FACS Flashcards
What is flow cytometry?
A laser-based lab test that can detect chemical and physical differences of cells or particles.
What is FACS?
Fluorescence Activated Cell Sorting
What are the two things FACS allows us to do with a sample?
Collect data on and sort our sample.
What are the three core systems of the Flow Cytometer
- Fluidics
- Optics
- Electronics
How are cells/particles carried to the laser intercept/light beam?
The fluidics system
Sheath fluids carry the cells/particles in a single file fashion.
What is the function of the fluidics system?
Fluidics system is responsible for transporting sample from the sample tube to the flow cell (and past the laser and detector).
What is the function of hydrodynamic focussing in the fluidics system?
Hydrodynamic focussing uses water to force the cells to travel at the same speed and to travel as single cells.
What makes up the fluidics system?
Flow cell
Sheath fluid
Droplet generator
Nozzle
What is the optics system of flow cytometry used for?
Responsible for illuminating cells and detecting the light signals they emit.
What are the components of the optics system of flow cytometry?
- Lasers
- Interrogation Point
- Optical filters
- Beam splitters
- Lenses
- Detectors
- Mirrors
What does the optics system convert?
Emitted photons into an electrical signal, a photocurrent for the electrical system.
What does the laser from the optics system do?
Provide focused light beams that excite fluorescent molecules (fluorophores) within the cells as they pass through the interrogation point.
What are the main two components making up the electronics system?
- Photo diodes
- Photo Multiplier Tubes (PMTs)
What is the role of the electronics system?
Responsible for digitising and processing the photocurrent from the detector for analysis.
What are the three main parameters measured in FACS?
- Forward light scatter (FSC)
- Side light scatter (SSC)
- Fluorescence emission signals
How does forward light scatter separate cells?
Based on particle/cell size
More forward light suggests bigger cell.
How does side scattered light separate cells?
Based on granularity and complexity of cells
How do fluorescence emission signals separate cells?
Based on presence or absence of specific proteins
How do fluorescence emission signals work?
Fluorophore (fluorescent dye) is used to label the protein of interest.
Cells pass through laser and dye absorbs light of a specific excitation wavelength.
Then emits light at a different emission wavelength
Fluorescing cell possesses interrogation point and creates pulse of photon emission.
What is this detected by?
And what do they convert the light signal into?
Photon multiplier tubes
Convert light signal into a small electrical signal (a voltage pulse)
Do the voltage pulse area and fluorescence intensity correlate?
Yes, the higher the fluorescence intensity, the higher the pulse area.
What is the purpose of channel numbers?
To help categorise and display different fluorescence levels.
How can we boost the intensity of and detect weaker signals?
Increasing the voltage
Why do we put cells in a cell suspension during sample preparation/
To avoid clogging the system with clumps.
What is flow cytometry’s greatest advantage?
Speed
Can sort 1000s of cells in seconds
What happens at the point of interrogation?
Cells pass through in a single stream.
Laser interacts with cells.
Light scatter and fluorescence signals are generated here.
What captures the scattered light?
Collection lenses
What do beamsplitters and filters in the optical system do?
Filters are used to select specific wavelengths.
Beamsplitters can direct light paths to different detectors.
How is flow cytometry data usually represented?
Via histograms or dot plots
What are isotope controls?
Primary antibodies lacking specificity to the target but match the class and type of the primary antibody used in the application.
Negative control to help differentiate non-specific background signal from specific antibody signal.
