List the optical components of a flow cytometry system
Excitation source
Flow cell/ chamber
Collection optics: filters and mirrors
Detectors
Summarise the types and functions of filter systems used for light signal manipulation before detection in a flow cytometer
Dichroic mirrors reflect only a fixed wavelength range but allow all other wavelengths through;
Shortpass filters
block wavelengths above a fixed threshold;
Longpass filters block wavelengths below a fixed threshold;
Bandpass
filters allow through wavelengths of a specific range (X±Y nm)
Identify the appropriate sensor (explain what features can be analysed by these) type for each kind of light signal
Photodiodes are good for forward scatter and side scatter measurement of high intensity source light where tunable
signal gain is redundant, to measure size and granularity of cells.
Photomultiplier (PMT) tubes are good for weak
fluorescent signal detection amplification e.g. from fluorophore labels for surface antigens, because of signal
amplification ability and tunable sensitivity
Give the 3 requirements for a flow cytometer
- Fluidics- system used to deliver particles individually to a specific point
- Optics- contains an excitation source, flow cell and data collection points
- Electronics- allow conversion of optical signals into electronic signals for data analysis
What is the excitation source in flow cytometry?
LASER- Light amplification by Stimulated Emission of Radiation)
Produces a coherent, plane-polarised, intense narrow beam of light
The light is monochromatic
How does LASER work?
Plasma tube contains gas under pressure which fluoresces under a current
Light emitted is reflected along the tube
When photons strike an atom in an excited state they release another photon of the same wavelength
Give the 2 common fluorochromes
FITC- Fluorescein Isothiocyanate
R-phycoerythrin (PE)
What are PMTs?
Photomultiplier Tubes
Most common detector in flow cytometry
High sensitivity but poor efficiency in red
Adjustable gain
Inexpensive
What is the role of PMTs?
- Detect light
- Amplify signals
What is a photodiode?
Newer technology
High effciency for vidsible spectrum
No adjustable
How is flow cytometry analysed?
Most common forms of display are frequency histogram and dotplot
Histogram- direct graphical representation of the number of events for each parameter
Dotplot- one parameter plotted against another for a single cell
What is gating?
the ability to select a population for analysis
Cells within the gate can be analysed for other parameters
What differences does Multi-colour flow cytometry have to normal flow cytometry?
More:
detectors, lasers, fluorochromes, information and data, expensive
Why would you use multi-colour flow cytometry?
Meaures many analytes in solution simultaneously
Use smaller precious specimens
Save time and reagents
Capable of collecitng large numbers of events more efficiently
What is a Luminex-ProcartaPlex?
Luminex, multiplex flow cytometry technology
Allows multiple analyses in one tube
Utilises macro spheres to which reagents can be bound to
What are macrospheres?
Utilises approx 6um polystyrene
Each batch is dyed with a uniqu combination of red and infre-red fluorochromes to give a unique signature
Allows definition of up to 100 beads
What is cell sorting?
A flow cytometer with added ability to physically sperate out a population described by a gate
Allows populations of cells or particles to be separated from the sample
How does cell sorting work?
Cells are injected in a flow cell
Drop generation is released then broken into droplets of 30-100,000 droplets/second
Drop is charged with a particle of interest
Charge drop is deflected then separated into waste and collection tube
How does electrostatic deflection work?
Hydrodynamic focusing in a nozzle, vibrates breaking stream into droplets
Laser interrogation and signal processing, followed by sort decision
Electronic delay until cell reaches its break of point
Charged droplets deflect by electrostatic field from plates held at ahigh voltage
Various collection devices can be attached
