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Side Scatter SSC (refracted/reflected light)

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.

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Side Scatter SSC (refracted/reflected light)

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  1. 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.

  2. Side Scatter SSC (refracted/reflected light) Forward Scatter FSC (diffracted light) Light Scattering The light is forced to deviate from a straight trajectory by an object Refraction + reflection + diffraction + … = Scattering

  3. Fluorescence-Activated Cell Sorter (FACS)

  4. Fluorescence-Activated Cell Sorter (FACS)

  5. 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) 1947 Photoelectronic 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 1969 Microscope-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

  6. The number of publications Cell Lab Quanta SC Beckman (2006) The NE Journal of medicine JAMA Lancet Nature Science Cell First fluorescence based flow cytometer Partec ICP 11 (1968)

  7. Refraction Reflection etc … Diffraction Cell - Light interaction

  8. Side Forward Cell - Light interaction Mie theory of SCATTERING Comparable with wavelength

  9. Data Aquisition Side Scatter (SSC) ≈ Granularity Forward Scatter FSC ≈ size 488 nm LASER Light Amplification by the Stimulated Emission of Radiation

  10. Data Aquisition Side Scatter (SSC) ≈ Granularity Forward Scatter FSC ≈ size 488 nm LASER Up to 10 000 cells/sc

  11. Data Interpretation

  12. Spectral Shift & Filters ( Stokes Shift) Sir G. Stokes: the first significant paper on fluorescence in 1852

  13. Fluorescence 530 nm Dichroic mirror Fluorescence 530 FSC ≈ size Dichroic: An optical device which can split a beam of light into two beams with differing wavelengths. Data Aquisition Scattering 488 nm 488 nm

  14. 488 nm LASER Data Aquisition SSC Fluorescence 530 Dichroic mirror 488 FSC

  15. FL1 FSC 488 nm LASER Data Aquisition SSC FL2 530 600 488

  16. Optical Design PMT 5 PMT 4 Sample PMT 3 Dichroic Filters Flow cell PMT 2 Scatter PMT 1 Laser Sensor Bandpass Filters

  17. FL1 FL1 FSC NOMBRE Data Presentation

  18. 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.

  19. R1 80% R2 20% R5 15% R4 3% R3 2% 0 1023 . 101 102 103 104 Data Presentation NOMBRE FL1

  20. 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.

  21. 50% 50% FL1 103 104 Median 2500 . 101 102 103 104 Geometric Mean 2750 Peak 3000 Data Presentation Peak 3000 Median 2500 Geometric Mean 2750

  22. Mean 68 Geometric Mean 44 Geometric Mean

  23. Geometric Mean Geometric Mean Mean LOG LIN

  24. 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…”

  25. Data Presentation Hijri: Nature 2005, 433: 160-163 Figure 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.

  26. Data Presentation R1 80% R2 20% Geometric Mean

  27. 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).

  28. Data Presentation ?

  29. 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

  30. Data Presentation G0/G1 G2/M S

  31. Data Presentation Luan: Transplantation 2002, 73: 1565-1572 Figure 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.

  32. Data Presentation Barreda: Dev Comp Immunol 2000, 24: 395-406 Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages

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