Inside the black box

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Inside the black box
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fluidics
electronics
light
optics
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Flow Cytometry Problems with early systems.
Detector
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Hydrodynamic focusing
V. Kachel, H. Fellner-Feldegg E. Menke -
MLM
Chapt. 3
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Fluidics Particle Orientation and Deformation
V. Kachel, et al. -
MLM
Chapt. 3
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The Flow Cell
The introduction of a large volume into a small
volume in such a way that it becomes focused
along an axis is called Hydrodynamic Focusing.
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Sample differential pressure
10 psi
10 psi
10 psi
10.2 psi
10.4 psi
10.8 psi

• If differential is too large, cells will no
  longer line up single file
• Results in wider CVs and increase in multiple
  cells passing through the laser at once.
  No more single cell analysis!

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What can we measure

• Scattered light
• Fluorescence
• Thats it

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Forward Angle Light Scatter
Laser
FALS Sensor
Intensity is said to be proportional to cell size
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90 Degree Light Scatter
Laser
FALS Sensor
Intensity is said to be proportional to
Granularity or internal complexity of the cell
90LS Sensor
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Lymphozyt
Basophiler
Monozyt
Neutrophiler
Eosinophiler
FACSAcademy
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Why Look at FSC v. SSC

• Since FSC size and SSC internal structure, a
  correlated measurement between them can allow for
  differentiation of cell types in a heterogenous
  cell population

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How to visualize a specific antibody
fluorescein
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Fluorescence
Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescnece Intensity
Wavelength
low energy
high energy
wavelength (nm)
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Spectra of Common Fluorochromes
Laser Lines (nm)
PE-Texas Red
Texas Red
PI
Ethidium
PE
FITC
cis-Paranaric Acid
300
400
500
600
700
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Fluorescence Detectors
Laser
FALS Sensor
Fluorescence detector (PMT3, PMT4 etc.)
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The photoreceptors of a flow cytometer
longpass filter
bandpass filter
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Flow Cytometry Optics dissection of fluorescence
PMT
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Dichroic Longpass filters
PMT
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Flow cell
PMT
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Bandpass
Filters
PMT
1
Laser
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Signal Detection - PMTsphoto multiplier tube
Secondary emission
Cathode
Anode
Amplified Signal Out
Photons in
End Window
Dynodes
Voltage
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The color blindness of a flow cytometer
Laser Lines (nm)
PE-Texas Red
Texas Red
PI
Ethidium
PE
FITC
cis-Paranaric Acid
300
400
500
600
700
Original from Purdue University Cytometry
Laboratories, Modified by R. Duggan
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Measurements of the Pulse
Voltage Intensity
Time
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A mixed cell population
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One parameter display (Histogram)
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Two parameter display (Dot Plot)
FSC SSC FL1 FL2 FL3 FL4 75 42 320 10 10 9
FSC SSC FL1 FL2 FL3 FL4 20 41 7 480 8 8
FSC SSC FL1 FL2 FL3 FL4 20 41 320 480 8 8
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Two parameter display (Dot Plot)
CD45 PE
CD14 FITC
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Two parameters displayS
dot plot
3D plot
contour plot
density plot
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Data Analysis Concepts
Regions A certain characteristic (blond, little,
man, woman) Gates Logical combination of
characteristics (blond women, little men)
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Characterization of subpopulations
Gate
Auswertung
CD45 PE
CD14 FITC
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11-color, 13-parameter flow cytometry
Identification of human naive T cells by
phenotype, function, and T-cell receptor
diversity S C De Rosa, L A Herzenberg, L A
Herzenberg M Roederer Nature Medicine 7, 245 -
248 (2001).
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http//www.cyto.purdue.edu/ Jean Paul Robinson