𝑓 − SiPM recovery time
�𝑁𝑁𝑓𝑓𝑖𝑖𝑓𝑓𝑓𝑓𝑓𝑓 −Average number of activated microcells
𝑁𝑁𝑜𝑜𝑜𝑜𝑜𝑜 − Total number of microcells
𝑁𝑁𝛾𝛾 − Number of photons in a pulse PDE − Photon detection efficiency
• 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
9/24/2019 Photodetection in Flow Cytometry
R
light
Photodiode or APD
𝑆𝑆𝑁𝑁
1. The lower bound of dynamic range is limited by the photodetector’s 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
9/24/2019 Photodetection in Flow Cytometry
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 𝐵𝐵𝑁𝑁
performance of the detection system.
Slide # 68
4. Summary and conclusions
9/24/2019 Photodetection in Flow Cytometry
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 Piatekpiatek@physics.rutgers.edu
Photodetection in Flow Cytometry
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