Principles of Flow Cytometry Flashcards
What is a flow cytometer?
- an instrument capable of simultaneous measurement of multiple physical characteristics (size/granularity/fluorescence) of a single cell
What is the rate of cell measurement in a flow cytometer?
- 500 to 4000 cells per second
What are the requirements for a flow cytometer?
- fluidics
- optics
- electronics
What is the function of fluidics?
- to deliver the particles individually to a specific point
- carries and aligns the cells to pass aligned through the light beam
How is the delivery of cells in fluidics achieved?
- 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
How does hydrodynamic focusing work?
- 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
What does the optical system consist of?
- an excitation source (usually laser) and data collection optics (photodetectors or photomultiplier tubes)
Arc lamps as an excitation source
- 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
Laser as an excitation source
- 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
When does fluorescence occur?
- when a molecule is excited by light of one wavelength returns to the ground state by emitting light of a longer wavelength
Application of fluorochromes in flow cytometry
- 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
Two common fluorochromes
- 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
What are the types of filters in a flow cytometer?
- dichroic mirrors (beam splitters)
- longpass filters
- shortpass filters
- bandpass filters
What do dichroic mirrors do?
- allow light of a certain wavelength to be reflected while the remaining wavelengths can pass through
What do longpass filters do?
- allow light ABOVE a specified wavelength through
What does shortpass filters do?
- allow light BELOW a specified wavelength through
What do bandpass filters do?
- only allows a specified range of light wavelengths through
Photodiodes as detectors
- newer technology
- high efficiency for visible spectrum
- no adjustable gain
- requires cooling
What is forward scatter?
- detects scatter along the path of the laser
- bigger the cell the larger the forward scatter
What is side scatter?
- 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
Photomultiplier tubes (PMT) as detectors
- 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
Fluorescence detectors
- usually PMT
- detect the presence of Fluorochromes
- in flow cytometry, usually place behind filters which determine the fluorochrome they are detecting
Frequency histograms
- most common form of display
- a direct graphical representation of the number of events for each parameter analysed
Isometric display
- 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
What is ‘gating’?
- series of subset extractions
- ability to select a population for analysis
- cells within the gate can be analysed for other parameters
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
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
Multi-colour flow cytometry
- increased lasers, thus increasing number of fluorochromes you can use
- increased number of required detectors
- higher cost
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
Multiplex flow cytometry
- Luminex Technology
- Allows multiple analyses in one tube (Maximum 100)
- Utilises microspheres to which reagents can be bound to
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
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
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
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
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
2 methods of cell sorting
- Electrostatic deflection of a stream in air
- Mechanical sorting within a flow cell
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
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
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
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
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
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
Advantage and disadvantage of Dako Mo Flo
- Advantage: Analyzes and sorts cells at 70,000 cells per second
- Disadvantage: Cost £250,000
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
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
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