流式细胞仪基本原理,技术要点及应用

Excitation/Emission Spectra
Common Fluorophores
• Blue（488nm）
• Violet（405nm）
– FITC – Alexa Fluor 488 – PE – PerCP – PerCP-Cy5.5 – PE-Cy5 – PE-Cy7 – PI
Forward Scatter (FSC) & Side Scatter(SSC)
Side Scatter
Side Scatter
Light Scatter Parameters
Side Scatter (SSC) Internal Structure or reflectivity of particle
4. Light less than 735 nm is reflected by the mirror, to the next detector.
2. Light > 735 nm passes through the dichroic mirror.
3. The bandpass filter allows only PE-Cy7 signal to enter the detector.
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Emission Spectra and Detector
Rules:
Correct spectra spillover
Correct spectra spillover
Compensation= PE- x% of FITC
Same Mean Value
As each cell passes through the laser beam, this leaves a pulse of voltage over time. The total pulse height and area is measured by the flow cytometer. This is known as an event. Emitted fluorescence intensity is proportional to the number of antibody binding sites.
Correct spectra spillover
Compensation= FITC- x% of PE
Same Mean Value
What Is Flow Cytometry?
• Flow ~ motion • Cyto ~ cell • Metry ~ measure
Measuring properties of individual cells while in a fluid stream (flowing) Including size, complexity, phenotypes (surface and intracellualr proteins) & viability
Forward Scatter (FSC ) particle size
FSC & SSC of Human PBMC
Neutrophils Monocytes
Lymphocytes
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Fluorescence Parameters
Fluorochromes on the cells are excited By the laser and their emissions are detected using optical filters to direct light to PMTs or PDs
Longpass dichroic mirror
Bandpass filter
PE-Cy7
incoming signals
PE-Cy5
empty
side scatter
PE
FITC
empty
PPEE-Cyy77
Electronics
Electronics
Electronics
How do you interpret the data?
BCI GalliosTM… 3 lasers
Miltenyi Biotec MACSQuantTM… 3 lasers
BD FACSCanto IITM… 3 lasers
544nm , Green ;
Optics and Detectors
Optics
Detectors
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Outline
• Learn the basic principles of flow. • Understand how a flow cytometer works. • Learn how data is interpreted. • Flow Cytometry Applications.
How do Fluorophores Work?
Fluorophores/Fluorochromes /Fluors/Dyes
Fluors can be excited by a particular wavelength of light and enter an excited state. Eventually, they come back down from their high, emitting a longer wavelength. This emitted wavelength is what we detect in flow.
PE-Cy5
650 LP
PE APC PE-Cy5
560 SP
505 SP
610 LP
signal objective
BD LSR II, LSR Fortessa, FACS Canto and FACS Aria detectors.
1. Mixed fluorescent signals are transmitted down the optical path.
PMT
<560 nm
560 nm short pass (SP) dichroic
530/30 nm bandpass fluorescein filter
>560 nm
PMT
585/42 nm bandpass PE filter
laser
488 nm
BD Biosciences FACS Calibur detectors
Red (633 nm) laser
3 colors
APC
APC-
APC-Cy7
Cy5.5
Violet (405 nm) laser
2 colors
BV421
BV510 BV570 BV605 BV650
BV711
BV780
400
500
600
700
800
900
Emission wavelength (nm)
• R–ed7（-A6A3D3nm）
– APC – APC-Cy7 – AF647 – AF700
– AmCyan – BrilliantViolet 421 – PacificBlue – BrilliantViolet 510 – BrilliantViolet 570 – BrilliantViolet 605 – BrilliantViolet 650 – BrilliantViolet 711 – BrilliantViolet 785 – Qdot605
Normally, we keep the sheath pressure FIXED, and adjust the sample pressure .
SHEATH PRESSURE
SAMPLE PRESSURE
LO
SHEATH PRESSURE
SAMPLE PRESSURE
HI
Cells too close!
• UV (360nm）
– DAPI
Sixteen color analysis is possible with only three laser wavelengths
6 colors
FITC
Blue-green
(488 nm)
laser
PE PE-TxRed
PE-Cy5 PE-Cy5.5
PE-Cy7
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技术部: 杨亚军
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Why Use Flow Cytometry?
• The use of flow in research has boomed since the mid-1980s
• Contributed to >140,000 published articles (Pubmed) in 2014
Stream too wide!
Lasers
Lasers
Lasers
Commercial flow cytometers are now routinely equipped with two or three solid state laser sources ( blue/red/violet ).
Histograms & Bivariate Dot Plots
Typical displays are composed of tens of thousands of event If only a single parameter is to be plotted, a histogram is used. Plotting two parameters yields a bivariate dotplot.
Controls
• Compensation controls - to correct spectra spillover
• Gating controls (e.g., FMO) -to define positive population
• Biological controls -Experience controls ( untreated vs treated) -Unstained controls -Isotype controls (for non-specific binding )
Long pass (LP)
460 500 540
Filters
Short pass (SP)
460 500 540
Band pass (BP)
460 500 540
LP 500
SP 500
BP500/50
Fluorescence detection
To detect both fluorescein and PE, we would separate the signals with a 560 SP dichroic, And filter the separated signals with 530/30 nm and 585/42 nm bandpass filters.
DPSS 488 nm 20 mW Red diode 642 nm 25 mW Violet diode 405 nm 50 mW
FITC / GFP PE PE-Texas Red PE-Cy5 PE-Cy5.5 PE-Cy7 APC APC-Cy5.5 APC-Cy7
Pacific Blue Pacific Orange Qdots
Four fluorescence detectors, three off the 488 nm, one off the 640 nm laser.
FITC SSC
side scatter
488/10 nm
PE
585/42 nm
APC
610 LP
FITC
530/30 nm
505 SP
560 SP
What is Flow Cytometer?
Lasers
Fluidics
Optics
Electronics & computer
Detectors
l
Fluidics System
Fluidics System
Flow Chamber
Sample Sheath Sheath
laser
Sheath versus sample pressure