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
2
Q
What is the rate of cell measurement in a flow cytometer?
A
- 500 to 4000 cells per second
3
Q
What are the requirements for a flow cytometer?
A
- fluidics
- optics
- electronics
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
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
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
7
Q
What does the optical system consist of?
A
- an excitation source (usually laser) and data collection optics (photodetectors or photomultiplier tubes)
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
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
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
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
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
13
Q
What are the types of filters in a flow cytometer?
A
- dichroic mirrors (beam splitters)
- longpass filters
- shortpass filters
- bandpass filters
14
Q
What do dichroic mirrors do?
A
- allow light of a certain wavelength to be reflected while the remaining wavelengths can pass through
15
Q
What do longpass filters do?
A
- allow light ABOVE a specified wavelength through
16
Q
What does shortpass filters do?
A
- allow light BELOW a specified wavelength through
17
Q
What do bandpass filters do?
A
- only allows a specified range of light wavelengths through
18
Q
Photodiodes as detectors
A
- newer technology
- high efficiency for visible spectrum
- no adjustable gain
- requires cooling
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
What is 'gating'?
- series of subset extractions
- ability to select a population for analysis
- cells within the gate can be analysed for other parameters
26
Intrinsic parameters measured by flow cytometry
- no reagents or probes required
- cell size (forward light scatter)
- cytoplasmic granularity (90 degree light scatter)
- pigment content e.g. Hb
27
Extrinsic parameters measured by flow cytometry
- 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
Multi-colour flow cytometry
- increased lasers, thus increasing number of fluorochromes you can use
- increased number of required detectors
- higher cost
29
Why use Multi-colour flow cytometry?
- 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
Multiplex flow cytometry
- Luminex Technology
- Allows multiple analyses in one tube (Maximum 100)
- Utilises microspheres to which reagents can be bound to
31
Microspheres
- 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
Colour-coded microspheres
- Unique microsphere sets are colour-coded using a blend of different fluorescent intensities of two dyes
- 100 colour-codes means 100 simultaneous tests
33
How does Multiplex flow cytometry work?
- 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
Luminex
- 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
What is a cell sorter?
- 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
2 methods of cell sorting
- Electrostatic deflection of a stream in air
- Mechanical sorting within a flow cell
37
Electrostatic deflection
- 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
Coincidence
- 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
Anti-Coincidence gating
- 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
Accuracy of droplet charging
- 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
Phase gating
- 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
Mechanical sorting
- 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
Advantage and disadvantage of Dako Mo Flo
- Advantage: Analyzes and sorts cells at 70,000 cells per second
- Disadvantage: Cost £250,000
44
Fluorescence-activated cell sorting (FACS)
- 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
What makes FACS a useful instrument?
- provides fast, objective and quantitative recording of signals from
individual cells as well as physical separation of cells of particular interest
46
How does FACS actually work?
- 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
