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FlowBasics2[1]
Flow cytometry is a technique that allows for the measurement of physical and chemical characteristics of single cells flowing through a stream of fluid. It enables the simultaneous multiparametric analysis of thousands of cells per second. Cells are labeled with fluorescent markers and passed individually through a laser beam, where detectors measure the cells' light scattering and fluorescent properties. This information can be used to identify and sort cell subpopulations. Flow cytometry provides high-speed analysis of multiple parameters at a single-cell level and is widely used in research, clinical diagnosis, and other applications.
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FlowBasics2[1]
- 2. Flow Cytometry * Isthe measurement (meter) of characteristics of single cells (cyto) in a suspended in stream of fluid. *It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus *This process is performed at rates of thousands of cells per second, and this information can be used to individually sort or separate subpopulations of cells
- 3. History » 1940s andearly 1950s with Wallace Coulter's invention of the Coulter counter which measured the volume of cells by displacement of an electrolyte and subsequent increase in electrical impedance. » Later M.Fulwyler, added detection capabilities - measurement of size and granularity by light scatter and measurement of fluorescence. » In 1972 L. Herzenberg (Stanford Univ.), coined the term Fluorescence Activated Cell Sorting - FACS. FACS was used and trademarked by one of the manufacturers (B-D) so this class of instrument is now commonly referred to as flow cytometers.
- 4. Fluorescence Activation Process (orImmunofluorescence) FITC FITC FITC FITC 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
- 5. Single dyes Fluorescentlabels Dye Laser excitation Line (nm) Maximal Absorbance (nm) Maximal Emission (nm) Fluorescence Color FITC 488 490 525 Green APC 633, 635, 647 650 661 Violet PerCp 488 490 675 Red PE 488 490, 565 578 Orange
- 6. Tandem dyes Fluorescentlabels Dye Laser excitation Line (nm) Maximal Absorbance (nm) Maximal Emission (nm) Fluorescence Color APC-Cy5 633, 635, 647 650 695 Violate APC-Cy7 633, 635, 647 650 785 Infrared PerCp- Cy5.5 488 496,546 695 Pink PE-Cy5 488 496,546 667 pink PE-Cy5.5 488 496,546 695 pink PE-Cy7 488 496,546 785 Infrared
- 7. Cellular Parameters Measuredby Flow Intrinsic • No reagents or probes required (Structural) – Cell size (Forward Light Scatter) – Cytoplasmic granularity (90 degree Light Scatter) – Photosynthetic pigments
- 8. Cellular Parameters Measuredby Flow • Extrinsic Reagents are required. – Structural • DNA content • DNA base ratios • RNA content – Functional • Surface and intracellular receptors. • DNA synthesis • DNA degradation (apoptosis) • Cytoplasmic Ca++ • Gene expression
- 9. Flow Cytometry Applications •Immunofluorescence • Cell Cycle Kinetics • Cell Kinetics • Genetics • Molecular Biology • Animal Husbandry (and Human as well) • Microbiology • Biological Oceanography • Parasitology • Bioterrorism
- 10. Clinical Applications of FlowCytometry • Diagnosis of Haematological Malignancies • Detection of MRD • Lymphocyte Subset Enumeration • Analysis of DNA Ploidy and Cell Cycle • Efficacy of Cancer Chemotherapy
- 11. Clinical Applications of FlowCytometry • Reticulocyte Enumeration • Platelet Function Analysis • Cell function Analysis • Applications of Transfusion Medicine • Applications in Organ Transplantation
- 12. - Flow cell- liquid stream (sheath fluid) - Light source - (mercury, xenon) high power water-cooled lasers Red - Blue (argon) dye laser - Detector and Analogue to Digital Conversion ( ADC) System » FSC & SSC » fluorescence signals. - Amplification system - linear or logarithmic - Computer for analysis of the signals.
- 13. Principles of FlowCytometry - First labeled with fluorescent dyes. - Forced through a nozzle in a single-cell stream passing through a laser beam - The laser is focused to a known wavelength - Excitation of a specific fluorochrome - Photo-multiplier tubes detect the scattering of light and emission from the fluorescent dye
- 14. Laser optics Laser Beam Flow chamber Sheath Sample Y X Z YZ X Cells are presented to the laser using principles of hydrodynamic focusing
- 15. PE FL FITC FL 488nmSct Laminar Fluidic Sheath Core Sheath Outer Sheath
- 16. • Each cellgenerates a quanta of fluorescence PE FL FITC FL 488nm Sct Confocal LensDichroic Lenses Photomultiplier Tubes (PMT’s) Discriminating Filters Forward Light Scattering Detector
- 17. Negative cells arealso detected PE FL FITC FL 488nm Sct Confocal Lens Dichroic Lenses Forward Light Scatter
- 18.
- 19. Flow Cytometry andsorting Ultrasonic Transducer 488nm Formard Light Scatter Detector Collimated Light Path Through Dichroic and Band Pass Filters SS FL2FL1 FL4 FL3 Pulse Height (0-10Volts) Time(useconds) Pressurized 1X PBS(Sheath) Pressurized Cell Sample Analog Data PMTs
- 20. From Fluorescence toComputer Display • Individual cell fluorescence is picked up by the detectors (PMT’s). • PMT’s convert light into electrical pulses. • These electrical signals are amplified and digitized using Analog to Digital Converters (ADC’s). • 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. • All events are individually correlated for all the parameters collected.
- 21. Light Scattering, 2Parameter Histogram Forward Light Scatter (FLS) 90 degree Light Scatter Bigger More Granular Live Cells Bigger Cells Dead Cells Apoptotic Cells X Axis Y Axis
- 22. 1 Parameter Histogram 12 3 4 6 7 150 160 170 .. 190 Channel Number Positive Negative BrighterDimmerCount 1 4 6 Fluorescence picked up from the FITC PMT cell count on the y-axis and the measurement parameter on x-axiscell count on the y-axis and the measurement parameter on x-axis.. Y Axis X Axis
- 23. 2 Parameter Histogram FITCFL PE FL Negative Population Single Positive FITC Population Single Positive PI Population Double Positive Population Two measurement parameters, on the x- and y-axes, and cell countTwo measurement parameters, on the x- and y-axes, and cell count height on a density gradientheight on a density gradient
- 24. Gating and Statistics •Data generated in flow cytometry is displayed using Multiparamater Acquisition and Display software platforms. • 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. • This information can be used to look at fluorescence expression within subpopulations of cells in a sample (gating).
- 25. Gating • Allow youto view cells of interest • lets you decide which data to view and which data to ignore or discard. • Gating can subsequently be changed when you analyze your data without any loss of information.
- 26. Data Display andAnalysis
- 27. Data Display andAnalysis
- 28. Data Display andAnalysis
- 29. Running Samples • Preparesamples. • One sample should be completely negative. This sample should be analyzed first. This sample is used for adjusting the PMT’s amplification voltage. • 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. • Once the instrument settings are optimized, run samples and collect data.
- 30. Compensation • Flow cytometersmeasure light emission, specifically, colour or wavelength • Unfortunately, the dyes we use to mark surface antigens share emission spectra • Compensation is a method to compute the contribution of a single fluorochrome to a measurement and compensate for spectral overlap
- 31. Compensation Compensated Uncompensated FL1 ~525nm FL2 ~575 nm FL3 ~620nm FL4 ~675 nm FITC ECFP EGFP EYFP PE DsRED PE-Texas Red Propidium Iodem PE-Cy5 APC PerCP FL1 ~525nm FL2 ~575 nm FL3 ~620 nm FL4 ~675 nm
- 32. Advantages • high speedanalysis (>100.000 s-1) • Measures single cells • Measures large number of cells • simultaneous analysis of multiple parameters (up to 20) • Identifies small subpopulations • quantification of fluorescence intensities • sorting of predefined cell populations (up to 70.000 s-
- 33. Disadvantages • very expensiveand sophisticated instruments • Need single particle • Tissue architecture is lost • Little information about intra-cellular distributions • Training continuously
- 34. Acknowledgements • Dr. PaulRobinson (Purdue University Cytometry Laboratories • Dr. Paul Wallace (Roswell Park Cancer Institute) • Dr. Mario Roederer (VRC - NIH) • BD Biosciences • National Taiwan University Hospital (NTUH) Lab. Med. Flow Dept.
- 35. NCI Sabratha/Libya- BDFACSCanto flow cytometer