Flow Cytometry and Cell Sorting Flashcards
What is flow cytometry used for?
To count and analyse the size, shape and properties of individual cells within a a heterogeneous population of cells
What does flow cytometry measure ?
The simultaneous measurement of multiple physical characteristics including size/ granularity/fluorescence of a single cell
Measurements made on what basis?
Per cell - 500 to 4000 cells per second in a moving fluid stream
Is flow cytometry data quantitative or qualitative?
Quantitative
What are the several key components of a flow cytometer?
The sample, fluidics, lasers, optics, detectors and a computer system
What do the fluidics do in a flow cytometer ?
Move the sample into the flow cytometer
what does the fluidics rely on?
light/fluorochromes etc
How is this fluidics achieved?
By injecting sample (clean single cell suspension) into the centre opening the close channel through which sheath fluid is flowing
What does the laser do?
Produces a coherent, plane-polarised, intense, narrow beam of light which is Monochromatic
Limitations of Laser
- Expensive
- Difficult to replace
- Require servicing
What do the optics do?
Gather the light, excitation source
Optics consists of what?
an excitation source and data collection optics
Optics consists of what?
an excitation source and data collection optics
What’s an arc lamp?
Glass bulb with 2 electrodes
glass envelope containing a gas or vapour at high pressure
An initial high voltage spark between two electrodes creates a plasma arc
Maintainedby the application of high current at a low voltage
Limitations of arc lamp
- Prone to flicker
- Average life span of clamps are short
What do the detectors do?
Sense the light
What does the computer system do?
Outputs the data into a form that can be analysed by the researcher
electronics allow for the conversion of what?
optical signals into electronic signals for data analysis
what’s the purpose of electronic data?
analyse and converts/records the data as computers can’t read it
Principle of laser
Password two contains gas under pressure, fluoresces under application of a current
The light emitted is reflected along the tube
When these protons strike an atom in an excited state, they release what?
another proton of the same wavelength
When these protons strike an atom in an excited state, they release what?
another proton of the same wavelength
A small percentage of light goes through the system, where does it go from here?
through the prism and sends light back in of one photon
Properties of laser light
Coherent radiation at discreet wavelengths