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AP0609 - Advanced Analytical Techniques > Principles of Flow Cytometry > Flashcards

Principles of Flow Cytometry Flashcards

1
Q

What is a flow cytometer?

A
  • an instrument capable of simultaneous measurement of multiple physical characteristics (size/granularity/fluorescence) of a single cell
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2
Q

What is the rate of cell measurement in a flow cytometer?

A
  • 500 to 4000 cells per second
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3
Q

What are the requirements for a flow cytometer?

A
  • fluidics
  • optics
  • electronics
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4
Q

What is the function of fluidics?

A
  • to deliver the particles individually to a specific point
  • carries and aligns the cells to pass aligned through the light beam
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5
Q

How is the delivery of cells in fluidics achieved?

A
  • by injecting the sample (clean single cell suspension) into the centre of an enclosed channel through which sheath fluid (carrier fluid which is filtered isotonic saline) is flowing
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6
Q

How does hydrodynamic focusing work?

A
  • when two streams of fluids with different flow rates are running side-by-side and in the same direction into a flow cell, then a laminar flow is created
  • sheath fluid pressure is constant while sample fluid is adjusted, manipulating the pressure differences gets the desired cross-sectional area (i.e., the diameter of a cell)
  • properly aligns your cells, one by one, at the junction where the analysis by lasers begins
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7
Q

What does the optical system consist of?

A
  • an excitation source (usually laser) and data collection optics (photodetectors or photomultiplier tubes)
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8
Q

Arc lamps as an excitation source

A
  • glass envelope containing a gas or vapour at high pressure
  • initial high voltage spark between 2 electrodes creates a plasma arc
  • plasma arc is maintained by application of high current at a low voltage
  • prone to flicker and average life of arc lamps is short
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9
Q

Laser as an excitation source

A
  • produces a coherent, plane-polarised, intense, narrow beam of light
  • the light is monochromatic
  • expensive
  • plasma tube contains gas under pressure which fluoresces under the application of current
  • the light emitted is reflected along the tube
  • when these photons strike an atom in an excited state they release another photon of the same wavelength
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10
Q

When does fluorescence occur?

A
  • when a molecule is excited by light of one wavelength returns to the ground state by emitting light of a longer wavelength
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11
Q

Application of fluorochromes in flow cytometry

A
  • the cells can be stained (the cell will bind a Fluorescent Dye)
  • And/or a fluorochrome conjugated with an antibody in an amount proportional to the quantity of the Binding Constituent (eg, DNA, RNA, Surface antigen)
  • The cell’s emitted fluorescence INTENSITY will then be PROPORTIONAL to the fluorescing CELLULAR CONSTITUENT
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12
Q

Two common fluorochromes

A
  • FITC: bright, absorption maximum close to emission lines from both the argon laser and a mercury arc lamp
  • R-phycoerythrin: can be excited at 488nm so only one laser required
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13
Q

What are the types of filters in a flow cytometer?

A
  • dichroic mirrors (beam splitters)
  • longpass filters
  • shortpass filters
  • bandpass filters
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14
Q

What do dichroic mirrors do?

A
  • allow light of a certain wavelength to be reflected while the remaining wavelengths can pass through
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15
Q

What do longpass filters do?

A
  • allow light ABOVE a specified wavelength through
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16
Q

What does shortpass filters do?

A
  • allow light BELOW a specified wavelength through
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17
Q

What do bandpass filters do?

A
  • only allows a specified range of light wavelengths through
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18
Q

Photodiodes as detectors

A
  • newer technology
  • high efficiency for visible spectrum
  • no adjustable gain
  • requires cooling
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19
Q

What is forward scatter?

A
  • detects scatter along the path of the laser
  • bigger the cell the larger the forward scatter
20
Q

What is side scatter?

A
  • measures scatter at a ninety-degree angle relative to the laser
  • provides data on internal structures; more internals structures – higher SS
  • data on cell surface characteristics; dead cells have a rougher surface – higher SS
21
Q

Photomultiplier tubes (PMT) as detectors

A
  • detect light
  • amplify signal so good for the detection of weak fluorescence
  • most common detector in flow cytometry
  • old well characterised technology
  • high sensitivity but poor efficiency in red (>650nm)
  • adjustable gain (sensitivity)
  • inexpensive
22
Q

Fluorescence detectors

A
  • usually PMT
  • detect the presence of Fluorochromes
  • in flow cytometry, usually place behind filters which determine the fluorochrome they are detecting
23
Q

Frequency histograms

A
  • most common form of display
  • a direct graphical representation of the number of events for each parameter analysed
24
Q

Isometric display

A
  • obtained by plotting the density or contour plot in 3D
  • the Z axis is now used to plot the frequency of events
  • can be tilted or rotated to provide clear viewing angles
25
Q

What is ‘gating’?

A
  • series of subset extractions
  • ability to select a population for analysis
  • cells within the gate can be analysed for other parameters
26
Q

Intrinsic parameters measured by flow cytometry

A
  • no reagents or probes required
  • cell size (forward light scatter)
  • cytoplasmic granularity (90 degree light scatter)
  • pigment content e.g. Hb
27
Q

Extrinsic parameters measured by flow cytometry

A
  • reagents are required
  • Structural: DNA content, DNA base ratios, RNA content
  • Functional: Surface and intracellular receptors, DNA synthesis, DNA degradation(apoptosis), Cytoplasmic Ca++, Gene expression
28
Q

Multi-colour flow cytometry

A
  • increased lasers, thus increasing number of fluorochromes you can use
  • increased number of required detectors
  • higher cost
29
Q

Why use Multi-colour flow cytometry?

A
  • More accurate population identification
  • Use smaller specimens as more parameters are available to test in one tube
  • Save time and reagents as fewer tubes are required to be tested
  • Capable of collecting large number of events more efficiently
30
Q

Multiplex flow cytometry

A
  • Luminex Technology
  • Allows multiple analyses in one tube (Maximum 100)
  • Utilises microspheres to which reagents can be bound to
31
Q

Microspheres

A
  • Utilises 5.6 μm polystyrene microspheres
  • Each microsphere is dyed with a combination of red and infra-red fluorochromes
  • This allows the definition of 100 different beads
  • Simple surface chemistry allows the coupling of antibodies, antigens,peptides, oligonucleotides or receptors
32
Q

Colour-coded microspheres

A
  • Unique microsphere sets are colour-coded using a blend of different fluorescent intensities of two dyes
  • 100 colour-codes means 100 simultaneous tests
33
Q

How does Multiplex flow cytometry work?

A
  • 2 lasers are used
  • precision fluidics align the microspheres in single file, and pass them through the lasers one at a time
  • one lase excites molecular tags; reactions are measured with fluorescent intensity and reported in real time
  • other laser excites microsphere; fluorescent intensity of microsphere identifies the reaction
34
Q

Luminex

A
  • Beads are incubated with sample
  • Beads are washed before addition of PE reporter
  • Samples are analysed on Luminex
  • Luminex has 96 well plate capability so high throughput is possible
35
Q

What is a cell sorter?

A
  • flow cytometer with the added ability to physically separate out a population described by a gate
  • E.g. a flow cytometer is capable of measuring the CD4 population of Lymphocytes, a cell sorter would also be capable of separating this population into a new tube
36
Q

2 methods of cell sorting

A
  • Electrostatic deflection of a stream in air
  • Mechanical sorting within a flow cell
37
Q

Electrostatic deflection

A
  • Hydrodynamic focusing in a nozzle vibrated by a transducer produces a stream breaking into droplets
  • Laser interrogation and signal processing followed by sort decision: white sort right, blue sort left, green or yellow no sort
  • Electronic delay until cell reaches break off point. Then the stream is charged :+ if white, - if blue
  • Charged droplets deflect by electrostatic field from plates held at high voltage (+/- 3000 volts)
  • Various collection devices can be attached :tubes, slides, multi-well plates
38
Q

Coincidence

A
  • At high sample event rates the possibility exists that cells not fulfilling the criteria maybe sorted
  • This occurs if two or more cells are detected in the time frame of droplet formation
  • Anti-Coincidence gating can be used to prevent this
39
Q

Anti-Coincidence gating

A
  • works by creating a time window around the particle of interest relating to droplet formation
  • If any other partial is detected in this window then the stream is directed to waste
40
Q

Accuracy of droplet charging

A
  • Although droplet formation is a stable process it can be effected by sheath temperature or sheath pressure (form faster of slower)
  • may lead to the charging pulse not being delivered to the correct droplet
  • To overcome this it is common to charge more than one (2 or 3) droplet
  • can decrease purity without anti-coincidence gating (enrich mode), or decrease yield with anti-coincidence gating on
41
Q

Phase gating

A
  • Determines if cell is in the centre or outside quarters of the droplet window
  • If the cell is not in the centre, the system can sort 2 drops rather than 1 to ensure recovery
  • Or the centre only collection can be applied when maximum purity is required
42
Q

Mechanical sorting

A
  • hydrodynamic focusing and interrogation takes place in a flow cell
  • when a sort decision is made, a ‘catcher’ tube moves into stream to collect the cell
43
Q

Advantage and disadvantage of Dako Mo Flo

A
  • Advantage: Analyzes and sorts cells at 70,000 cells per second
  • Disadvantage: Cost £250,000
44
Q

Fluorescence-activated cell sorting (FACS)

A
  • specialised type of FCM
  • sorting a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell
45
Q

What makes FACS a useful instrument?

A
  • provides fast, objective and quantitative recording of signals from
    individual cells as well as physical separation of cells of particular interest
46
Q

How does FACS actually work?

A
  • cell suspension is entrained in the centre of a narrow stream of liquid
  • the flow is arranged so that there is a large separation between cells relative to their diameter
  • a vibrating mechanism causes the stream of cells to break into individual droplets
  • Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character
    of interest of each cell is measured
  • Charge is applied directly to the stream, and the droplet retains
    charge of the same sign as the stream; the stream is then returned to neutral.
  • The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge

AP0609 - Advanced Analytical Techniques (10 decks)

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