Flow Cytometry Basic Training

A Look Inside the Box

James Marvin Flow Cytometry Facility Northwestern University

Section I

Background Information on Flow Cytometry

The Many Parts of Flow

Experimental design Specific Applications Courses Sample preparation Choosing the proper instrument Flow Basics Setting up the instrument Collecting the proper data Data Interpreting the data Analysis Graphics presentation and publication Sorting

What Is Flow Cytometry?

Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream

Cytometry vs. Flow Cytometry

Cytometry Localization of antigen is possible Poor enumeration of cell subtypes Limiting number of simultaneous measurements Flow Cytometry. Cannot tell you where antigen is. Can analyze many cells in a short time frame. Can look at numerous parameters at once.

Uses of Flow Cytometry

It can be used for

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

Medline Publications citing "Flow Cytometry"

5000
Publications

4000 3000 2000 1000 0

19 1960 1965 1970 1975 1980 1985 1990 2095 00

The use of flow in research has boomed since the mid-1980s

Year

Background Info Summary

Flow Cytometry is a quickly expanding technology Has continually increased in popularity since the mid 1980s. Gives us the ability to analyze many properties of many cells in very little time

Section II
The 4 Main Components of a Flow Cytometer

What Happens in a Flow Cytometer?

Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is filtered and collected then converted to digitized values that are stored in a file Which can then be read by specialized software.

Fluidics

Interrogation

Electronics

Interpretation

What Happens in a Flow Cytometer (Simplified)

The Fluidics System

Cells in suspension flow single file

You need to have the cells 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 (50um300um)

Fluidics Schematic
Sample Tube

Sheath Pressure Waste Tank Sheath Tank

(Constant)

Sample Pressure
(Variable)

Vacuum

Line Pressure

How The Flow Cell Works

The cells from the sample tube are injected into the sheath stream Flow in a flow cell is laminar. Hydrodynamic focusing pushes the cells to line up single file along their long axis. The shape of the flow cell provides the means for hydrodynamic focusing.

Fluidics
Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.

Notice also how the position of the inner ink stream is influenced by the position of the ink source.
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

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

The Flow Cell

Sheath Cell Sample Stream

The introduction of a large volume into a small volume in such a way that it becomes focused along an axis is called Hydrodynamic Focusing.

Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Sample

Sample

Sheath Sheath Sheath

Laser Focal Point

Sample Core Stream

Incoming Laser

Low Differential

High Differential

Sample Differential
10 psi 10.2 psi 10.4 psi 10.8 psi 10 psi 10 psi

Difference in pressure between sample and sheath This will control sample volume flow rate The greater the differential, the wider the sample core. If differential is too large, cells will no longer line up single file Results in wider CVs and increase in multiple cells passing
through the laser at once. No more single cell analysis!

300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 0 1024

G0/G1 CV= 2.42

Count

Low pressure

68.70

19.16

9.56

S phase G0/G1 G2/M

2048 FL3

3072

4096

High pressure

340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 0

GO/G1 CV= 7.79

74.85 9.12 15.84

Count

1024

2048 FL3

3072

4096

Fluidics Recap

Purpose is to have cells flow one-by-one past a light source. Cells move out of tube because there is slightly greater pressure on the sample than on the sheath Cells are focused due to hydrodynamic focusing and laminar flow.

What Happens in a Flow Cytometer?

Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is filtered, collected and converted to digitized values that are stored in a file Which can then be read by specialized software.

Fluidics

Interrogation

Electronics

Interpretation

Interrogation

Light source needs to be focused on the same point where cells are focused.
Light source
On

all flow lab instruments-Lasers

Lasers
Light amplification by stimulated emission of radiation

Lasers can provide a single wavelength of light (monochromatic) They can provide milliwatts to watts of power Also provide coherent light All help to create a stable and reliable signal

Coherent: all emmiting photons have same

wavelength, phase and direction as stimulation photons

Light Scatter

When light from a laser interrogates a cell, that cell scatters light in all directions. The scattered light can travel from the interrogation point down a path to a detector.

Forward Scatter

Light that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel. The intensity of this signal has been attributed to cell size, refractive index (membrane permeability) Forward Scatter=FSC=FALS=LALS

Forward Scatter

Laser Beam
FSC Detector

Original from Purdue University Cytometry Laboratories

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, etc.) Side Scatter=SSC=RALS=90 degree Scatter

Side Scatter
Laser Beam
FSC Detector

Collection Lens

SSC Detector
Original from Purdue University Cytometry Laboratories

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
Granulocytes

Lymphocytes
SSC

Monocytes

RBCs, Debris, Dead Cells

FSC

Fluorescence Channels

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 Those photons pass through the collection lens and are split and steered down specific channels with the use of filters.

Fluorescence Detectors
Laser Beam
FSC Detector

Collection Lens
Fluorescence Detector A, B, C, etc
Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Filters

Many wavelengths of light will be scattered 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

Long Pass Filters

Transmit all wavelengths greater than specified wavelength

Example:

500LP will transmit all wavelengths greater than 500nm

Transmittance 400nm

500nm

600nm

700nm

Original from Cytomation Training Manual, Modified by James Marvin

Short Pass Filter

Transmits all wavelengths less than specified wavelength

Example:

600SP will transmit all wavelengths less than 600nm.

Transmittance 400nm

500nm

600nm

700nm

Original from Cytomation Training Manual, Modified by James Marvin

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)
Transmittance 400nm

500nm

600nm

700nm

Original from Cytomation Training Manual, Modified by James Marvin

Dichroic Filters

Can be a long pass or short pass filter Filter is placed at a 45 angle to the incident light Part of the light is reflected at 90 to the incident light, and part of the light is transmitted and continues on.
Detector 1

Detector 2 Dichroic Filter

Optical Bench Layout

To separate scatter and multiple fluorescence wavelengths simultaneously from each cell, The design of a multi-channel layout must consider
Spectral

Properties of the fluorochromes used The appropriate positioning of filters

Spectra of Common Fluorochromes

Laser Lines (nm)
350 457 488 514 610 632

PE-Texas Red
Texas Red PI Ethidium PE FITC cis-Paranaric Acid
300 400 500 600 700
Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Example Channel Layout

Detector#4

Detector#3

Bandpass Filters
Detector#1

Detector#2

Original from Purdue University Cytometry Laboratories

Channel Layout Model

SSC <505nm PE-Cy5 >605nm FITC 505nm-555nm

555nm-605nm PE

Compensation

Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more) Depending on filter arrangement, a detector may see some 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

Compensation-Practical Eg.

Detectors

There are two main types of photo detectors used in flow cytometry
Photodiodes
o

Used for strong signals, when saturation is a potential problem (eg. FSC detector)

Photomultiplier
o

tubes (PMT)

More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes.

Photodiodes and PMTs

Photo Detectors usually have a band pass filter in front of them to only allow a specific band width of light to reach it Therefore, each detector has a range of light it can detect, once a filter has been placed in front of it.

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 shape 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 Specific wavelengths are then detected by PMTs that have a filter in front of them

What Happens in a Flow Cytometer?

Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software.

Fluidics

Interrogation

Electronics

Interpretation

Electronics

Detectors basically collect photons of light and convert them to current The electronics must process that light signal and convert the current to a digitized value/# that the computer can graph

What Happens in the PMT

A voltage is applied to the detector which makes electrons available for the photons to pick up As the number of photons increase, more and more electrons are picked up yielding a greater current output from the detector Also, as the voltage applied to the detector increases the same amount of photons will have a greater current output

Photoelectric Effect
Einstein- Nobel Prize 1921

Electronics Schematic
Detector
Current out Photons in
Photons

Linear Amplification

or
Log Amplification

Voltage
Time

PMT Voltage Input 150V-999V

Original from Becton Dickinson Training manual, Modified by James Marvin

Threshold

When the laser interrogates an object, light is scattered. If the amount of light scattered surpasses a threshold, then the electronics opens a set window of time for signal detection The threshold can be set on any parameter, but is usually set on FSC

Threshold
FSC Detector

Threshold (eg. 52) Time FSC Detector

Threshold (eg. 52) Time

Photons In ~ Voltage Out

Detector
Photons converted to no. of electrons

Current out Photons in

Linear Amplification

or
Log Amplification

Voltage
Time

PMT Voltage Input 150V-999V

Original from Becton Dickinson Training manual, Modified by James Marvin

The Voltage Pulse

As the cell passes through the laser, more and more light is scattered until the cell is in the center of the laser (maxima) As the cell leaves the laser, less and less light is scattered After a set amount of time, the window closes until another object scatters enough light to be triggered.

The Pulse

Time

10

10
1

256
Channel Number 196

10,000
1000 100 Relative Brightness

6.21 volts

(Volts)

.1

(Volts)

128 64 0

3.54 volts

.01
1.23 volts

10
1

.001 (1mV)

Pg 255

Measurements of the Pulse

Voltage Intensity Pulse Area Width Height

Time

Linear and Log Amplifiers

The current exiting the detector passes through either a linear or log amplifier where it is converted into a voltage pulse. You can adjust the intensity of the voltage by amplifying it on a linear scale or converting it to a logarithmic scale The use of a log amp is beneficial when there is a broad range of fluorescence as this can then be compressed; this is generally true of most biological distributions. Linear amplification is used when there is not such a broad range of signals e.g. in DNA analysis and calcium flux measurement.

Analog to Digital Converters

An ADCs takes the voltage pulse and converts it to discrete binary numbers depending on total resolution The binary signal generated is converted to a relative bin number Those relative bin numbers are acquired as a list of values from each detector for each event (cell) and are eventually plotted on a graph.

Analog to Digital Conversion

8-bit binary code

ADC
Voltage Intensity

11001100

27+26+25+24+23+22+21+20
204
Time

Relative bin number

10-bit binary code

ADC

0011100100

29+28+27+26+25+24+23+22+21+20
228
Relative bin number

List Mode File

Param 1 Param2 Param 3 Param 4 Param 5 Event # FSC SSC FITC PE APC
1 2 3 4 100 110 90 95 500 505 480 490 10 700 720 15 650 700 670 720 4 6 10 15

Electronics Recap

The varying number of photons reaching the detector are converted to a proportional number of electrons The number of electrons exiting a PMT can be multiplied by making more electrons available to the detector (increase Voltage input) The current generated goes to a log or linear amplifier where it is amplified (if desired) and is converted to a voltage pulse The voltage pulse goes to the ADC to be digitized The values are placed into a List Mode File

What Happens in a Flow Cytometer?

Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software.

Fluidics

Interrogation

Electronics

Interpretation

Interpretation

Once the values for each parameter are in a list mode file, specialized software can graphically represent it. The data can be displayed in 1, 2, or 3 dimensional format Common programs include

CellQuest Flowjo WinMDI FCS Express

Creation of a Histogram
Event # 1 2 3 4 Param 1 FSC 100 110 90 95 Param2 SSC 500 505 480 490 Param 3 FITC 10 700 720 15 Param 4 PE 650 700 670 720 Param 5 APC 4 6 10 15

0101001000.10000

Types of Plots

Single Color Histogram

Fluorescence intensity (FI) versus count

Two Color Dot Plot

FI of parameter 1 versus FI of Parameter 2

Two Color Contour Plot

FI of P1 versus FI of P2. Concentric rings form around

populations. The more dense the population, the closer the rings are to each other

Two Color Density Plot

FI of P1 versus FI of P2. Areas of higher density will

have a different color than other areas

Plots
Contour Plot
Density Plot

Greyscale Density

Dot Plot

www.treestar.com

Gating

Is used to isolate a subset of cells on a plot Allows the ability to look at parameters specific to only that subset Can use boolean logic to include or exclude multiple gates

Gating Example

Blue Detector

Red Detector

PE detector

Blue Detector

Red Detector

PE detector

Important Points on Analysis

What kind of data are you looking for?

How much fluorescence? What percent are positive? How much more positive is x than y? What is the ratio between param1 and param2 MFI (geometric or arithmetic) %-ages CV Median Anything you can do with a list of numbers

What kind of statistics are available

Everythings Relative

The relative bin numbers are just thatrelative. Saying your cells have a mean fluorescence intensity of 100 means absolutely nothing until you compare it to a negative. The fact that everything is relative allows you to compare 2, 3, or 20 samples using the same instrument settings.

What Happens in a Flow Cytometer?

Cells in suspension flow single file past a focused laser where they scatter light and emit fluorescence that is collected, filtered and converted to digitized values that are stored in a file Which can then be read by specialized software.

Fluidics

Interrogation

Electronics

Interpretation

References

Numerous References available in the Flow Lab

Cytometry Current Protocols in Flow Cytometry Many more reference books available

Purdue University Cytometry Laboratories website: http://www.cyto.purdue.edu/

Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 powerpoint slides

The Scripps Research Institute Flow Cytometry Core Facility: http://facs.scripps.edu/

Flow Lab Contact Info

James Marvin, Managing Director

[email protected] 908-1294

Jeff Nelson, Sr. Research Technologist

[email protected] 908-1294

Paul Mehl, Research Tech

[email protected] 908-1294

Location:
Main Lab: Olson Bldg 8505