optics, fluorescent excitation/emission & compensationdepts.washington.edu/flowlab/shoreline...
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Principles of flow cytometry: optics, fluorescent excitation/emission &
compensation
Shoreline Community College BIOL 288
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Components of a Flow Cytometer
Fluidics Optics Electronics
Sheath Sample
Light source (lasers) Optical path (filters & mirrors) Light collection (detectors)
Detector signal processing Computer interface Data storage & output
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• The optics are the heart of the cytometer.
There are three major components to the optical system of a cytometer.
Lasers – Excitation Light Source
Mirrors and Filters – Direct and focus light to the interrogation point and to the light collection point
Detectors – collect the light energy
Optics
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Optics
Laser sources
Steering Optics
BD LSR II
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Optics
Beam focusing
Flow Cell
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Optics
Flow Cell
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Fluorescence & Side scatter
Sheath fluid
Focused Laser Beam
Forward scatter
Sample Injector Tip
Fluidics Optics
Flow Cell
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Optics
Fluorescence and Side Scatter (SSC)
Forward Scatter (FSC)
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Optics
Laser sources
Steering Optics
BD LSR II
Fiber Optic Launch
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Optics
Fiber optic cables
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Flow Cytometer
488nm Blue laser
633nm Red laser FL1
FL2 FL3
FL4
Fluorescent detectors
Flow Cell
Sample Intake Probe (SIT)
Sheath fluid Cleaning and Waste
Optical System
Fluidic System
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Detectors or Photomultiplier Tubes (PMT’s) collect light energy not colors.
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Filters
The filters and mirrors direct specified wavelength of light to the detector with the final filter in front of the detector the most selective and determines what “color” is seen
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530/30 nm
530 +/- 15 {515 – 545} nm range
Filters
488nm SP
Wavelength above 488nm
Wavelength below 488nm
Bandpass Filter
Shortpass Filter
Longpass Filter
650nm LP
Wavelength below 650nm
Wavelength above 650nm
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Optics
Fiber optic cable
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Absorption
Excitation
Emission
The excitation wavelength is always a higher energy than the emission wavelength
Lower energy=longer wavelength Higher energy=shorter wavelength
?? What is the difference between Absorption and Excitation??
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Common Fluorescent Markers
Dye or Protein
Excitation Maximum
Emission Maximum
Pacific Blue 405 455
Pacific Orange 405 551
BV605 405 605
PE 480;565 578
PE-Cy7 conjugates 480;565 767
PE-Texas Red 480;565 613
FITC 495 519
PerCP-Cy5.5 490 694
EGFP 484 507
EYFP 514 527
mCherry 587 610
APC 650 660
APC-Cy7 conjugates 650;755 767
Alexa Fluor 680 679 702
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Spectral Spillover
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Compensation
uncompensated
How does the software calculate Compensation?
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Compensation
Single stained controls
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Compensation
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Compensation
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FSC Sensor
Compensation
Compensated data will have spreading error
The use of FMO’s helps for gating on dim positive events
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Panel Design Pair the dimmest marker to your brightest fluorochrome. Utilize the Staining Index (SI) to determine brightness of dyes.
But remember minimizing spillover especially when trying to detect a dim population in combination with other highly expressed ones may require alternate dye choices