Flow Cytometry Basics James Marvin Director Flow Cytometry

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

Seventeen-colour flow cytometry: unravelling the immune system Nature Reviews Immunology, 2004 # of colors 1 2 3 4 5 6 7 8 9 10 # of plots 1 1 3 6 10 15 21 28 36 45

Ø Ø Ø Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential Ion flux Cell viability Intracellular protein staining p. H changes Cell tracking and proliferation Sorting Redox state Chromatin structure Flow Cytometry Applications ØTotal protein ØLipids ØSurface charge ØMembrane fusion/runover ØEnzyme activity ØOxidative metabolism ØSulfhydryl groups/glutathione ØDNA synthesis ØDNA degradation ØGene expression ØPhagocytosis ØMicroparticle analysis ØRNA detection • The uses of flow in research has boomed since the mid-1980 s, and is now the gold standard for a variety of applications

Section I Background Information on Flow Cytometry

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

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

Section II The 4 Main Components of a Flow Cytometer

What Happens in a Flow Cytometer? Interpretation Fluidics Electronics Interrogation

Hydrodynamic Focusing

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

Low pressure High pressure

Acoustic focusing uses ultrasonic radiation pressure (>2 MHz) to transport particles into the center of the sample stream

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

What Happens in a Flow Cytometer? Fluidics Interpretation Fluidics Interrogation Electronics Interpretation

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

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

The amount of light scattered by any particle is directly proportional to the diameter of the particle and inversely proportional to the wavelength of the light being used to detect it. Noise/ Debris 110 nm particles V-SSC vs SSC

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

Spectra of Common Fluorochromes with Typical Filters CYTOF

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

Compensation example

Compensation matrix • Compensation matrix for 8 color analysis

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

Blue Laser Excitation 530/20 Blue No Compensation Applied 585/20 Yellow 585/20 Blue Spatial separation Blue and Yellow Laser Excitation 530/20 Blue

Interrogation Recap l l l 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

What Happens in a Flow Cytometer? Fluidics Interpretation Fluidics Interrogation Electronics Interrogation Interpretation

Electronics l The “electronics” process light signal intensities and convert them to a proportional digitized value/# that the computer can graph

Photons -> Photoelectrons -> Electrons Photoelectric Effect Einstein- Nobel Prize 1921 A photomultiplier tube, useful for light detection of very weak signals, is a photoemissive device in which the absorption of a photon results in the emission of an electron. These detectors work by amplifying the electrons generated by a photocathode exposed to a photon flux.

Detector names

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

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

Electronics Recap l Photons Electrons Voltage pulse Digital #

What Happens in a Flow Cytometer? Fluidics Interpretation Fluidics Interrogation Electronics Interpretation

See you at Data Analysis