1. Flow Cytometry Basics James Marvin Director, Flow Cytometry Core Facility University of Utah Health Sciences Center Office 801-585-7382 Lab 801-581-8641 jmarvin@cores.utah.edu

2. New Instrumentation iCyte/Sony Eclipse -4 lasers -5 color detection -Electronic volume BD FacsCanto -4 lasers -8 color detection

3. Seventeen-colour flow cytometry: unravelling the immune system Nature Reviews Immunology, 2004 “This ain’t your grandma’s flow cytometer” # of colors 1 2 3 4 5 6 7 8 9 10 # of plots 1 3 6 10 15 21 28 36 45

4. Flow Cytometry Applications  Immunophenotyping  DNA cell cycle/tumor ploidy  Membrane potential  Ion flux  Cell viability  Intracellular protein staining  pH changes  Cell tracking and proliferation  Sorting  Redox state  Chromatin structure  Total protein  Lipids  Surface charge  Membrane fusion/runover  Enzyme activity  Oxidative metabolism  Sulfhydryl groups/glutathione  DNA synthesis  DNA degradation  Gene expression  Phagocytosis  Microparticle analysis The uses of flow in research has boomed since the mid-1980s, and is now the gold standard for a variety of applications

5. Section I Background Information on Flow Cytometry

6. Experimental Design “One on One” Instrumentation “Flow Basics” Analysis “Data Analysis” Presentation “Data Analysis” Sample Procurement Sample preparation Fix/Perm Which Fluorophore Controls Isotype? Single color FMO Appropriate Lasers Appropriate Filters Instrument Settings Lin vs Log Time A, W, H Doublet discrimination Interpretation Mean, Median % + CV SD Signal/Noise Gating Histogram Dot Plot Density Plot Overlay Bar Graph Many components to a successful assay

7. What Is Flow Cytometry? Flow ~ motion Cyto ~ cell Metry ~ measure Measuring both intrinsic and extrinsic properties of cells while in a moving fluid stream

9. Cytometry vs. Flow Cytometry Cytometry/Microscopy Localization of antigen is possible Poor enumeration of cell subtypes Limiting number of simultaneous measurements Flow Cytometry. No ability to determine localization (traditional flow cytometer) Can analyze many cells in a short time frame. (30k/sec) Can look at numerous parameters at once (>20 parameters)

10. Section II The 4 Main Components of a Flow Cytometer

11. What Happens in a Flow Cytometer? Cells in suspension flow single file Cells in suspension flow single file through a focused laser where they scatter light and emit fluorescence that is filtered, through a focused laser where they scatter light and emit fluorescence that is filtered, measured, thenconverted to digitized values that are stored in a file measured, then converted to digitized values that are stored in a file which can then be analyzed and interpreted within specialized software. which can then be analyzed and interpreted within specialized software. Interrogation Fluidics Electronics Interpretation

12. The Fluidics System “Cells in suspension flow single file” Cells must flow one-by-one into the cytometer to do single cell analysis Accomplished through a pressurized laminar flow system. The sample is injected into a sheath fluid as it passes through a small orifice (50um-300um)

13. Sheath and Core Sheath Core

14. Hydrodynamic Focusing V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3 Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions. PBS/Sheath Sample/cells/core Laminar flow Hydrodynamic Focusing Laminar flow occurs when a fluid flows in parallel layers, with no disruption between the layers

15. Particle Orientation and Deformation a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow. b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s. V. Kachel, et al. - MLM Chapt. 3

16. What Happens in a Flow Cytometer (Simplified) Cell flash. swf Flow Cell- the place where hydrodynamically focused cells are delivered to the focused light source

17. Incoming Laser Sample Sheath Sample Core Stream Low Differential High Differential or “turbulent flow” Laser Focal Point Gaussian- A “bell curved” normal distribution where the values and shape falls off quickly as you move away from central, most maximum point.

18. Low pressure High pressure

19. Fluidics Recap Purpose is to have cells flow one-by-one past a light source. Cells are “focused” due to hydrodynamic focusing and laminar flow. Turbulent flow, caused by clogs or fluidic instability can cause imprecise data

20. What Happens in a Flow Cytometer? Cells in suspension flow single file Cells in suspension flow single file through a focused laser where they scatter light and emit fluorescence that is filtered, through a focused laser where they scatter light and emit fluorescence that is filtered, measured, andconverted to digitized values that are stored in a file measured, and converted to digitized values that are stored in a file Which can then be read by specialized software. Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation

21. Interrogation Light source needs to be focused on the same point where cells are focused. Light source  99%=Lasers

22. Lasers Light amplification by stimulated emission of radiation Lasers provide a single wavelength of light (monochromatic) They can provide milliwatts to watts of power Low divergence Provide coherent light Gas, dye, or solid state Coherent: all emmiting photons have same wavelength, phase and direction as stimulation photons

23. Light collection Collected photons are the product of laser light scattering or bouncing off cells 488nm Information associated with physical attributes of cells (size, granularity, refractive index) Scatter Fluorescence VS Collected photons are product of excitation with subsequent emission determined by fluorophore 350nm-800nm Readout of intrinsic (autofluorescence) or extrinsic fluorescence (intentional cell labeling)

24. Forward Scatter FSC Detector Laser Beam Original from Purdue University Cytometry Laboratories.5 0 -8 0

25. Forward Scatter The intensity of forward scatter signal is often attributed to cell size, but is very complex and also reflects refractive index (membrane permeability), among other things Forward ScatterFSC Forward Scatter=FSC=FALS=LALS FSC

26. Side Scatter FSC Detector CollectionLens SSCDetector Laser Beam Original from Purdue University Cytometry Laboratories

27. Side Scatter Laser light Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, drug delivery nanoparticles.) Side ScatterSSC Side Scatter=SSC=RALS=90 degree Scatter

28. Why Look at FSC v. SSC Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells LIVE Dead

29. Fluorescence Energy Absorbed exciting light Emitted fluorescence S0 Ground State Excited higher energy states S1 S2 S3 As the laser interrogates the cell, fluorochromes on/in the cell (intrinsic or extrinsic) may absorb some of the light and become excited As those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength Stokes shift- the difference in wavelength between the excitation and the emission

30. Optical Filters Many wavelengths of light will be emitted from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other. 3 types of filters  Long Pass filter  Short Pass filter  Band Pass filter

31. Long Pass Filters Transmit all wavelengths greater than specified wavelength  Example: 500LP will transmit all wavelengths greater than 500nm 400nm 500nm 600nm 700nm Transmittance

32. Short Pass Filter Transmits all wavelengths less than specified wavelength  Example: 600SP will transmit all wavelengths less than 600nm. 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual, Modified by James Marvin

33. Band Pass Filter Transmits a specific band of wavelengths  Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20) 400nm 500nm 600nm 700nm Transmittance

34. Dichroic Filters Can be a long pass or short pass filter Depending on the specs of the filter, some of the light is reflected and part of the light is transmitted and continues on. DichroicFilter Detector 1 Detector 2

35. Spectra of Common Fluorochromes with Typical Filters

36. Compensation Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more) A detector may “see” fluorescence from more than 1 fluorochrome. (referred to as bleed over) You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome

38. Compensation-Practical Eg.

39. Multi-laser Instruments and pinholes Implications- -Can separate completely overlapping emission profiles if originating off different lasers -Significantly reduces compensation

40. Spatial separation Blue Laser Excitation Blue and Yellow Laser Excitation 585/20 Yellow 585/20 Blue 530/20 Blue

41. Interrogation Recap A focused light source (laser) interrogates a cell and scatters light That scattered light travels down a channel to a detector FSC ~ size and cell membrane integrity SSC ~ internal cytosolic structure Fluorochromes on/in the cell will become excited by the laser and emit photons These photons travel down channels and are steered and split by dichroic (LP/SP) filters

42. What Happens in a Flow Cytometer? Cells in suspension flow single file Cells in suspension flow single file Through a focused laser where they scatter light and emit fluorescence that is filtered, measured Through a focused laser where they scatter light and emit fluorescence that is filtered, measured andconverted to digitized values that are stored in a file and converted to digitized values that are stored in a file Which can then be read by specialized software. Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation

43. Electronics Detectors basically collect photons of light and convert them to an electrical current The “electronics” must process that light signal and convert the current to a digitized value/# that the computer can graph

44. Detectors There are two main types of photo detectors used in flow cytometry  Photodiodes  Used for strong signals, when saturation is a potential problem (eg. FSC detector)  Photomultiplier tubes (PMT)  Used for detecting small amounts of fluorescence emitted from fluorochromes.  Incredible Gain (amplification-up to 10million times)  Low noise

45. Detector names

46. Photoelectric Effect Einstein- Nobel Prize 1921 Photons -> Photoelectrons -> Electrons Electric pulse generation

47. Measurements of the Pulse Pulse Height Pulse Width Pulse AreaTime Measured Current at detector

48. 0 10 (Volts) Relative Brightness 6.21 volts 1.23 volts 3.54 volts 10 1 10 2 10 3 10 4 1 ADC Analog to Digital Conversion Count

49. Does voltage setting matter? Voltage=362 292 272 252 522 -Voltage doesn’t change sensitivity or laser power -All your doing is changing the amplification of the signal -Caveat- there is large “sweet spot” of PMT voltage, outside of this range you may run the risk of non linear amplification

50. FSCSSC FITC PE APC APC-Cy7 FCS File or List Mode File

51. Electronics Recap Photons  Electrons  Voltage pulse  Digital # The varying number of photons reaching the detector are converted to a proportional number of electrons The number of electrons exiting a PMT can be multiplied by making more electrons available to the detector (increase Voltage input) The current generated goes to a log or linear amplifier where it is amplified (if desired) and is converted to a voltage pulse The voltage pulse goes to the ADC to be digitized The values are placed into a List Mode File

52. What Happens in a Flow Cytometer? Cells in suspension flow single filepast Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered a focused laser where they scatter light and emit fluorescence that is collected, filtered andconverted to digitized values that are stored in a file and converted to digitized values that are stored in a file Which can then be read by specialized software. Which can then be read by specialized software. Interrogation Fluidics Electronics Interpretation

53. See you at Data Analysis May 2 nd 10am

54. Antibody Antigen binding site

55. Immunophenotyping

56. roGFP Redox senstitive biosensor

57. Ca 2+ Flux Indo-1 Ca2+ free Emission=500nm Indo-1 Ca2+ bound Emission=395nm

58. Apoptosis Live Apoptotic Annexin V MTR PI FLICA

59. Cell cycle

60. Sorting Last attached droplet