Flow Cytometry 101. Jodi Moore, PhD Jan 2012 Goldenson 113

Transcription

1 Flow Cytometry 101 Jodi Moore, PhD Jan 2012 Goldenson 113

2 Origins:? Dye Chemistry s Electronics Microscopy Automated Clinical Cytology ID Apps Computers

3 Cytometry vs. Flow Cytometry Microspectrophotometry Confocal Microscopy Scanning Cytometry Image Analysis LocalizaBon of anbgen is possible Poor enumerabon of cell subtypes within a populabon Limited number of simultaneous measurements? LocalizaBon of anbgen is not possible Detailed subset idenbficabon and enumerabon MulBple correlated parameters measured simultaneously Can analyze many cells rapidly

4 Why is Flow Cytometry a Powerful Research Tool?? Single Cell pro caspase caspase EGF Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Stat Stat Stat JAK Correlated Measurements of: Lineage/Phenotype Cellular FuncBon Apoptosis/Necrosis Enzyme AcBvaBon Intracellular Proteins DNA Content/Ploidy Cell Cycle Protein PhosphorylaBon Protein- Protein InteracBons Gene Expression SorBng Highly Pure PopulaBon

5 Requirements for Flow Cytometry Any suspended parbcle or cell from uM is suitable for analysis. Sample must be in a single- cell suspension! Samples are labeled with fluorescent probes or fluorophore- conjugated anbbodies, and the free reagent is washed away. ExcitaBon sources (lasers) must match the excitabon maxima of the fluorochromes, and collecbon opbcs must correlate with the emission spectra of the fluorochromes.

6 What Does a Flow Cytometer Do? Rapid detecbon and quanbtabon of light sca7er and rela8ve fluorescence intensity of a parbcle or cell. Sca_er properbes (FSC and SSC) give us informabon about the relabve size and internal complexity of the parbcle, respecbvely. Fluorescence can be generated from a number of sources, intrinsic and extrinsic to cells.

7 How Does a Flow Cytometer Work? Fluidics: Introduce and focus the cells for interrogabon. Op8cs:? ExcitaBon- Laser and lenses, which shape and focus the laser beam. CollecBon- CollecBon lens (fibers) and a system of opbcal mirrors and filters that route specified wavelengths of collected light to opbcal detectors. Electronics: Convert the opbcal signals to proporbonal electronic signals and digibze them for computer analysis. Monitor and control the operabon of the cytometer.

8 Overview

9 Fluidics: Sheath Flow Principle 1. HYDRODYNAMIC FOCUSING: Refers to the restricbon of the cells to the center of the stream. It is facilitated by the reducbon in the cross- secbonal area of the vessel. Furthermore, the increased speed of the sheath stream versus that of the sample core stream at the injecbon point impinges on the core stream, thereby reducing its cross- secbonal width. FLOW CELL 2. LAMINAR FLOW: CondiBon whereby two adjacent, non- turbulent streams of fluid maintain their separabon with minimal mixing. Sheath Sheath Sample Central Core

10 OpBcs ExcitaBon OpBcs: Lasers which excite fluorochromes, fluorescent proteins, detecbon reagents in the assay Standard flavors: 488nm, 405nm,644nm and UV More exobc flavors: 532nm, 561nm, 592nm, 457nm Fiber opbc cables that carry beams to steering prisms, which then direct the beams to the flow cell and fluid stream; unless air launched CollecBon OpBcs: Fiber opbc cables that direct the emi_ed light to the appropriate emission block Filters/Mirrors that direct the signals in the emission block to the appropriate PMT

11 Laser Delays cell enters and leaves the first laser intercept blue t=0 cell enters and leaves the second laser intercept violet t= cell enters and leaves the third laser intercept Bme (μsec) yellow t= SETTING: TIME DELAY

12 OpBcal Filters Longpass Shortpass Bandpass LP 500 SP /50 Transmits all wavelengths greater than specified wavelength Transmits all wavelengths less than specified wavelength Transmits a specific band of wavelengths i.e. 500/50 = nm (500 +/- 25)

13 Dichroic Lenses Can be a long or short pass filter Filter is placed at a 45 o angle to the incident light Part of the light is reflected at 90 o to the incident light, and part of the light is transmi_ed Detector 1 Detector 2 Dichroic Lens

14 BD s Detector Arrays: Default ConfiguraBon Lowest Energy InnovaBve design for increased sensibvity is based on the principle that light reflecbon is more efficient than light transmission, resulbng in detecbon with minimum light loss.

15 Photons In How Is Fluorescence Detected? Photocathode Dynodes Anode e- Focusing Electrode Secondary Emission Amplified Signal Out: Current The role of the PMT is to convert an opbcal signal, comprised of photons, into a proporbonate amplified electrical signal, or current PhotomulBplier tubes (PMTs) ublize the photoelectric effect, whereby photons striking the photocathode sbmulate the producbon of photoelectrons, which are mulbplied as they pass through a series of electrodes (dynodes) in a process known as secondary emission. At the anode, the accumula8on of charge results in a sharp current pulse, which is transmi7ed to the electronics. This signal is then processed though log or linear amplifiers and converted to a voltage pulse. PMTs are ublized to detect SSC and all FL signals. FSC is detected by a less sensibve photo diode.

16 CreaBon of a Voltage Pulse Laser InterrogaBon Stream Voltage A voltage pulse is created when a cell or parbcle enters the laser beam and starts to sca_er light or fluoresce. The PMT converts light into an electrical pulse. Laser InterrogaBon Laser InterrogaBon Voltage Voltage Time Time Max. Height The highest part of the pulse occurs when the cell is in the center of the beam and the maximum amount of sca_er or fluorescence is achieved. The magnitude of the voltage pulse depends on the number of photons detected, the PMT voltage, and the amplifier gain. Time

17 Electronics Convert analog signals to proporbonal digital signals Compute area and height for each pulse Perform compensabon, calculate rabos and width Interface with the computer for data transfer Perform sorbng decisions including gabng, conflict resolubon, and drop charging

18 Pulse Measurements voltage area height The pulse processors measure pulses by three characterisbcs: height, area, and width. Pulse height is the maximum digibzed intensity measured Pulse area is an integrabon of the digibzed measures over Bme Pulse width calculates: Area/height x 64,000 threshold Bme window gate: width

19 Current produced by the PMT generates a voltage pulse; these electrical signals are digibzed using Analog to Digital Converters (ADCs). Data CollecBon & Display DigiBzed signals are processed by addibonal electronics that calculate peak, area, and width signals.. and generates histograms, dot plots, and FCS 3.0 files (Flow Cytometry Standard). Time FL1 Time Data Processor FL2 Time FL3

20 Signal GeneraBon in Flow Light Sca_er Signals Forward Sca_er Side Sca_er Fluorescence

21 What are Light Sca_er ProperBes? Side Scatter Detector α Cell Complexity Incident Light Source Forward Scatter Detector α Cell Surface Area* Forward Sca_er (FSC/small angle sca_er) diffracted light Detected along axis of incident light in the forward direcbon (Angles o ) Related to relabve cell surface area and refracbve index of the cell; not a quanbtabve biological equivalent to cell size* Oten used as a threshold to trigger signal processing Side Sca_er (SSC/wide angle sca_er) reflected and refracted light Detected at 90 to the laser beam (Angles o ) Related to cell granularity and internal complexity Occurs at any interface where there is a change in the refracbve index

22 PopulaBons in Flow Cytometry Each event that we measure is a single cell in flow cytometry, however, what we actually evaluate in flow cytometry is POPULATIONS or DISTRIBUTIONS of cells. We employ pa_ern recognibon regularly in discriminabng flow cytometry data.

23 Typical Sca_er Display Increasing Size Side Sca_er ApoptoBc Cells Dead Cells/ Noise Larger Cells Increasing Granularity (SSC) Live Cells *RelaBve Differences THRSHLD Forward Sca_er (FSC)

24 What InformaBon Can be Gained from Measurement of Sca_er ProperBes? Side Scatter Neutrophils Monocytes Lymphocytes Dead Viable Forward Scatter Cellular Subset DifferenBaBon/ RELATIVE Size/Internal substructure Cell Viability

25 When we return... Fluorescence

26 Fluorescence: Electronic Energy TransiBon HEAT, VIBRATION OUT (S n ) Excited Singlet State (S 1 ) Relaxed Singlet State E Excitation E Emission LIGHT IN HIGHER ENERGY LIGHT OUT LOWER ENERGY Ground State (S 0 )

27 Fluorescence Stoke s Shit = Δλ peak 525nm- 480nm=45nm λ= 488nm λ= 525nm Ex. Max = 480nm Em. Max = 525nm Incident Laser Light FITC Emi_ed Fluorescence CharacterisBc excitabon and emission spectra for each fluorochrome ExcitaBon and emission curves depict the probability of absorpbon or emission of a photon by the fluorochrome molecule at any given wavelength

28 Fluorescence Quantum Yield (Φ) Φ = Number of photons emitted Number of photons absorbed Defines the efficiency of the fluorescence process Maximum yield is 1.0 (100%) Measured compared to a standard; the quinine salt quinine sulfate in a sulfuric acid solubon is a common fluorescence standard, Φ = 0.58 GFP Φ = 0.79, AF488 Φ = 0.92, mcherry Φ = 0.22

29 Molar ExBncBon Coefficient (ε) Measures of the light absorbing capacity of a dye at a given wavelength per molar concentrabon Dyes with large molar exbncbon coefficients are efficient absorbers GFP ε = 55,000 M - 1 cm - 1, AF488 ε = 71,000 M - 1 cm - 1 BRIGHTNESS is proporbonate to the product of the exbncbon coefficient and quantum yield Brightness = ε*φ/1000

30 Typical Fluorescence Display Count NegaBve PopulaBon Dimmer PosiBve PopulaBon Brighter Channel Number (x- axis: represents the parameters signal value in channel numbers)

31 250 Emitted Fluorescence Intensity Binding Sites # Cells Number of Events FITC FITC FITC FITC FITC FITC FITC FITC FITC FITC 50 0 Negative GFP 90.4 Positive GFP <FITC-A>: GFP Mean/Median Fluorescence Intensity (x- axis: Mean or Median Fluorescence Intensity = MFI)

32 MulBple Correlated Parameters Single PosiBve PE PopulaBon Double PosiBve PopulaBon PE FL NegaBve PopulaBon FITC FL Single PosiBve FITC PopulaBon

33 Typical Fluorescence Display Log amplification is normally used for fluorescence displays because it expands weak signals and compresses strong signals, resulting in a distribution that is easy to display on a histogram. Linear scaling is preferable where there is not such a broad range of signals.

34 Data Display Histograms (Single Parameter) Overlays Contour Plot Density Plot Dot Plot (Dual Parameters)

35 Cell Cycle 1000 G 0 /G # Cells S G 2 M FL2-A Dean/Jett/Fox: %G1 = 55.8; %S = 32; %G2 = Cell Cycle DNA Dye Cycling Control Event Count: 8105 A stoichiometric DNA dye is quantitatively representative of the amount of DNA present. The Encyclopedia of Science Online

36 GaBng P1 SINGLETS P1 SINGLETS Used to idenbfy subsets of data or populabons. Only looking at data from your populabon of interest. Gate- defined populabons can be used to generate stabsbcs and limit the number of events displayed, collected (stopping gate) or saved (storage gate). GaBng is only used as a visualizabon tool during acquisibon or analysis; all events are saved unless using a storage gate to limit events saved.

37 ReporBng Values in Flow Cytometry Data # Cells 400 # Cells APC-A Ungated Specimen_001_DAPI only.fcs Event Count: APC-A Ungated Specimen_001_bTUB 10DF.fcs Event Count: % % = 91% APC+

38 ReporBng Values in Flow Cytometry Data The median is less sensibve to extreme scores than the mean and this makes it a be_er measure than the mean for highly skewed distribubons % of Max /68 = 13- fold increase in MFI (median fluorescence intensity) APC-A Specimen_001_DAPI only.fcs 68.2 Specimen_001_bTUB 10DF.fcs 862

39 ReporBng Values in Flow Cytometry Data 10 5 Hypodiploid 31.5 G1A 24.7 SA 21.7 G2MA DsRed-A 10 3 DsRed-A G1 3.5 S 2.75 G2M K 100K 150K 200K 250K Pacific Blue-A Singlets EL4_6h 50uM Viable VV & YOPRO 10df.fcs Event Count: FITC-A Ungated Specimen_001_GFP,3a,Venus,3a,Cherry,3a,Cerulean,3a,Sapphire.fcs Event Count: 30000

40 ReporBng Values in Flow Cytometry Data # Cells FL2-A Dean/Jett/Fox: %G1 = 55.8; %S = 32; %G2 = Cell Cycle Cycling Control Event Count: 8105

41 ReporBng Values in Flow Cytometry Data 250K 200K Ratio: FITC-A/PerCP-A 250K 200K 150K 100K 50K Time live 05 FURA X Calcium_VEGF1,2f,Iono.fcs Event Count: K FITC-A: FLUO4 PerCP-A: FURA RED 150K 100K 50K 0 250K 200K 150K 100K 50K Time Time % Cells Responding Ratio: FITC-A/PerCP-A 200K 150K 100K 50K Time live EC1 05 FURA X Calcium_VEGF1,2f,Iono.fcs Event Count: Ratio: FITC-A/PerCP-A Time Sample 05 FURA X Calcium_VEGF1,2f,Iono.fcs EC1 05 FURA X Calcium_VEGF1,2f,Iono.fcs

42 Common Fluorochrome Emission Spectra PacBlue AF405 ViViolet (V) FITC AF488 Sytox Blue (V) PE AF430 ViAqua (V) PE- TR PE- AF610 PI (V) 7- AAD (V) AF647 APC PE- Cy5 PacOrange APC- Cy5.5 AF700 Cy5 PE- Cy5.5 APC- Cy7 APC- AF750 PE- Cy7

43 Spectral Overlap/Spillover Occurs because fluorescent materials emit over a fairly broad range of wavelengths. Filters do not completely block spillover into adjacent detectors; therefore, a process to subtract the spectral overlap must be performed.

44 Filters: FITC: 530/30 PE: 575/26 PCPCy5.5: 695/40 FITC Spillover Filters: PECy7: 780/60 APC: 660/20 AF700: 730/45 Filters: APCCy7: 780/60 PB: 450/40 FITC Beads Posi8ve Beads = Green Unstained Beads = Blue Spillover = Red

45 FITC/PE Compensation-Log Display FITC only PE only x mean = 4.0 y mean = 40.0 Raw Data y mean = 2.0 x mean = 2.0 Compensated y mean 2.0 x mean 2.0

46 CompensaBon Refers to the process of mathemabcal subtracbon of dye spectral spillover into adjacent collecbon channels (detectors) Serves to align stained populabons in dye space without bias from spectral overlap Requires a NEGATIVE and SINGLE- COLOR controls for each fluorochrome/fp in the assay The posibve control must have enough posibves for an accurate count versus the negabve (at least 25% posibve) and must be as bright (MFI) as your brightest sample 150 Beads versus Cells Hardware (Pre- AcquisiBon Comp) Sotware (Post- AcquisiBon Comp) # Cells Negative Positive PE-A

47 FITC/PE Compensation Log versus Bi-exponential Transformation Also known as logicle displays Incorporates linear scaling for low and negative values together with log scaling for high values Bi-exponential transformation only changes the visualization of the data; doesn t effect gating or change the overlap of two populations Facilitates data display in an aesthetic, intuitive manner

48 Data Spread As A Result of CompensaBon (Digital Data) 575nm 695nm 780nm 719nm 660nm 660nm 660nm 660nm Bright fluorescent signals tend to spread in the dimensions against which they are compensated. Since dim (photon- deficient) fluorescent populabons have higher variance than bright (photon- rich) populabons, they will exhibit more data spread. Symmetry about the axes of the populabons indicates that they are properly compensated. CounBng errors are influenced by signal intensity and the degree of spillover, and are minimized by using bright dyes with as li_le spectral overlap as possible in cells with coordinately expressed markers.

49 NegaBve Values in Flow? How? Fluorescence from which an esbmated value of background light and electronic noise is subtracted, in conjuncbon with measurement error. Photon counbng error in photon- dim channels affects fluorescence spillover and contributes to negabve data spread following compensabon.

50 Facility InstrumentaBon

51 BD FACSAriaIIu 3 Lasers: 405, 488 & 594nm Simultaneous 9- Color Analysis/SorBng 1-4 way sort Temperature control of sample and collecbon tubes SorBng into plates with ACDU AutomaBc CompensaBon calculabon Digital Electronics High Speed/Variable Pressure SorBng

52 BD SORP LSRII w/hts 3 Lasers: 405, 488 & 594nm 2 Sca_er Parameters 12 FL Parameters Tube, 96- & 384- well plate input

53 BD FACSCalibur 2 Lasers: 488nm & 633nm 2 Sca_er Parameters 4 FL Parameters 12x75mm tube input.

54 Stratedigm S1000EX: 4 Lasers, 13 Colors!"#$%&'$()*+*#,%*)-&.).,(/0) Legend: T-Molding perimeter Top shelf with 3 supports Integrated bench light Integrated power strip, 8 outlets Integrated, adjustable monitor arm Deluxe, adjustable keyboard & mouse Tray 7. Integrated roll out shelf for fluidics tanks 8. Drawers Dimensions: Height to bench top: 30 Height to top shelf: 67 Length : 96 Depth: 36 The Future of Flow Cytometry

55 Vocabulary Configuration: The physical location of dichroic mirrors and filters in your detector array; if more than one, the others are given names that identify changes that have been made to the default configuration Acquisition: When you are viewing OR recording events in your sample Stopping Gate: Can be numeric (# of events) or time (800 seconds) that designates when sample recording stops Parameters: The name that designates each PMT detector PMT Voltages: The numbers that adjust the mean channel number or MFI of your population; these are usually adjusted with regard to a negative sample QC: Quality control; procedures performed on the instrument to ensure reproducible, peak performance Morphologic Gates: Gates that can be defined by FSC and SSC clustering patterns Pre-Acquisition Compensation, also Automatic Compensation: Hardware compensation performed pre-acquisition by the instrument Post-Acquisition Compensation: Compensation, done after data is acquired, by a software package CV: Coefficient of variation; a statistical measure of the dispersion of data points; used in QC applications MFI: Mean or Median Fluorescence Intensity, represented by a linear/log channel number Threshold: Establishes a level by which you have determined that an event represents a cell, rather than electronic noise; triggers the cytometer to display events at and above that level FCS File: Flow Cytometry Standard file format (Current v3.0)

56 Ready for a break??