Lecture 4 - Flow Cytometry Flashcards
What is flow cytometry?
Use of focused light (lasers) to interrogate cells delivered by a fluidics system
Why do we use flow cytometry?
- Allows isolation of specific cell sub-populations
- Essential for diagnostics and research
- Analyses individual cells
Parameters that the flow cytometer takes into account
- Size
- Granularity
- Surface molecules
- Cytoplasmic & nuclear molecules
FACS
- Fluorescence Activated Cell Sorting
- Discovered by Lou Herzenberg in 1972
Components of flow cytometer
- Fluidics
- Optics
- Electronics
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
How fluidics works
- 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
Components of fluidics system
Sheath flow:
sheath tank»_space; sheath filter»_space; sheath interior reservoir»_space; bubble filter
Sample flow:
test tube»_space; sample injection tube (SIT)
Sheath flow and sample flow merged together:
Flow cell»_space; interrogation point (laser works here)»_space; waste interior reservoir»_space; waste tank
How optics works
Consist of:
- Lasers (BD Canto II can have 3 lasers)
- Fiber optic cables = carry beams to steering prisms
- Steering prisms = direct laser beams to the fluid stream
Collection optics = Direct emitted light that will be processed as useful data
Collection optics consists of:
- Fiber optic cables = direct emitted light to appropriate emission block
- Filters = direct signals in emission block to appropriate detectors
Detectors (in optics)
- 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
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
Summary of fluidics
- Sample = single cells = test tube
- Liquid drawn up & pumped into flow chamber
- Cells flow through flow chamber - one at a time
500-2000 cells per second
Summary of optics
- Laser beam of bright light hits cells
- Light bounced off each cell = information about the cell
- Fluorochromes absorb light & emit specific color
- Filters - send emitted light to color detectors
- Light detector: Processes light signals & sends information to the computer
- Color detectors: Collect different colors of light emitted by fluorochromes
Summary of electronics
Computer – data from the light detector and the color detectors
Forward scatter channel = size
- 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)
- Proportional to cell size: Bigger cell = more light scattered = higher detected signal
Side scatter channel = complexity
- 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
- SSC proportional to cell complexity: More organelles = more light scatter = higher detected signal
FACS: size & granularity
Light scatter:
- Differentiation of size = FSC
- Differentiation of complexity (granules) = SSC
- Each dot = one event = one cell
Fluorescent labelling
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
- Each fluorescent compound has its own emission wavelength
- Fluorescein isothiocyanate (FITC) emits light at ~530nm when excited by a 488nm (blue) laser
Fluorescence
- Some molecules absorb light energy»_space; higher energy state = excited state
- The energy of the excited state, “decays” or decreases»_space; emission of light energy
- This process is called fluorescence
- To “fluoresce” means to emit light via this process
Fluorophore in ground state
- A fluorophore is a molecule that is capable of fluorescing
- In its ground state, it is in a low-energy, stable configuration»_space; does not fluoresce
- ground state = low energy
Absorption of light
• When light from an external source hits the fluorophore»_space; absorbs the light energy
- happens in ground state (low energy)
Excitation
- If the energy absorbed is sufficient»_space; higher-energy state, called an excited state.
- This process is known as “excitation”
Energy loss (after excitation)
• Fluorophores are unstable at high-energy configurations» lowest-energy excited state, which is semi-stable
Emission
• Fluorophores then rearrange from semi-stable excited state»_space; ground state
» excess energy emitted as light
• Light energy emitted is of a longer wavelength than light energy absorbed, due to energy lost during the transient excited lifetime
- energy drops down to almost ground state
Visible light spectrum
• Fluorophores absorb a range of wavelengths of light energy, and also emit a range of wavelengths
• Within these ranges are the excitation maximum and the emission maximum
» absorbed and emitted light are different colors on the visible spectrum
Visible light spectrum (ranges and sections)
400nm: short wavelength, high frequency
700nm: long wavelength, low frequency
Violet: 400 - 455 nm Blue: 455 - 492 nm Green: 492 - 577 nm Yellow: 577 - 597 nm Orange: 597 - 620 nm Red: 620 - 700 nm
Alexafluor graphs
- point of excitation is at the wavelength written in the title of the graph, and point of emission is slightly afterwards
Fluorescent labeling
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
The importance of controls (flow cytometry)
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
- after this, multiple stained cells can be run
Gating
Stained with monoclonal antibodies against:
- CD45 labelled with FITC
- CD3 labelled with PE-Cy5 (gated on lymphocytes)
- CD4 labelled with PE
What properties of a cell or particle can be measured by a flow cytometer?
- Size
- Granularity
- Surface molecules
- Cytoplasmic & nuclear molecules
What light source is used in most flow cytometers?
- focused light (lasers)
- LASER = Light Amplification by Stimulated Emission of Radiation
- fluorescent light
What are the 3 main systems in a flow cytometer?
- Fluidics
- Optics
- Electronics
Light emitted from a particle is collected by:
.
Fluorescent compounds have defined absorption and _____________ spectra
emission
Spectral spillover is corrected by using:
.
(T/F) Particles must be in single-cell suspension before flow cytometric analysis
True
(T/F) A dot plot can be used to display two parameters.
True
(T/F) A gate can be used to restrict the analysis to a specific population within the sample
True
