2. Overview What is flow cytometry? Development of flow cytometry Components of Flow Typical applications Flow data

3. Flow Cytomtery Measurement (cytometry) of single cells in suspension that pass by (flow) a laser beam Not appropriate for analysis of cell clumps or tissues Discrete measurements from each cell in the sample, providing a distribution rather than an average of the measured characteristics in the cell sample Simultaneous measurement of multiple parameters Size (volume) Granularity (internal complexity) Fluorescence Light scatter signals Derived from fluorescent labels

4. Basic Outline of a Flow Cytometer Fluidics Optics Electronics

5. Commercial History First commercial particle analyzer: Model A Coulter counter (1950) First commercial fluorescence analyzer: Partec (1969) First commercial cytometer, the Cytograph – the Cytofluorograph – Kamentsky in 1970 First commercial cell sorter: Becton & Dickinson FACS-1 (1974, tradename) Hertzenberg Epics series 1977-79 by Coulter First benchtop analyzers about 1981 3 Colors available 1985 and 4 colors in 1986 First Benchtop Sorters 1992 First commercial high-speed cell sorter: Cytomation MoFlo (1994)

6. Advantages of Flow Cytometry Flexibility of the data acquisition Speed of measurement Thousands of cells can be analyzed in seconds Statistical information immediately available Ability to reanalyze with new gates gives us new information from old acquisitions

7. Light Scatter All objects passing through a laser beam in a cytometer will scatter light Large objects will scatter more light in the forward direction than small objects Forward Angle Light Scatter (FSC), roughly indicates size Forward light scatter, FALS, FS, FSC Side Scatter near 90° (SSC), structure dependent - “reflective” qualities, or granularity of a particle SS, SSC, 90° light scatter Actual laser spot is obscured and the light at 2° - 20° off the straight laser line is what is measured Measurements in Flow Cytometry

8. Fluorescence Excitation light energy is absorbed by fluorescent molecule, and molecule is “excited” As excited molecule returns to unexcited ground-state, a specific wavelength is emitted. Fluorescence emission is always of a longer wavelength (lower energy ) than the excitation wavelength. The longer the wavelength the lower the energy The shorter the wavelength the higher the energy e.g.. UV light from sun causes the sunburn not the red visible light

9. Emission Accomplished! Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence. Fluorophore Excitation / Absorbance Wavelength dependent Fluorophore Emission / Fluorescence The light given off or emitted is at a longer wavelength – but lower energy

10. Human eye can “see” 380nm-680nm Visible Light Region of the Electromagnetic Spectrum Spectrum is often shown this way

11. Ethidium PE PI FITC 600 nm300 nm500 nm700 nm400 nm 514488 Selected Laser Lines Dyes

12. (FITC)

13. Where is Fluorescence in Flow Cytometry Coming from?  Intrinsic fluorescence  Genuine feature of the cell  “autofluorescence”  tryptophan, tyrosine, pigment content, hemoglobin,  green fluorescent protein (GFP) - transfection assays  static  Extrinsic fluorescence  Experimentally added to the cell  Fluorescent probes/dyes - FITC, PE, PI, etc  Static  Kinetic

14. Common Applications Immunophenotyping Made possible with the advent of Monoclonal antibodies Large majority of the uses of flow Determination of cell surface antigens and after permeabilization for intracellular stains Clinically important for disease prognosis and diagnosis The number of subsets of cells that can be recognized is growing yearly. DNA quantification Intercalating dyes like propidium iodide (red fluorescent) Functional assays Calcium probes, probes for oxidative burst (DHR), membranes, phagocytosis assays, and many more Y

15. Monoclonal Antibodies Immunization Isolation of B-cells Fusion with metabolically deficient myeloma cell Selection Cloning by limited dilution

16. Example: Lymphocyte Typing

17. Following the Sample From the sample tube Through the aspiration rod Through the flow Cell Down the stream Into Waste or Sort collection tubes Through the tubing inside the instrument Intersecting the laser

18. Following the Cytometer signal path Cytometer lens computer sort module pulses PMT’s Cell diff amps linear amps PD log amps signal processing amplified signals Slide Courtesy of Joe Trotter, Director, Flow Cytometry Facility The Scripps Research Institute Trigger signal Stream Laser

19. Histogram IgM IgD

20. Statistics What types of statistics are we interested in?? Percentages of populations How bright those are – indicates how MUCH antigen is present Do those change? Is there a reaction to a stimulus?

21. Example MICR 304 S2008

22. TUTORIAL http://www.invitrogen.com/site/us/en/home/support/Tu torials.html

23. Acknowledgement This lecture has been drawn from a Dakocytomation training PowerPoint presentation Credit to Andrew Beernink (abeernink@novasite.com); Susan DeMaggio MS BSMT(ASCP)Qcym (flocyte@cox.net)abeernink@novasite.com