Flow Cytometry Basics

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 1 3 6 10 15 21 28 36 45

4. Flow CytometryApplications • 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. Many components to a successful assay Instrumentation “Flow Basics” Analysis “Data Analysis” Presentation “Data Analysis” ExperimentalDesign “One on One” • 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

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/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) Cytometry vs. Flow Cytometry

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

11. Fluidics Electronics Interpretation Interrogation What Happens in a Flow Cytometer? • Cells in suspension flow single file • 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 • which can then be analyzed and interpreted within specialized software.

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 Core Sheath

14. Hydrodynamic Focusing 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 V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

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) Flow Cell- the place where hydrodynamically focused cells are delivered to the focused light source Cell flash. swf

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

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. Fluidics Electronics Interpretation Interrogation What Happens in a Flow Cytometer? • Cells in suspension flow single file • 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 • Which can then be read by specialized software.

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

22. LasersLight 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. Fluorescence Scatter VS Light collection • Collected photons are product of excitation with subsequent emission determined by fluorophore • 350nm-800nm • Readout of intrinsic (autofluorescence) or extrinsic fluorescence (intentional cell labeling) • 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)

24. Laser Beam FSCDetector Forward Scatter .50-80 Original from Purdue University Cytometry Laboratories

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 Scatter=FSC=FALS=LALS FSC

26. Laser Beam FSCDetector Collection Lens SSC Detector Side Scatter Original from Purdue University Cytometry Laboratories

27. Side Scatter • 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 Scatter=SSC=RALS=90 degree Scatter

28. Granulocytes Lymphocytes SSC Monocytes RBCs, Debris, Dead Cells FSC 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 Dead LIVE

29. Fluorescence Excited higher energy states 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 S2 Energy S1 Emitted fluorescence Absorbed exciting light S0 Ground State 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. Transmittance 400nm 700nm 600nm 500nm Long Pass Filters • Transmit all wavelengths greater than specified wavelength • Example: 500LP will transmit all wavelengths greater than 500nm

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

33. Transmittance 400nm 700nm 600nm 500nm 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)

34. Detector 1 Detector 2 Dichroic Filter 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.

35. Spectra of Common Fluorochromes with Typical Filters

36. Compensation • Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more) • Adetector 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 Blue 585/20 Yellow 530/20 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. Fluidics Interrogation Electronics Interpretation What Happens in a Flow Cytometer? • Cells in suspension flow single file • 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 • Which can then be read by specialized software.

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

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

48. ADC Analog to Digital Conversion 10 104 6.21 volts 103 102 Relative Brightness (Volts) 3.54 volts 1.23 volts 101 0 1 Count

49. Does voltage setting matter? 292 272 Voltage=362 252 522 -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. APC-Cy7 PE APC FSC SSC FITC FCS FileorList Mode File