Flow basics2.ppt2


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Flow basics2.ppt2

  1. 1. Basic Principles in Flow Cytometry Prepared by Hector Nolla Manager CRL Flow Cytometry Lab University of California, Berkeley
  2. 2. Flow Cytometry <ul><ul><ul><ul><ul><li>Flow Cytometry is the technological process that allows for the individual measurements of cell fluorescence and light scattering. This process is performed at rates of thousands of cells per second. </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>This information can be used to individually sort or separate subpopulations of cells. </li></ul></ul></ul></ul></ul>
  3. 3. History <ul><li>Flow cytometry developed from microscopy. Thus Leeuwenhoek is often cited in any discussion regarding it’s history. </li></ul><ul><li>F.T. Gucker (1947)build the first apparatus for detecting bacteria in a LAMINAR SHEATH stream of air. </li></ul><ul><li>L. Kamentsky (IBM Labs), and M. Fulwyler (Los Alamos Nat. Lab.) experimented with fluidic switching and electrostatic cell sorters respectively. Both described cell sorters in 1965. </li></ul><ul><li>M. Fulwyler utilized Pulse Height Analyzers to accumulate distributions from a Coulter counter. This feature allowed him to apply statistical analysis to samples analyzed by flow. </li></ul>
  4. 4. History <ul><li>In 1972 L. Herzenberg (Stanford Univ.), developed a cell sorter that separated cells stained with fluorescent antibodies.The Herzenberg group coined the term Fluorescence Activated Cell Sorter (FACS). </li></ul>
  5. 5. Fluorescence Activation Process (or Immunofluorescence) FITC FITC Antibodies recognize specific molecules in the surface of some cells But not others When the cells are analyzed by flow cytometry the cells expressing the marker for which the antibody is specific will manifest fluorescence. Cells who lack the marker will not manifest fluorescence Antibodies are artificially conjugated to fluorochromes Antibodies FITC FITC FITC FITC
  6. 6. Cellular Parameters Measured by Flow <ul><li>No reagents or probes required ( Structural ) </li></ul><ul><ul><li>Cell size(Forward Light Scatter) </li></ul></ul><ul><ul><li>Cytoplasmic grabularity(90 degree Light Scatter) </li></ul></ul><ul><ul><li>Photsynthetic pigments </li></ul></ul><ul><li>Reagents are required. </li></ul><ul><ul><li>Structural </li></ul></ul><ul><ul><ul><li>DNA content </li></ul></ul></ul><ul><ul><ul><li>DNA base ratios </li></ul></ul></ul><ul><ul><ul><li>RNA content </li></ul></ul></ul><ul><ul><li>Functional </li></ul></ul><ul><ul><ul><li>Surface and intracellular receptors. </li></ul></ul></ul><ul><ul><ul><li>DNA synthesis </li></ul></ul></ul><ul><ul><ul><li>DNA degradation (apoptosis) </li></ul></ul></ul><ul><ul><ul><li>Cytoplasmic Ca++ </li></ul></ul></ul><ul><ul><ul><li>Gene expression </li></ul></ul></ul>Intrinsic Extrinsic
  7. 7. Flow Cytometry Applications <ul><li>Immunofluorescence </li></ul><ul><li>Cell Cycle Kinetics </li></ul><ul><li>Cell Kinetics </li></ul><ul><li>Genetics </li></ul><ul><li>Molecular Biology </li></ul><ul><li>Animal Husbandry (and Human as well) </li></ul><ul><li>Microbiology </li></ul><ul><li>Biological Oceanography </li></ul><ul><li>Parasitology </li></ul><ul><li>Bioterrorism </li></ul>
  8. 8. <ul><li>Flow cytometry integrates electronics, fluidics, computer, optics, software, and laser technologies in a single platform. </li></ul>
  9. 9. Y X Z Y Z X Cells are presented to the laser using principles of hydrodynamic focusing Laser optics Laser Beam Flow chamber Sheath Sample
  10. 10. Laminar Fluidic Sheath Core Sheath Outer Sheath PE FL FITC FL 488nm Sct
  11. 11. <ul><ul><ul><li>Each cell generates a quanta of fluorescence </li></ul></ul></ul>PE FL FITC FL 488nm Sct Confocal Lens Dichroic Lenses Photomultiplier Tubes (PMT’s) Discriminating Filters Forward Light Scattering Detector
  12. 12. Negative cells are also detected PE FL FITC FL 488nm Sct Confocal Lens Dichroic Lenses Forward Light Scatter
  13. 13. Optical Bench Schematic Flow Cell Laser Beam FS Sensor Fluorescence Pickup Lens SS Sensor FL1 Sensor 525BP FL2 Sensor 575BP FL3 Sensor 620BP FL4 Sensor 675BP 488DL 488BK 550DL 600DL 645DL
  14. 14. From Fluorescence to Computer Display <ul><li>Individual cell fluorescence quanta is picked up by the various detectors(PMT’s). </li></ul><ul><li>PMT’s convert light into electrical pulses. </li></ul><ul><li>These electrical signals are amplified and digitized using Analog to Digital Converters (ADC’s). </li></ul><ul><li>Each event is designated a channel number (based on the fluorescence intensity as originally detected by the PMT’s) on a 1 Parameter Histogram or 2 Parameter Histogram. </li></ul><ul><li>All events are individually correlated for all the parameters collected. </li></ul>
  15. 15. Light Scattering , 2 Parameter Histogram Forward Light Scatter (FLS) 90 degree Light Scatter Bigger More Granular Live Cells Bigger Cells Dead Cells Apoptotic Cells X Axis Y Axis
  16. 16. 1 Parameter Histogram 1 2 3 4 6 7 150 160 170 .. 190 Channel Number Positive Negative Brighter Dimmer Count 1 4 6 Fluorescence picked up from the FITC PMT
  17. 17. 2 Parameter Histogram FITC FL PE FL Negative Population Single Positive FITC Population Single Positive PI Population Double Positive Population
  18. 18. Gating and Statistics <ul><li>Data generated in flow cytometry is displayed using Multiparamater Acquisition and Display software platforms . </li></ul><ul><li>Histograms corresponding to each of the parameters of interest can be analyzed using statistical tools to calculate percentage of cells manifesting specific fluorescence, and fluorescence intensity . </li></ul><ul><li>This information can be used to look at fluorescence expression within subpopulations of cells in a sample (gating). </li></ul>
  19. 19. Flow Cytometry Data Smaller Region, Live cells mostly Larger Region includes all cells
  20. 20. Running Samples <ul><li>Prepare samples. </li></ul><ul><li>One sample should be completely negative. This sample should be analyzed first. This sample is used for adjusting the PMT’s amplification voltage. </li></ul><ul><li>Adjust the PMT Voltage until you can see a population peak in the first decade of your 1 parameter and or your two parameter plot.These samples are used for adjusting Spectral Overlap . </li></ul><ul><li>Once the instrument settings are optimized, run samples and collect data. </li></ul>
  21. 21. Flow Cytometry and sorting
  22. 22. Criticism: <ul><li>Content </li></ul><ul><li>There are too much text on the slide. </li></ul><ul><li>The content was not fully organized. </li></ul><ul><li>The information was not enough. </li></ul><ul><li>Design </li></ul><ul><li>There are no designs at all. </li></ul><ul><li>There is no other font color. </li></ul><ul><li>There is no effect and even background. </li></ul>
  23. 23. Criticism: <ul><li>Style </li></ul><ul><li>The arrangement of font style was not good. </li></ul><ul><li>The text was not synchronized. </li></ul><ul><li>The layout was unorganized. </li></ul><ul><li>The pictures are just pictures, no style, effect and even design. </li></ul>
  24. 24. Prepared by: Vivian C. Sareno 3sed-sc