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Flow Cytometry
Tipologia: Notas de estudo
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Introduction to
Flow Cytometry
By Misha Rahman, Ph.D.
Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc.
Principles
Data analysis
Protocols
Troubleshooting
Preface
How can I explain what flow cytometry is to someone that knows nothing about it? Well, imagine it to be a lot like visiting a supermarket. You choose the goods you want and take them to the cashier. Usually you have to pile them onto a conveyor. The clerk picks up one item at a time and interrogates it with a laser to read the barcode. Once identified and if sense prevails, similar goods are collected together e.g. fruit and vegetables go into one shopping bag and household goods into another. Now picture in your mind the whole process automated; replace shopping with biological cells; and substitute the barcode with cellular markers – welcome to the world of flow cytometry and cell sorting! We aim to give you a basic overview of all the important facets of flow cytometry without delving too deeply into the complex mathematics and physics behind it all. For that there are other books (some recommended at the back). Instead, we present a guide that will be invaluable to beginners of flow cytometry or act as a fact-packed synopsis for those of you interested in teaching others about the virtues of this powerful application. At AbD Serotec, we specialize in flow cytometry reagents and offer the largest range of antibody markers commercially available, together with accessory buffers. Our guiding principle is to manufacture products for use with every known flow cytometry instrument. We offer no bias towards only one machine but are more interested in maximizing scientific performance. Confidence in our reagents is shown by our quality promise that states that if any product does not perform as described on its datasheet we will replace the reagent with a fresh vial or offer you credit. Please contact us or view our website at www.ab-direct.com for our ever- increasing range of flow cytometry reagents. You can see and print out detailed datasheets for every product online. But should you need any further information, just contact our Technical Services Team (see the back cover for your nearest office) and they will be happy to assist. I hope this booklet will prove useful to you. If you like it, let us know and we’ll produce similar guides for the research applications we support.
Morris Gillis Marketing Manager
Without hydrodynamic focusing the nozzle of the instrument (typically 70 μM) would become blocked, and it would not be possible to analyze one cell at a time.
After hydrodynamic focusing, each particle passes through one or more beams of light. Light scattering or fluorescence emission (if the particle is labeled with a fluorochrome) provides information about the particle’s properties. The laser and the arc lamp are the most commonly used light sources in modern flow cytometry. Lasers produce a single wavelength of light (a laser line) at one or more discreet frequencies (coherent light). Arc lamps tend to be less expensive than lasers and exploit the color emissions of an ignited gas within a sealed tube. However, this produces unstable incoherent light of a mixture of wavelengths, which needs subsequent optical filtering. Light that is scattered in the forward direction, typically up to 20 o^ offset from the laser beam’s axis, is collected by a lens known as the forward scatter channel (FSC). The FSC intensity roughly equates to the particle’s size and can also be used to distinguish between cellular debris and living cells. Light measured approximately at a 90 o^ angle to the excitation line is called side scatter. The side scatter channel (SSC) provides information about the granular content within a particle. Both FSC and SSC are unique for every particle, and a combination of the two may be used to differentiate different cell types in a heterogeneous sample. Fluorescence measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules such as DNA and cytokines. Flow cytometers use separate fluorescence (FL-) channels to detect light emitted. The number of detectors will vary according to the machine and its manufacturer. Detectors are either silicon photodiodes or photomultiplier tubes (PMTs). Silicon photodiodes are usually used to measure forward scatter when the signal is strong. PMTs are more sensitive instruments and are ideal for scatter and fluorescence readings. The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting (passing) others. There are three major filter types. ‘Long pass’ filters allow through light above a cut-off wavelength, ‘short pass’ permit light below a cut-off wavelength and ‘band pass’ transmit light within a specified narrow range of wavelengths (termed a band width). All these filters block light by absorption (Figure 2).
When a filter is placed at a 45 o^ angle to the oncoming light it becomes a dichroic filter/mirror. As the name suggests, this type of filter performs two functions, first, to pass specified wavelengths in the forward direction and, second, to deflect blocked light at a 90 o^ angle. To detect multiple signals simultaneously, the precise choice and order of optical filters will be an important consideration (refer to Figure 3).
When light hits a photodetector a small current (a few microamperes) is generated. Its associated voltage has an amplitude proportional to the total number of light photons received by the detector. This voltage is then amplified by a series of linear or logarithmic amplifiers, and by analog to digital convertors (ADCs), into electrical signals large enough (5–10 volts) to be plotted graphically. Log amplification is normally used for fluorescence studies 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 e.g. in DNA analysis. The measurement from each detector is referred to as a ‘parameter’ e.g. forward scatter, side scatter or fluorescence. The data acquired in each
Long pass^500 transmits > 500^ nm long pass nm
560 nm short pass transmits < 560 nm
630/15 nm band pass permits everything in the 615–645 nm band width
Light passed
Blocked light absorbed
Blocked light deflected
Short pass
Band pass
Dichroic mirror
FIGURE 2 Different types of optical filters
The speed of flow sorting depends on several factors including particle size and the rate of droplet formation. A typical nozzle is between 50–70 μM in diameter and, depending on the jet velocity from it, can produce 30,000–100,000 droplets per second, which is ideal for accurate sorting. Higher jet velocities risk the nozzle becoming blocked and will also decrease the purity of the preparation.
Laser interrogation
Break-off point (charging)
Voltage plates
- - -
+
+
+
FIGURE 4 Electrostatic flow sorting
Principles of fluorescence
Fluorochromes are essentially dyes, which accept light energy (e.g. from a laser) at a given wavelength and re-emit it at a longer wavelength. These two processes are called excitation and emission. The process of emission follows extremely rapidly, commonly in the order of nanoseconds, and is known as fluorescence. Before considering the different types of fluorochrome available for flow cytometry, it is necessary to understand the principles of light absorbance and emission. Light is a form of electromagnetic energy that travels in waves. These waves have both frequency and length, the latter of which determines the color of light. The light that can be visualized by the human eye represents a narrow wavelength band (380–700 nm) between ultraviolet (UV) and infrared (IR) radiation (Figure 5). Sunlight, for example, contains UV and IR light that, although invisible to the eye, can still be felt as warmth on the skin and measured scientifically using photodetectors. The visible spectrum can further be subdivided according to color, often remembered by the mnemonic ‘ROY G BV’ standing for red, orange, yellow, green, blue and violet. Red light is at the longer wavelength end (lower energy) and violet light at the shorter wavelength end (higher energy).
When light is absorbed by a fluorochrome, its electrons become excited and move from a resting state (1) to a maximal energy level called the ‘excited electronic singlet state’ (2). The amount of energy required will differ for each
Higher energy
Lower energy
Ultraviolet
Visible spectrum
Infrared
400 nm 500 nm 600 nm 700 nm
FIGURE 5 The electromagnetic spectrum
Wavelength (nm)
(blue light)
Excitation laser line Peak or maximum
Excellent signal (green)
Weak signal (orange)
Spectrum
400 500 600 700
Rel
ative
absorb
an
ce
400 500 600 700
Rel
ative fluores
cen
ce emission
A B Peak or maximum
These optimal conditions are termed maximal absorbance and maximal emission wavelengths. Maximal absorbance usually defines the laser spectral line that is used for excitation. In the case of FITC, its maximum falls within the blue spectrum. Therefore, the blue Argon-ion laser is commonly used for this fluorochrome, as it excites at 488 nm, close to FITC’s absorbance peak of 490 nm. FITC emits fluorescence over the range 475–700 nm peaking at 525 nm, which falls in the green spectrum. If filters are used to screen out all light other than that measured at the maximum via channel A (see Figure 7), FITC will appear green. Hence, ‘fluorescence color’ usually refers to the color of light a fluorochrome emits at its highest stable excited state. However, if FITC fluorescence is detected only via channel B (see Figure 7), it will appear orange and be much weaker in intensity. How the flow cytometer is set up to measure fluorescence will ultimately determine the color of a fluorochrome.
FIGURE 7 (^) Light absorbance (LEFT) and light emission (RIGHT) of FITC
The purpose of a fluorescent probe, such as a fluorochrome-conjugated antibody, is to directly target an epitope of interest and to allow its biological and biochemical properties to be measured more easily by the flow cytometer. Fluorescent probes are useful in a wide range of applications including: identifying and quantifying distinct populations of cells, cell surface receptors or intracellular organelles; cell sorting; immunophenotyping; calcium influx experiments; determining nucleic acid content; measuring enzyme activity, and for apoptosis studies. By changing the excitation light and using more than one fluorochrome, it is possible to analyze several parameters of the sample at any one time. This forms the basis of multicolor fluorescence studies.
There are dozens of fluorescent molecules (fluorochromes) with a potential application in flow cytometry. The list is ever growing but it is not the scope of this booklet to cover them all. Instead, some of the most useful fluorochromes for surface or intracellular epitope detection are described on page 13, including the very latest in fluorescent probe technology – tandem dyes. There is enough variation in the two tables to cover most researchers’ needs.
Single dyes: Some of these single dyes e.g. FITC have been in use for the past 30 years but are now facing competition from alternatives like Alexa Fluor ®^ dyes, which offer the user greater photostability and increased fluorescence.
Tandem dyes: In a tandem dye, a small fluorochrome takes a ‘piggy-back’ ride on another larger fluorochrome. When the first dye is excited and reaches its maximal singlet state, all its energy transfers to the second dye (an acceptor molecule), located in close proximity. This activates the second fluorochrome, which then produces the fluorescence emission. The process is called FRET (fluorescence resonance energy transfer). It is a clever way to achieve higher Stokes Shifts and, therefore, increase the number of colors that can be analyzed from a single laser wavelength. The majority of tandem dyes have been manufactured for the standard 488 nm laser, which is found in most flow cytometers. Tandem dyes are very useful for multicolor fluorescence studies especially in combination with single dyes. For example, Alexa Fluor®^ 488, Phycoerythrin, PerCP-Cy5.5 and PE-Cy7 can all be excited at 488 nm, but will produce green, yellow, purple and infrared emissions respectively, which can be measured using separate detectors.
One consideration to be aware of when performing multicolor fluorescence studies is the possibility of spectral overlap. When two or more fluorochromes are used during a single experiment there is a chance that their emission profiles will coincide, making measurement of the true fluorescence emitted by each difficult. This can be avoided by using fluorochromes at very different ends of the spectrum e.g. Alexa Fluor ®^ 405 and Phycoerythrin; however, this is not always practical. Instead, a process called fluorescence compensation 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. Figure 8 helps to explain the concept. The graphs show the emission profiles of two imaginary fluorochromes ‘A’ and ‘B’ which are being detected in FL-1 and FL-2 channels respectively. Because the emission profiles are so close together, a portion of fluorochrome A spills over into FL-2 (red shade) and conversely, some of fluorochrome B reaches FL-1 (dark blue shade). To calculate how much compensation needs to be applied to the dataset if both dyes are used simultaneously, some control readings must first be taken. Fluorochrome A should be run through the flow cytometer on its own and the % of its total emission that is detectable in FL-2 (spillover) determined. The procedure should be repeated with fluorochrome B, except that this time FL- is spillover. Suppose the results are:
This means that when the two fluorochromes are used for a dual-color experiment, the true reading for fluorochrome A in FL- = (total fluorescence measured in FL-1) minus (5% of fluorochrome B’s total fluorescence) Similarly, the true reading for fluorochrome B in FL- = (total fluorescence measured in FL-2) minus (17% of fluorochrome A’s total fluorescence) Fortunately, modern flow cytometry analytical software applies fluorescence compensation mathematics automatically, which simplifies matters considerably.
Spillover Fluorescence FL-1 FL- Fluorochrome A N/A 17% Fluorochrome B 5% N/A
Spectral properties of two imaginary fluorochromes, 'A' and 'B'. is measured in the FL-1 channel and in the FL- channel.
Wavelength
FL-
Fluores
cen
ce
A
B
A
B
A
B
FL-
Wavelength
Spectral overlap. Dark blue shade represents the proportion of that overlaps into the FL-1 channel. Red shade represents the proportion of that interferes with FL-2 channel measurements.
FL-
Fluores
cen
ce
A
B
FL-
FIGURE 8 Fluorescence compensation
On the density plot, each dot or point represents an individual cell that has passed through the instrument. Yellow/green hotspots indicate large numbers of events resulting from discreet populations of cells. The colors give the graph a three-dimensional feel. After a little experience, discerning the various subtypes of blood cells is relatively straightforward. Contour diagrams are an alternative way to demonstrate the same data. Joined lines represent similar numbers of cells. The graph takes on the appearance of a geographical survey map, which, in principle, closely resembles the density plot. It is a matter of preference but sometimes discreet populations of cells are easier to visualize on contour diagrams e.g. compare monocytes in Figure 9. Newer gating strategies utilize fluorescence parameters along with scatter parameters. Once again, blood can be used to demonstrate this principle.
Above on the left is a FSC/SSC plot for human lysed whole blood using smaller numbers of cells than in Figure 9. The lymphocytes, monocytes and granulocytes have been gated as region 1 (R1), region 2 (R2) and region 3 (R3), respectively. ‘Region’ simply refers to an area drawn on a plot displaying flow cytometry data. On the right the same cells are now plotted as SSC on the y-axis versus CD fluorescence on the x-axis. CD45 is a marker expressed on all white blood cells at varying intensities but is absent on red blood cells. In relative terms, lymphocytes have a low SSC and high CD45 count (R4), granulocytes have a high SSC and low CD45 count (R6), while monocytes are somewhere in between the other two (R5). The major difference between the lymphocytes
FIGURE 10 Lysed whole blood analysis using scatter and fluorescence
gated in R1 and those gated in R4 is the absence of red blood cells in the latter, making it a much purer preparation. This highlights the usefulness of gating strategies that combine a scatter parameter with a fluorescence parameter.
These are graphs that display a single measurement parameter (relative fluorescence or light scatter intensity) on the x-axis and the number of events (cell count) on the y-axis.
The histogram in Figure 11 looks very basic but is useful for evaluating the total number of cells in a sample that possess the physical properties selected for or which express the marker of interest. Cells with the desired characteristics are known as the positive dataset. Ideally, flow cytometry will produce a single distinct peak that can be interpreted as the positive dataset. However, in many situations, flow analysis is performed on a mixed population of cells resulting in several peaks on the histogram. In order to identify the positive dataset, flow cytometry should be repeated in the presence of an appropriate negative isotype control (see Figure 12).
FIGURE 11 A single-parameter histogram
