Slide 1: Photodetection in Flow Cytometry
Photodetection in Flow Cytometry Slawomir Piatek New Jersey Institute of Technology and Hamamatsu Photonics, Bridgewater, NJ, USA 06.4. 2020 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 1
Slide 2: Index
Index ■ Introduction to Flow Cytometry ■ Photodetection ■ How are Scattered Plots Affected? 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 2
Slide 3: Motivation
Motivation SSC-A 106 FSC-A 106 How does photodetection affect the science depicted in the scatter plots? 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 3
Slide 4: Introduction to Flow Cytometry
Introduction to Flow Cytometry 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 4
Slide 5: Flow Cytometer Consists of Three Components
Flow Cytometer Consists of Three Components: Fluidics Optics Photodetectors bridge these two components Electronics 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 5
Slide 6: Fluidics
Fluidics 1. Hydrodynamic focusing creates a narrow flow, known as the core, where the cells arrange in a one-by-one file 2. The diameter of the core depends on the pressure difference between the sheath fluid and the core fluid. 3. Only one cell at the time should be passing through the Interrogatio
Slide 7: Optics
Optics DM Fluorescence-tagged cell DM FD Data collection SCD & analysis Beam mixer Laser 1 FSD Laser 2 Legend: FSD – forward-scatter detector SCD – side-scatter detector FD – fluorescence detector DM – dichroic mirror Flow 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 7
Slide 8: Interrogation Point
Interrogation Point Beam profile (typically Gaussian) cylindrical lens before cylindrical lens (not to scale) interrogation point Beam profile at the interrogation point (not to scale). The long ~20 μm axis is perpendicular to the flow. ~100 μm 🄫 Hamamatsu Photonics K.K. and its affiliates. All…
Slide 9: Light Sources
Light Sources 1. Solid-state lasers are the most common illuminators in modern flow cytometers 2. These lasers are compact, light weight, and can provide up to 150 mW of output power 3. Typical wavelengths [in nm]: 488, 505, 514, 532, 552, 561, 594 🄫 Hamamatsu Photonics K.K. and its affiliates. All
Slide 10: Interaction of Light with the Cell: Signal Generation
Interaction of Light with the Cell: Signal Generation reflection diffraction/scattering cell absorption refraction fluorescence 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 10
Slide 11: Spurious Light
Spurious Light Spurious light can be due to: Impurities 𝑛1 𝑛2 Index of refraction differences Flow turbulence 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 11
Slide 12: Signal Formation
Signal Formation 100 μm 20 μm 10-μm cell 1. Suppose we have 20-mW laser with 𝜆 = 488 nm 2. About 10% of the power (or 2 mW) will illuminate a 10-μm cell as it passes through the interrogation point. The amount will be proportionally larger/smaller for a cell that is larger/smaller 3. The implied…
Slide 13: Signal Formation
Signal Formation 100 μm 20 μm 10-μm cell 4. Thus the 10-μm cell is illuminated with 4.9×1015 photons per s. 5. If the interrogation rate is 1,000 cells per second, a cell will scatter a total of about 4.9×1012 photons Reference: “Practical Flow Cytometry” by Shapiro 🄫 Hamamatsu Photonics K.K. and…
Slide 14: Anatomy of a Pulse
Anatomy of a Pulse Power Peak power lens Pulse of light time 𝜏 𝜏 Area of the pulse = total energy in the pulse Pulse duration 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 14
Slide 15: Forward-Scatter Detection
Forward–Scatter Detection Iris Filter Flow cell Lens λcenter = λlaser Beam blocker Photodetector typically a photodiode 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…
Slide 16: Side-Scatter Optical Setup
Side-Scatter Optical Setup Data processing 1. Light is a mixture of scattered laser light and fluorescence dichroic mirrors 2. Light level is much lower than in the forward scatter APD/PMT/SiPM 3. Multiple fluorescence wavelengths can be present collector and collimator 4. Need to use photodetectors
Slide 17: The Role of the Photodetector
The Role of the Photodetector light level voltage to A/D converter time time photodetector electronics A photodetector + electronics convert the input light signal (a pulse) into electrical pulse (voltage as a function of time) 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 17
Slide 18: Current to Voltage Conversion
Current to Voltage Conversion virtual ground! 𝑅𝐹 i 𝑉𝑜𝑢𝑡 = 𝑖𝑅 i − 𝑉𝑜𝑢𝑡 = −𝑖𝑅𝐹 PD + 𝑅 PD Resistive termination Transimpedance amplifier (pre-amplifier) + Simple circuit and inexpensive + Virtual ground, no loading of the PD + Well-behaved frequency response − Complex noise and…
Slide 19: What is Measured?
What is Measured? Output voltage from preamplifier Peak FWHM time Area 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. 🄫 Hamamatsu Photonics K.K. and its…
Slide 20: Detection and Data Processing Chain
Detection and Data Processing Chain PD 𝑅 - 𝑣𝑜 electronics to measure peak, area, or FWHM A/D + Pre-amplifier 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 20
Slide 21: Scatter Plots
Scatter Plots Fluorescence ch. 2 Side-scatter signal Forward-scatter signal Fluorescence ch. 1 Scatter plots are ubiquitous to flow cytometry 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 21
Slide 22: Histograms
Histograms No. of cells (events) Signal, e.g. side scatter Histograms of a given measured quantity are also common 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 22
Slide 23: Photodetection
Photodetection 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 23
Slide 24: Point Photodetectors used in Flow Cytometry
Point Photodetectors used in Flow Cytometry PMT PD APD SiPM PMT – photomultiplier tube APD – avalanche photodiode PD – photodiode SiPM – silicon photomultiplier 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 24
Slide 25: Photomultiplier Tube
Photomultiplier Tube 𝐾 − cathode 𝐷1 , … 𝐷6 − Dynodes 𝑃 − anode Light 𝐾 𝑃 𝐼 𝑒− 𝑃 𝑣𝑜 𝐷1 𝐷2 𝐷3 𝐷4 𝐷5 𝐷6 𝑅𝑙 Gain: 104 − 108 𝐼𝐾 𝐶1 𝐶2 𝐶3 𝑅1 𝑅2 𝑅3 𝑅4 𝑅5 𝑅6 𝑅7 𝑉~1000 V There are two essential phenomena involved in the operation of a PMT: extrinsic photoelectric effect and…
Slide 26: Solid-State Photodetectors
Solid-State Photodetectors Si PIN photodiode Si APD SiPM 𝑉𝐵𝐼𝐴𝑆 𝑉𝐵𝐼𝐴𝑆 𝑉𝐵𝐼𝐴𝑆 hν hν hν hν p p i n n 𝑅 𝑅 𝑅 𝑣𝑜 𝑣𝑜 𝑣𝑜 Gain =1 Gain up to ~100 Gain ~106 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 26
Slide 27: Detection Characteristics: Photosensitivity
Detection Characteristics: Photosensitivity PD E light Can we detect the “red” pulse? A photodetector’s effective photosensitivity depends on its quantum efficiency (function of wavelength) and intrinsic gain. 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 27
Slide 28: Detection Characteristics: Gain Variations
Detection Characteristics: Gain Variations PD E 1. Random gain variation of a photodetector increases noise, which translates into a larger scatter of the measured quantity 2. All photodetectors with intrinsic gain exhibit gain variation. The contribution to noise is expressed with excess noise fact
Slide 29: Gain and Excess Noise
Gain and Excess Noise 𝜇 𝐹 5 7 𝛿 PMT 10 − 10 ~1.2 𝐹≈ 𝛿−1 APD 1−100 ~3 − 4 𝐹 ≈ 𝜇 0.3 SiPM 105 − 106 ~1.1 𝐹 ≈ 1 + 𝑃𝑐𝑡 Legend 𝜇 − Intrinsic gain of a photodetector 𝐹 − Excess noise factor 𝛿 − Gain of the first dynode in a PMT (typically ~4) 𝑃𝑐𝑡 − Probability of crosstalk in a SiPM…
Slide 30: Detection Characteristics: Dark Current
Detection Characteristics: Dark Current PD E 1. Photodetector’s dark current results in the output signal offset from zero 2. The variation around the mean is noise 3. The magnitude of dark current depends on the photodetector’s bias voltage and temperature 🄫 Hamamatsu Photonics K.K. and its…
Slide 31: Detection Characteristics: Bandwidth
Detection Characteristics: Bandwidth PD E Is there an output signal pileup? 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 🄫 Hamamatsu Photonics K.K. and…
Slide 32: Bandwidth and Noise
Bandwidth and Noise PD E Too much bandwidth adds noise (indicated by the thicker line) but does not improve fidelity 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 32
Slide 33: Bandwidth and Noise
Bandwidth and Noise 100 kHz bandwidth 10 MHz bandwidth 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 33
Slide 34: Detection Characteristics: Linearity and Dynamic Range
Detection Characteristics: Linearity and Dynamic Range saturation PD E Is the output proportional to the input? Linearity and dynamic range depend on the properties of the photodetector and detection electronics 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 34
Slide 35: Detection Characteristics: Stability
Detection Characteristics: Stability PD E Does the output vary for a constant input? There are numerous factors that can affect stability (at different time scales): gain variation, temperature drift, 1/f noise,… 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 35
Slide 36: Detection Characteristics: Temperature Drift
Detection Characteristics: Temperature Drift increasing temperature PD E 1. If uncompensated, temperature drift will cause gain change of the photodetector. This effect is very strong in APDs and SiPMs 2. Temperature drift also affects noise characteristics 🄫 Hamamatsu Photonics K.K. and its…
Slide 37: Signal-to-noise ratio
Signal-to-noise ratio 𝑆 𝑆 𝑁 𝑁 mean (Signal) time 1. 𝑆Τ must be greater than 1 for detection to contain useful information 𝑁 2. 𝑆Τ depends on many factors such as incident light power, photodetector’s sensitivity, detection 𝑁 bandwidth, type of frontend electronics, and more… 🄫 Hamamatsu…
Slide 38: Signal-to-Noise Ratio
Signal-to-Noise Ratio 𝑅 = 10 𝑘Ω 𝜆 = 520 𝑛𝑚 PMT SiPM APD PD 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 38
Slide 39: How are Scattered Plots Affected
How are Scattered Plots Affected 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 39
Slide 40: How are Scatter Plots Affected?
How are Scatter Plots Affected? Reference (Ideal) Power variation due to flow Side-scatter signal Forward-scatter signal Side-scatter signal Forward-scatter signal 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 40
Slide 41: How are Scatter Plots Affected?
How are Scatter Plots Affected? Side-scatter signal Reference (Ideal) Higher photosensitivity Side-scatter signal Forward-scatter signal Forward-scatter signal 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 41
Slide 42: How are Scatter Plots Affected?
How are Scatter Plots Affected? Reference (Ideal) Higher random noise Side-scatter signal Side-scatter signal Forward-scatter signal Forward-scatter signal 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 42
Slide 43: How are Scatter Plots Affected?
How are Scatter Plots Affected? Reference (with random noise) Gain drift (Detector with gain) Side-scatter signal Side-scatter signal Forward-scatter signal Forward-scatter signal (Detector without gain) 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 43
Slide 44: How are Scatter Plots Affected?
How are Scatter Plots Affected? Reference (with random noise) Limited dynamic range Side-scatter signal Side-scatter signal Forward-scatter signal Forward-scatter signal 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 44
Slide 45: How are Scatter Plots Affected?
How are Scatter Plots Affected? 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 45
Slide 46: Hamamatsu in flow cytometry
Hamamatsu in flow cytometry Manufactures all types of photodetectors used in flow cytometry Designs and manufactures customized detection circuits and ASICs Manufactures a variety of optical components for flow cytometry Conducts R&D to quickly respond to the changing needs in flow cytometry
Slide 47: Hamamatsu in flow cytometry
Hamamatsu in flow cytometry Manufactures all types of photodetectors (APD, SiPM, SPAD, Cameras and PMT) used in flow cytometry Custom integrated optical assemblies from front-end electronics to complete ASICs Manufactures a variety of optical components for flow cytometry Work with customers
Slide 48: Closing Remarks
Closing Remarks 1. Flow cytometry is a versatile technique to study cells and microparticles 2. Photodetector is an indispensable component of every flow cytometer 𝑆 3. The choice of the photodetector should be based on the best performance of the detection 𝑁 system 4. Limitations of the…
Slide 49: Thank you for listening
Thank you Thank you for listening Contact information: piatek@njit.edu 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 49
Slide 50: Join Us for Weeks of FREE Photonics Webinars (17 Topics)
Join Us for 10 Weeks of FREE Photonics Webinars (17 Topics) Week # Weekly Topics # of Talks Talk #1 Date Talk #2 Date 1 Introduction to Photodetectors 2 26-May-20 28-May-20 2 Emerging Applications - LiDAR & Flow Cytometry 2 2-Jun-20 4-Jun-20 3 Understanding Spectrometer 2 9-Jun-20 11-Jun-20 1 Weeks
Slide 51: 🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved.
🄫 Hamamatsu Photonics K.K. and its affiliates. All Rights Reserved. 51