DefiniAons. Flow cytometry. History of flow citometry. Development of flow cytometry 5/21/12. What funcaons does a flow cytometer be able to do?

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1 DefiniAons Flow cytometry Balázs Visegrády Biophysics lecture Flow cytometry Process or measurement method can measure discrete properaes physical, chemical, biochemical, biological parameters of separate paracles, e.g. biological cells or chromosomes in flow. Flow Sor-ng or FACS: Fluorescence- Ac-vated Cell Sor-ng Cells or paracles separaaon by measured parameters What funcaons does a flow cytometer be able to do? ParAcles cells - count in cell suspension. Biological and non biological separaaon. Live and dead paracles, cells separaaon paracles measured in 1 minute. Measurement of scarering and/or inner fluorescence of paracles. SorAng: separaaon of discrete paracles. History of flow citometry Flow cytometer to detect bacteria in aerosol (1947) Wallace Coulter - It can detect the fluctuaaon of the electric conductance (resistance) whilst suspended cells pass through across a thin slit in buffer. Kamentsky sorter (1965) Development of flow cytometry The principles of the measurements The paracles pass through a hydrodynamically focused flow in an exciang light beam (laser, Hg- vapour lamp, etc.). ScaRering signals detecaon. Specific fluorescence detecaon MulAparameter data analysis. ElectrostaAc or mechanic paracles separaaon: sor$ng. 1

2 Technical setup PMT 5 Light source Flow system OpAcs DetecAon system Data collecaon Data analysis SorAng Sample Flow cell Scatter Sensor Dichroic Filters Bandpass Filters PMT 1 PMT 4 PMT 3 PMT 2 Laser Hydrodynamic Systems Sample in Sheath Piezoelectric crystal oscillator Hydrodynamic focusing At he outer sheath (diam.: 200 μm) contains faster flowing fluid. Flow cytometer cell Signal direcaon Flow cell Laser beam Sheath in Fluorescence Sensors Scatter Sensor It creates a massive drag effect on the narrowing central chamber making a single file of paracles (20 μm) and is called hydrodynamic focusing. Sheath Core Under opamal condiaons (laminar flow) the fluid in the central chamber will not mix with the sheath fluid. Flow Chamber OpAcal components Forward ScaRer Light: ExciAng/illuminaAng system Lasers ( , 420, 457, 488, 514, 532, 600, 633 nm) Argon ion, Krypton ion, HeNe, HeCd, Yag, solid state Arc- lamps Mercury- vapour, Hg- Xenon (right lines) DetecAng system PhotomulAplier tubes (PMTs) Formerly 1-2 tubes Currently tubes. Photodiods Forward scaqer (FSC) measurement 2

3 Side ScaRer photodiod scaqer SSC side scarer is related to complexity of the cells Light scarer Fluorescent signal detecaon The emired fl. light is detected in 90 to the excitaaon light The emired light intensity is (usually) proporaonal to the number of the fluorophores in the cells Light intensity FSC forward scarer is related to size MulAple fluorophores OpAcal system MulAple fluorophores can be be excited simultaneusly or separately MulAple fluorophores can be be excited simultaneusly or separately The emired light will be separated using dichroic and and bandpass filters 3

4 Setup of the equipment The signals from PMTs will be converted to digital signals using analog/digital converters (ADC) Analog signal Digitális signals FSC SSC FITC APC Laser delay Laser delay How to convert the numbers to parameters? Signal width : Peak height : Area : No deadame The number of the sample The highest value The sum of the numbers area signal width height Cell sorang or FACS analysis Ajer the sample is hydrodynamically focused, each paracle is probed with a beam of light. The scarer and fluorescence signal is compared to the sort criteria set on the instrument. If the paracle matches the selecaon criteria, the fluid stream is charged The nozzle is vibrated at high frequency (piezoelectric vibrator (30-40 khz)) The droplets eventually pass through a strong electrostaac field, and are deflected lej or right based on their charge Surface marker test- immunofluorescence The leucocytes and (other cells) express specific molecule combinaaon on a surface of a cell, which depends on: types of the cells, Immunofluorescence labeling (direct) Cell stadium of differenaaaon of cells, acave and inacave state of cells other funcaonal state of cells, Single fluorescence dye- labeled anabody pathological mutaaons, differenaaaon mechanisms. Typically detecaon happen by monoclonal anabodies. The anabodies are labeled by fluorescence dyes for flow cytometry. 4

5 Fluorescence labeling (direct vs. indirect) Fluorescence stains must meet the following requirements: Cell Cell Emits the wavelength, where the auto fluorescence is small. The emission spectrum has to have minimal overlap with the spectrum of the other stains (compensaaon). Have to have high quantum efficiency, absorpaon coefficient, so they must generate strong, detectable fluorescence (sensiavity). Have to have small nonspecific binding. Have to have a simple protocol to label the proteins (anabodies). Do not modify the properaes of labeled molecule significantly. Have to be cheap Fluorescence compensaaon Saving data, data analysis FITC PE PE- TR PE- CY5 FCS (flow cytometry standatrd) file contains all the data measured = list mode file cells list mode file When two or more fluorochromes are used during a single experiment there is a chance that their emission profiles will coincide A process called fluorescence compensaaon is applied during data analysis, which calculates how much interference (as a %) a fluorochrome will have in a channel that was not assigned specifically to measure it. Number of events (cell count) Single- parameter histograms The graphs that display a single measurement parameter (relaave fluorescence or light scarer intensity) on the x- axis and the number of events (cell count) on the y- axis. Intensity of parameter (e.g. fluorescence) Forward Scatter Light Scatter Gating Side Scatter Projection Neutrophils Monocytes Lymphocytes Degree Scatter Human white blood cells Forward Scatter Projection 5

6 GaAng The principle of flow cytometry data analysis is to selecavely visualize the cells of interest while eliminaang results from unwanted paracles e.g. dead cells and debris. This procedure is called gaang. Total FL4 cells measured. Flow cytometry measurements GaAng SCATTER FLUORESCENCE IMAGE L M G Gated FL4 cells Data analysis Plosng methods: 1. Dotplot: 2 axis, 1 parameter on each axis, one data regards to one cell 2. Density plot: the color of the dots means the number of the cells 3. Contour plot: dots with the same values are bound with lines. 4. 3D (surface) plot: the cell numer is on the z axis Sample preparaaon: cell culture, fixaaon, fluorescence labeling Applying controls set the parameters of the measurement GaAng Measurement: collecang the appropriate set of date of the cells of interest, sorang Data analysis (can be made any later Ame, different ways) Experimental steps Possible errors in the measurements Asymmetric cells (e.g. blood cells) can give orient dependent signal further study is needed Without controls false posiave / negaave results DiagnosAc applicaaons of flow cytometry Control of central dogma Is the fluorescent signal always proporaonal to the fluorophore bound to the molecules? 6

7 Flow Cytometric Immunotyping CharacterizaAon of leukemia AIDS diagnosacs (CD4+ T cell count based) Cellcycle and DNA content analysis 1. FixaAon and permeabilizaaon of cells (immobilize the anagens, maintaining the cellular and subcellular architecture 2. The fluorophore penetrates into the nuclei (f.e. propidium jodid) bindingto the DNA 3. The fluorescence intensity measured proporaonal to the DNA content of the nuclei ApplicaAon field: Cancer cells Have higher DNA content higher S and G2/M raao apoptoac cell separaaon Chromosome analysis apoptoac cells normal G0/G1 cells Most of the human chromosomes can be separated by f.c. DiagnosAc applicaaons, summary DetecAng the raaos of filamentous/ monomeric acan in cells The raao of F/G- acan causes malfuncaons in myopathy desease We applied acan mutants in cell cultures We applied Marina- Blue- conjugated ana- mouse secondary anabody for the mutant acan detecaon, Alexa Fluor 488- conjugated DNaseI (green) for G- acan and Texas- Red- X- conjugated phalloidin for F- acan detecaon. RaAos of filamentous/monomeric acan in cells expressing selected mutants B Visegrády, L. Machesky

8 Flow cytometry, FACS Hydrodynamic focusing Summary Light scarering, fluorescence signal detecaon Study of high number of cell applying mulavariate data analysis Wide range of diagnosac, scienafic applicaaons Thank you! References: Damjanovich S. Fidy J. Szöllősi J. (szerk.), Orvosi biofizika, Medicina Kiadó, 2006 Howard M. Shapiro: PracAcal Flow Cytometry, 4th EdiAon, ISBN: J.Paul Robinson, lecture,

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