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

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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