Flow Cytometer 原理（PDF）

Flow Cytometry
Principles and Concepts
Beckman Coulter Taiwan Branch
流式細胞儀 產品專員
楊人泰
Tim Yang
Watching the Cell: Microscopy
• True Profile of the Cell
• Labor-intensive
• Arbitrary results
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Can we design an instrument to see cells…….
• more fast, >100 events per second
• with the capability to acquire large amount of cell
numbers to improve the accuracy of statistics.
• more objectively between you and me
• more sensitively than eyes
• still with multi-parameters of cell information
That’s Why we use Flow Cytometry, FCM,
or FACS (Fluorescence Activated Cell Sorter)
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讓細胞在管路中排路隊
Hydrodynamic Focusing（流體動力聚焦）
Scatter Signals
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What kinds of light source used in Flow
• Laser (more power but expensive)
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488 nm Blue (Could cover >90% applications)
633 nm Red
405/407/408 nm Violet
532 nm Green
325 nm UV or ML UV
• Lamp (cheaper UV but optical device needed)
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High angle scatter :
Reflection & refraction.
Cell structure.
Low angle scatter :
Diffraction. Cell size.
Laser
Direct beam stop.
Light
Fluorescence signal for
specific lableing
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What Flow Cytometry Give You
• Light Scatter for cell morphology
– Forward Angle Light Scatter (FS) for cell size
– Side Scatter (SS) for cell complexity
• Fluorescenses (FL) for specific labeling
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FL 1 : (525 nm BP) for green dyes
FL 2 : (575 nm BP) for yellow dyes
FL 3 : (615 nm BP) for orange dyes
FL 4 : (675 nm BP) for red dyes
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Side Light Scatter：How Granular is that Cell?
Larger Side Scatter
LASER
Smaller Side Scatter
LASER
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Flow Cytometry Is Made Of
• Fluid System
• Optical System
• Electronic System
• Computer System
• Sorting System
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Fluid System: Hydrodynimic Focusing（流體動力聚焦）
Sample
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Waste
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Fluid System: Hydrodynimic Focusing（流體動力聚焦）
Sample Pressure
Low
Sample Pressure
High
Sample
Sheath
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Fluid System: Hydrodynimic Focusing（流體動力聚焦）
Sample
Flow Cell
Sheath
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Optical System & Light Detector
FL3
FL2
FL1
SS
FL4
FS
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• Electronic System: PMT & High Voltage （放大訊號）
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• Electronic System: Analog to Digital
Cell
FS
SS FL1 FL2
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543
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• Electronic System: Leaner Scale Convert to Log Scale
Lin
Log
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Compensation（進行多重染色時需設定螢光補償）
Beckman-Coulter
System:
FITC/PE/ECD/PC5
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Compensation（進行多重染色時需設定螢光補償）
FL1 PMT：92＝90+2
FL2 PMT：100＝85+15
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Compensation（螢光補償）
單染FITC：調整 FL2-%FL1
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Compensation（螢光補償）
單染PE：調整 FL1-%FL2
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Compensation（螢光補償）
FITC / PE 雙染
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Flow Raw Data: List Mode Data
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• Computer System: Data Display
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• Computer System: Data Display
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• Computer System: Gate & Analysis
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• Computer System: 多色實驗、多層次Gating
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What’s Sorting ?
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機械式 Sorter vs. 電子式 Sorter
• Sorting Rate: 100~300 cells/sec
• Sorting Rate: 15000~30000 cells/sec
• Purity: < 80%
• Purity: < 99%
• Cell viability: < 60%
• Cell viability: < 97%
• Time consuming
• Time efficient
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Important terms
Instrument Components:
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Fluid System
Optical System
Electronic System
Computer System
Sorting System
Concepts:
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Hydrodynimics Focusing
Filter Set
PMT & High Voltage
Lin Data & Log Data
Color Compensation
List Mode Data
Gating and Analysis
Sorting (電子式、機械式)
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Some Limitation of Classical Flow
• Analyzer vs. Sorter
• UV Light Source
• Scatter in a Problem
• No Absolute Counting
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Sorter
Bench Top Analyzer
‧ 操作繁複，需專人負責
‧ 操作簡易，無須調教
‧ 液流壓力高，管徑較窄，可提供
高速分選工作所需
‧ 液流壓力低，管徑較寬，可提供
穩定之流速與分析環境
‧ 配置空間大
‧ 配置空間較小
‧ 適合進行大量檢體分析
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‧ 建議專職進行分選工作
‧ 可更換多種內徑Flow Cell
‧ 可加裝多重光源
傳統UV光源：採用大瓦數氣體雷射，可提供 200mW 以上UV光源
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水銀弧燈：需配置光學鏡頭及濾片才可使用
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以散射光量測細胞大小，會因細胞方向產生較大誤差
Light Scatter for size measurement
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散射光量測的細胞大小與實際細胞大小不成線性
Linearity
Forward Scatter
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傳統Flow 以氣體壓力進樣，無法控制進樣體積
新式Flow 以Syringe 進樣，準確控制進樣體積
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Data Analysis: Plot & Statistics
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Categories of data plot
• Single parameter histogram
• Dual parameter histogram
– Dot plot
– Contour plot
– Density plot
– Isometric plot (+ Count)
• Three parameter histogram(Tomogram)
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Single parameter histogram
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Single parameter histogram
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Single overlay
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Dual parameter histogram (1) --Dot plot
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Dual parameter histogram (2) --Contour plot
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Dual parameter histogram(3)--Density plot
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Dual parameter histogram(4)--Isometric ( Surface Plot )
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Three parameter histogram (Tomogram)
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Statistics in Common use
1. Percentage: % Total & %Gate
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2. Mean (MFI): X-mean and Y-mean
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Applications of Flow Cytometry: Overview
• DNA analysis: cell cycle analysis,
tumor monitoring,
detection of ploidy,
• Understanding Apoptosis:
• Cell- surface marker immunofluoresecnce analysis:
Stem cells tracking and enumeration;
Analysis of platelets;
Leukocyte Immunophenotyping;
HIV infection
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Applications of Flow Cytometry: Overview
• Cell function assays:
• calcium kinetic studies：Ca++ Con., Ca++ kinetic
• mitochondria membrane potential：DioC6, JC1
• cellular protein content measurements：GFP, YFP….
• cell proliferation：CFSE
• analysis of intracellular proteins：intracellular cytokine
• cellular RNA and DNA content：reticulocyte, SCSA
• intracellular pH, proton pump activity
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Applications of Flow Cytometry: Overview
• Detection of soluble protein： beads based multiplex assay
• Enzyme kinetics
• Detection of intracellular virus and viral products
• Environmental microrganism detection, aquatic-organism
studies
• Chromosomes analysis and sorting
• Sex specific sperm sorting
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Studies in Pollen / Sporangia
Differentiation of Phytophthora infestans Sporangia
from other ariborne biological particles
Blue FL
Pollen differentiation
Red FL
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Ploidy Analysis
Ploidy analysis of maize kernel endosperm in different days after pollination (DAP)
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Genome size estimation
Estimation of absolute genome size in Ensete gilletii
DNA amount of E. gilletii was
estimated as 1.21 pg
The peak mean=0.484.
( 2C=2.5 pg DNA ) saved as internal
reference. The peak mean=200
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Events
DNA Content Analysis: PI vs. DAPI
PI
DAPI
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Flow analysis of Phytoplankton
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Cell Function : Mitochondria Membrane Potential (Dym)
DioC6: Dym Stainer
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Sorting of GFP+ Yeast
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Flow analysis of bacteria
Mixed culture (1:1) of
M. luteus (gram-positive cocci)
and E. coli (gram-negative rods)
Isometric flow-cytometric plots. (A) E. coli; (B) M. luteus; (C) mixed culture (1:1) of E. coli and M. luteus.
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