physics presentation(1)

7/31/2019 Physics Presentation(1)

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Suggested Day 1


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An Introduction to Flow

CytometryBy

Max Parker-Shames,

Phoebe Parker-Shames,

Marie Keil, Natalie Edson

This Publication was made possible by Grant #024094 from NIAID.

Its contents are solely the responsibility of the authors and do not necessarily represent

the official views of the NIH.


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Abdcerotec.com


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What is Flow Cytometry?

Lets break it down:

Cyto = Cell Metry = Measure

So Cytometry= measure cells


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Basic Flow Cytometers

Flow Cytometers aremachines that measuremultiple aspects of

single cells

The cells areinterrogated (examined)by lasers

They are interrogatedas they flow by in astream of fluid.

http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/


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What are Flow Cytometers

used for? Medical Research (especially in cancer

research and immune functions)

Medical Diagnoses (For detecting ormonitoring diseases like HIV/AIDS or formonitoring transplants)

Marine Biology Protein Engineering

Pathology


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How do we measure things?

Human Senses

Sight

Sound

Smell

Touch

Taste


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What does flow cytometry

measure about cells? Size

Shape (Granularity)

Makeup (Surface proteins)

Density


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HOW?


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Flow Cytometers are made of

three basic parts Optics (where the cells are analyzed by

the lasers)

Electronics (where light is translatedinto electrons and the cells are sorted)

Computer Analysis (where the data is

analyzed using software like Flowjo)


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Lets look briefly at how they

work

http://www.unsolvedmysteries.ore

gonstate.edu/flow_06
http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06


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Cytometers work by examining

fluorescent markers They are like dyes, and are added to samples

before they are run through the cytometer.

Most are proteins. They bind to cells and give off light whenstimulated by a laser.

Some common ones used are FITC,

TRITC, NHS, and PE. We graph the relative amounts of these

markers to distinguish cells.


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Luciferin

(derived from fireflies)

Fluorescein Isothiocyanate

(FITC)

http://en.wikipedia.org/


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Amount of Blue Markers

AmountofYe

llow

Markers


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BUT

In real-world examples, the graphs willlook a lot more complicated.

Most cytometry samples containthousands of cells, not just four.

As long as you remember that they

follow the same principal, youll do fine.

Lets see an example:


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Forward Scatter (ForSc)

SideScatter(

OrthSc)

Each one of these dotsrepresents a single cell!


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Suggested Day 2


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Cell Sorting

The Fluidic System


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Fluidic System

In order to do analyses in FlowCytometers, the machine needsto analyze cells individually

To accomplish this, cells are run

through the machine in fast-moving stream of fluid (hencethe name flowcytometry)

This process is calledHydrodynamic Focusing

http://www.bdbiosciences.com


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The sample fluid stream is directed through the laser by thesheath fluid

The sample fluid is always a higher pressure than the sheath

fluid The relative pressure in the sample fluid controls the velocity of

the stream as it flows through the laser beam, or interrogationpoint

Low samplepressure, lowaverage cellcount. Thisgives greater

accuracy, buttakes longer

High samplepressure, highaverage cellcount. Thisgives lessaccuracy, butis much faster



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A Review of Properties of

LightWavelength, color, etc


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Wavelength,

Sinusoidal wave

v = f

Can be sound, light,or water waves

http://en.wikipedia.org/


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Colors

488 nm wavelength is the most commonly used type of laser inFlow Cytometers

http://antonine-education.co.uk/


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Lasers

Light Amplification byStimulated Emission ofRadiation

Lasers are almostalways a coherent lightsource (they haveuniform wavelength)

Usually, multiple lasersare used in FlowCytometers to analyzecells

http://plaza.ufl.edu/


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The Optics System

Excitation


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Excitation

Multiple Lasers are used inFlow Cytometers to excitethe cells

Fluorescent markers absorb

and reemit differentwavelengths

Different types of cellsscatter different colored light,this helps identify what kind

of cells they are



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Lenses collect emitted light and filters route

specific wavelengths into detectors It is important to keep this as exact as

possible to avoid overlapping colors

Light Sorting



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Electrostatic Flow

Sorting

After the cells are

interrogated by thelaser, vibrationsseparate the samplestream into dropletscontaining either oneor zero cells, called thebreak-off point

http://cyto.perdue.edu/


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At the point at which thestream breaks into droplets,it passes through anelectrically charged ringwhich charge cells based onthe results detected by thecytometers laser and

detector system

The cells then pass bycharged plates which sortthe cells based on thecharge that they have been

given



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Suggested Day 3


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Lets review what weve

learned and look at how theprocess works

T-Shirt sorting activity!


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Electronics System

So what happens after the cells

are interrogated by the laser andsorted by type?


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What does the electronics

system do? Converts the reflected

light into electrons

Converts analogsignals to digital

Performs

compensation Transfers data to the

computer



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The electronics system takes

the data from collection tocomputer



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Photomultipliers and

Photodiodes These are thetwo types ofdetectors thatconvertphotons intoelectricalsignals

They control

sensitivity byadjustingvoltage



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Compensation

There is some overlap between the colors emitted by differentfluorescent markers, therefore mathematical compensation isused to reduce overlapping results

A veryimportantfunction of theelectronics

system is toperformcompensation



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Suggested Day 4


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Heres a video overview of

Flow Cytometryhttp://www.youtube.com/watch?v=

nAfL4FXju1s
http://www.youtube.com/watch?v=nAfL4FXju1shttp://www.youtube.com/watch?v=nAfL4FXju1shttp://www.youtube.com/watch?v=nAfL4FXju1shttp://www.youtube.com/watch?v=nAfL4FXju1s


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Analyzing Flow Cytometry

Data


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Where does the data come

from?

Cell samples are collected for use in the flow cytometer.

Cells come from non-solid sources, like blood.

Sensors pick up the light emitted or reflected by eachparticle as the laser hits them.

The sensors transmit the data to a computer where it canbe analyzed.


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What does the data say?

Thats what were trying to find out!

Flow Cytometry data can showmedical researches whether newcures are having an affect, whether

a person has AIDS or not, whether aperson is rejecting a transplantedorgan, and many other things inother fields as well


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How do we analyze the data?

By using software called FlowJo

FlowJo allows you to analyze raw data from a flowcytometer graphically and numerically.


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Setting parameters

Different parameters (the variables on a graph) tell you

different things

For example, forward scatter indicates size of the cell,while side scatter indicates granularity (how much stuff is

inside)

Other parameters that you can observe on a graph includethe different fluorescent markers used to stain cells


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Each fluorescent marker used becomes a parameter in

FlowJo

Graphing two fluorescent markers against each other cantell you which parts of the data were positive for one, both, orneither of the markers

Since certain types of cells bind to certain kinds of markers,this shows you what kind of cells they are

Fluorescent markers as

parameters

Remember this?


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Amount of Blue Markers

A

mountofYe

llow

Marker

s

Remember this?

12

3 4


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BUT

Again, most real-world cytometrysamples contain thousands of cells, not

just four In these cases, you can look at areas of

greater density to identify different types

of cells Lets see that example again:


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Forward Scatter (ForSc)

S

ideScatter

(OrthSc)

Each one of these dotsrepresents a single cell!


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Histograms

There are manydifferent types ofgraphs in Flow

Cytometry softwarelike FlowJo

A histogram is aspecial type of graphthat shows the

frequency of cellsalong the spectrum ofa given parameter

QuickTime and a

decompressorare needed to see this picture.


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Pseudocolor

This is usually thedefault view forsamples

http://www.flowjo.com


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Contour Plots

These are probabilitycontour plots that oftenresemble topographical

maps These will usually be

the best display option

Their biggest downsideis that they do not

usually display outliers,however, in FlowJothere is an option todisplay outliers



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Suggested Day 5


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Intro to Gating

Candy sorting activity!


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More gating

Like you saw in the activity you just did, cells can beseparated into different subgroups, while remaining

part of the larger group.

This is very handy, as you can gate a group with oneset of parameters, and then gate the subgroups withdifferent parameters. An example of this is shown onthe next slide.

SampleSample/singlets


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QuickTime and a

decompressorare neede d to see this picture. QuickTime and a


QuickTime and a


QuickTime and a


Sample/singlets/CD3/CD4+ Sample/singlets/CD3


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Statistical analysis

Once weve gated the data, FlowJo has

some handy tools for doing statisticalanalysis.

FlowJo can compute median, mean,geometric mean, mode, and many otherthings.

FlowJo can also find whats calledFrequency of Parent, Grandparent, or Total,which is basically the percent the subgroupis of one of the groups above it.


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Another screenshot here

QuickTime and a



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This is analysis in very basic

terms.

In reality, it is much more complicated.

There can be hundreds of parameters and controlsin a single sample, and many samples perexperiment.

It is also possible to find out much more than justthe types of cells in the experiment; however, its

really hard to understand and ever harder to do.


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Good Information Resources

http://www.youtube.com/watch?v=nAfL4FXju1s

http://www.abdserotec.com/uploads/Flow-Cytometry.pdf

http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/


http://www.unsolvedmysteries.oregonstate.edu/flow_04

http://www.unsolvedmysteries.oregonstate.edu/flow_06

Wikipedia.org
http://www.youtube.com/watch?v=nAfL4FXju1shttp://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.unsolvedmysteries.oregonstate.edu/flow_04http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_04http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.youtube.com/watch?v=nAfL4FXju1s


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Image Resources

http://www.Abdcerotec.com



http://www.antonine-education.co.uk/

http://www.plaza.ufl.edu

http://cyto.perdue.edu/Abdcerotec.com


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