W2L4 - Flow Cytometry Flashcards
Flow Cytometry
Use of focused light (lasers) to interrogate cells delivered by a fluidics system
LASER = Light Amplification by Stimulated Emission of Radiation
Single cell basis
Allows isolation of specific cell subpopulations
Characterisation of different cell-associated parameters
- size
- granularity
- surface molecules
- cytoplasmic & nuclear molecules
What does FACS stand for?
Fluorescence Activated Cell Sorting
Basics of Flow Cytometry
Fluidics - cells in suspension - flow single-file - focuses the cells for 'interrogation' Optics - generates light signals - scatter light and emit fluorescence - light collected & filtered Electronics - processes optical signals - converts them to proportional digital values - stored on a computer
Fluidics
For accurate measurements cells must: - be measured one at a time - travel single-file through a stream at the point of laser interrogation Accomplished by injecting sample into sheath fluid as it passes through a small (50-300µm) orifice When conditions right sample fluid : - flows in central core - does not mix with sheath fluid - Hydrodynamic focussing
Flow Chamber Blockage
A single human hair Salt crystals Sample protein Beads Large or 'sticky' cells These block flow cell channel and disrupt flow
Optics
Excitation optics = generate excitation photons
Consist of:
1. Lasers (BD Canto II can have 3 lasers)
2. Fiber optic cables = carry beams to steering prisms
3. Steering prisms = direct laser beams to the fluid stream
Collection optics = direct emitted light that will be processed as useful data
Consist of:
1. Fiber optic cables = direct emitted light to appropriate emission block
2. Filters = direct signals in emission block to appropriate detectors
Detectors
Light must be converted from photons into volts to be measured
Use photodiodes for forward scatter
Use photomultiplier tubes (PMTs) for fluorescence and side scatter
Blue laser signals
- emitted scattered light from blue laser
- sent via fiber optic cables
- different light wavelengths separated by filters
- collected by appropriate detectors
Photomultiplier Tube
Amplifies signal for detection
Voltage applied to the dynodes changes the parameter/setup
Increases in log scale
Voltage applied also linked to compensation setup
Forward Scatter
Proportional to cell size
- bigger cell = more light scattered = higher detected signal
Majority of photons pass through stream unobstructed
Some photons contact cell membranes & diverge from their path
Light scattered in forward direction
Detector in line with laser path (opposite side of stream)
”Scattered” light collected in Forward Scatter channel (FSC)
Side Scatter
Proportional to cell complexity
- more organelles = more light scatter = higher detected signal
Cells translucent
Many photons pass through cytoplasm
Photon strikes organelle = photon reflected > angle than generated by FSC
Light scattered to side (perpendicular to axis laser light is traveling)
Detected in the side scatter channel by Side Scatter (SSC) detector
Fluorescence
Absorption of light:
- causes an electron in the fluorescent compound to be raised to a higher energy
- level = excitation
The excited electron:
- quickly decays to its ground state
- emits the excess energy as a photon of light
- this transition of energy is called fluorescence
Fluorescence Steps
A fluorophore is a molecular capable of fluorescing
1. In its ground state, it’s in a low energy, stable configuration -> does not fluoresce
2. When light from an external source hits the fluorophore -> absorbs the light energy
3. If the energy absorbed is sufficient -> higher-energy state, called an excited state (known as excitation)
4. Fluorophores are unstable at high-energy configurations -> lowest-energy excited state, which is semi-stable
5. Fluorophores then rearrange from semi-stable excited state -> ground state
- excess energy emitted as light
6. Light energy emitted is of a longer wavelength than light energy absorbed, due to
energy lost during the transient excited lifetime
Fluorescent Labelling
Cells pass through stream
Laser light excites fluorescent tag
Emit photons of light at higher wavelength
Fluorescence emitted by each fluorochrome
Detected in a wavelength-specific detector
Recorded as voltages for analysis
Importance of Controls
Must have unstained cells - check for autofluorescence - set up negative area Must have single stained cells - check for spillover - set up compensations - set up positive regions
Compensation
Compensation is required to fix spectral spillover when using 2 or more fluorophore
