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Basics of Flow Cytometry. Holden Maecker. Outline. Definitions, what can be measured by flow cytometry Fluidics: Sheath and sample streams, flow cells, sorting Optics: Lasers, filters Electronics: PMTs, signal processing Fluorochromes: spectra, spillover

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Presentation Transcript
outline
Outline
  • Definitions, what can be measured by flow cytometry
  • Fluidics: Sheath and sample streams, flow cells, sorting
  • Optics: Lasers, filters
  • Electronics: PMTs, signal processing
  • Fluorochromes: spectra, spillover
  • Data analysis: FSC files, gating, statistics
definitions
Definitions
  • Flow cytometry: study of cells as they move in fluid suspension, allowing multiple measurements to be made per cell.
  • FACS™: fluorescence-activated cell sorting
what measurements can be made
What measurements can be made?
  • Forward light scatter (FSC): proportional to cell size
  • Side light scatter (SSC): proportional to cell granularity
  • Fluorescence:
    • Binding of fluorescent-labeled antibodies
    • Ca++-sensitive dyes within cells
    • Fluorescent proteins expressed by cells
    • Binding of DNA dyes
scatter profile of lysed whole blood

largest and most granular population

1000

Granulocytes

800

600

400

Monocytes

200

smallest and least granular population

Lymphocytes

0

0

200

400

600

800

1000

Scatter profile of lysed whole blood

Side Scatter

Forward Light Scatter

fluorescence data display
Fluorescence data display

Negative control histogram

PE

Number of Events

FITC Fluorescent Intensity 

FITC

major components of a flow cytometer
Major components of a flow cytometer
  • Sample intake port
  • Sheath and waste reservoirs
  • Flow cell
  • Laser(s)
  • Optical filters
  • PMTs (photomultiplier tubes) or photodiodes
  • Signal processor
cytometer fluidics create laminar flow
Cytometer fluidics create laminar flow

Sample stream

Flow Cell

Sheath stream

Laser beam

Cell

typical 2 color cytometer configuration
Typical 2-color cytometer configuration

FL1

PMT

488/10 nm

band pass filter

530/30 nm band

pass filter

SSC

PMT

1% ND front

surface mirror

FL2

PMT

560nm short pass

dichroic mirror

585/42 nm

band pass filter

488nm band pass filter

488nm laser beam

FSC PD

flow cell

background and autofluorescence
Background and autofluorescence
  • All cells have a certain level of background fluorescence, due to:
    • Autofluroescence: from pigments and fluorescent moieties on cellular proteins
    • Non-specifically bound antibodies, and free antibody in the sample stream
  • The level of autofluorescence varies with the wavelength of excitation and collection:
    • Highest in FITC, PE detectors; lowest in far red (APC, Cy7) detectors
fluorescence sensitivity
Fluorescence sensitivity
  • Detection Efficiency (Q): number of photoelectrons generated per molecule of fluorophore
    • Dependent upon fluorophore, filters, PMT sensitivity, voltage gain setting, etc.
  • Background (B): non-specific signal intrinsic to the system
    • Dependent upon autofluorescence, unbound fluorophore, stray light, etc.
fluorescence spillover
Fluorescence spillover

Emission of FITC in PE channel

compensating for spillover

1650 - 185

  • 3540 - 125
Compensating for spillover

compensated

uncompensated

FITC mean fluorescence PE mean fluorescence

---------------------------- ----------------------------

negative positive negative positive

----------- ---------- ----------- ----------

uncompensated 125 3540 185 1650

compensated 125 3560 135135

% Spillover =

X 100

fcs files
FCS files
  • FCS 2.0 and FCS 3.0 conventions
  • Often referred to as list-mode files
  • Contain all of the measurements (FSC-H, FSC-A, SSC-H, SSC-A, FL1-H…) for each individual cell processed in a given sample
web reference tools
Web reference tools
  • BD Spectrum Viewer:

http://www.bdbiosciences.com/spectra

  • Maecker lab weblog:

http://maeckerlab.typepad.com

(protocols, manuscripts, literature updates)