side scatter ssc (refracted/reflected light)
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Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light.
The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity.
These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.
Side ScatterSSC
(refracted/reflected light)
Forward ScatterFSC
(diffracted light)
Light ScatteringThe light is forced to deviate from a straight trajectory by an object
Refraction + reflection + diffraction + … = Scattering
Fluorescence-Activated Cell Sorter (FACS)
Fluorescence-Activated Cell Sorter (FACS)
Torbjorn Caspersson at the Karolinska Institute (Stockholm, Sweden), 1930-xx Microspectrophotometers
Measurements of nucleic acid and protein content based on the intrinsic ultraviolet absorption of these substances near 260 and 280 nm
Gucker et al. Northwestern University (Evanston, IL) 1947Photoelectronic counter for colloidal particles.
injecting the air stream containing the sample into the center of a larger sheath stream of flowing air that passed through the focal point of a dark-field microscope.
Kamentsky in Caspersson’s laboratory 1969Microscope-based flow cytometer
that used a transmission measurement at visible wavelengths to estimate cell size and a 260 nm UV absorption measurement to estimate nucleic acid content
1970-801981-85 1986-90 1991-95 1996-00 2001-06
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The number of publications
The NE Journal of medicineJAMALancet
NatureScience Cell
First fluorescence based flow cytometerPartec ICP 11 (1968)
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Cell Lab Quanta SC Beckman (2006)
Cell - Light interaction
Diffraction
RefractionReflection
etc …
Cell - Light interactionMie theory of SCATTERING
Comparable with wavelength
Forward
Side
Side Scatter (SSC)
Data Aquisition
LASER488 nm
Forward ScatterFSC
≈ size
≈ Granularity
LightAmplification by theStimulatedEmission ofRadiation
Side Scatter (SSC)
≈ Granularity
Forward ScatterFSC
≈ size
Data Aquisition
Up to 10 000 cells/sc
LASER488 nm
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Data Interpretation
Spectral Shift & Filters( Stokes Shift)
Sir G. Stokes: the first significant paper on fluorescence in 1852
Fluorescence
FSC ≈ size
530 nm
Data Aquisition
488 nm
Scattering488 nm
Fluorescence530
Dichroic:An optical device which can split a beam of light into two beams with differing wavelengths.
Dichroicmirror
530
Fluorescence
FSC
SSC
LASER488 nm
Data Aquisition
488
Dichroicmirror
FL2SSC FL1
FSC
Data Aquisition
LASER488 nm
530
600
488
PMT 1
PMT 2
PMT 5
PMT 4
DichroicFilters
BandpassFilters
Laser
Flow cell
PMT 3
Scatter
Sensor
Sample
Optical Design
FL1
FSC
FL1
NOMBRE
Data Presentation
Hoebe: Nature 2005, 433: 523-527 Figure 4 A nonsense mutation in CD36 is responsible for the oblivious phenotype. a, b, Surface expression of CD36 on peritoneal macrophages (a) and platelets (b) derived from wild-type, obl/+ and/or obl/obl mice. Cells were labelled with a primary monoclonal antibody against mouse CD36 or with an isotype control, then exposed to a secondary anti-mouse IgA coupled to phycoerythrin. c, Comparison of peritoneal macrophage responses between /….***…./. Untreated, shaded histogram; LTA (10 ng ml-1)-treated, unshaded histogram.
Capparelli : Cytometry 2005, 63A: 108-113
Figure 5. Gliadin recovery in artificial mix of gliadin with gluten-free flour. One part per million of product (1 ppm) was easily detected by the one-site assay. C, control (no gliadin added); 1, 10, 50, 100, flow cytometric profiles of 1 mg of gluten-free flour mixed with 1, 10, 50, and 100 ng of gliadin and then extracted with 1 ml of 60% ethanol. Numerical range, 100 to 104. Type of scale, logarithmic.
Data Presentation
FL1
NOMBRE
0 1023
R180%
R220%
R43%R3
2%
R515%
. 104101 102 103
Data Presentation
Laugwitz: Nature 2005, 433: 647-653
Figure 4 Amplification, characterization and myocytic differentiation of isl1+ cardioblasts in vitro. c, d, Histograms of FACS-sorted [beta]-gal+-C12FDG-labelled cells in cardiac mesenchymal fractions after 5 days (c) and 14 days (d) in culture.
Data Presentation
FL1
. 104101 102 103
104103
Peak3000
Peak 3000
Median 2500
Geometric Mean2750
Geometric Mean 2750
Median2500
50% 50%
Data Presentation
Geometric Mean 44
Mean 68
Geometric Mean
0
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6
0 20 40 60 80 100
0
1
2
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5
6
0 20 40 60 80 1000
1
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10 100
MeanGeometric Mean
LOG LIN
Geometric Mean
1879:
Galton, F: The geometric mean, in vital and social statistics.Proceedings of the Royal Society 29, 365-367
“…many variables in the life sciences /*/ are subject to multiplicative, rather than additive variation…”
McAlicter, D: The law of the geometric mean.Proceedings of the Royal Society 29, 367-376
“…such variables tend to follow the log-normal distribution…”
Geometric Mean
Hijri: Nature 2005, 433: 160-163Figure 2 Measurements of fluorescence intensity for estimating the nuclear DNA content of G. etunicatum. a-c, Histograms from one of the three replicate experiments showing fluorescence intensities /…/ DNA content per nucleus of G. etunicatum. The dotted line shows the geometric mean of fluorescence of G. etunicatum nuclei and the corresponding DNA content.
Data Presentation
R1 80%
R2 20%
Geometric Mean
Data Presentation
Ollinger: Circulation 2005, 112: 1030-1039 Figure 5. Bilirubin exerts its antiproliferative effect on VSMCs through inhibition of p38 MAPK phosphorylation. D, Cell cycle progression in VSMCs at 24 hours after 10% FCS stimulation in the presence or absence of SB203580 (20 µmol/L).
Data Presentation
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Data Presentation
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DECONVOLUTION : processus itératif de filtrage faisant appel aux transformations de Fourier et visant à rendre à une image brouillée une netteté aussi grande que possible.
D
G0/G1
S
G2/M
Data Presentation
Luan: Transplantation 2002, 73: 1565-1572Figure 3. (E) Rapamycin inhibits G1 to S transition of renal cancer cells. Renal cancer cells were incubated with 10 ng/mL rapamycin (Rapa-treated cells) or without rapamycin (control cells) for 24 hours, and the relative DNA content was quantified by flow cytometric analysis.
Data Presentation
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Barreda: Dev Comp Immunol 2000, 24: 395-406Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages
Data Presentation
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