Week 2 Lecture 2 - Flow Cytometry

Question 1

3 Components of Flow Cytometry

Answer 1

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

Question 2

Fluidics

Answer 2

For accurate measurements particles must:
- be measured one at a time
- travel single-file to be interrogated by the laser -> interrogation point
Accomplished by injecting sample into sheath fluid as it passes through a small (50-300µm) orifice
Differential pressure between sheath fluid and sample fluid
- sample pressure higher than sheath
- sample flows in central core
- does not mix with sheath fluid

Question 3

Hydrodynamic Focussing

Answer 3

Can adjust sample pressure to change how fast/slow the sample is acquired
Low flow rate = small core stream -> sample runs slower
- good for smaller particles and DNA analysis
High flow rate = broader core stream -> sample runs faster
- data is less resolved as cells are less in line
- good for qualitative measurements such as immunophenotyping

Question 4

Excitation Optics and Collection Optics

Answer 4

Excitation optics
- lasers
- fiber optic cables = carry beams to steering prisms
- steering prisms = direct laser beams to the fluid stream
Collection optics = emission signal
- fiber optic cables = direct emitted light to appropriate emission block
- filters = direct signals in emission block to appropriate detectors

Question 5

Collection Optics: Filters

Answer 5

Emitted scattered light from blue laser
Sent via fiber optic cables
Measure 4 fluorescent parameters + SSC
Wavelengths separated by filters

Question 6

Electronics - Detectors

Answer 6

Signal is converted from photons into volts to be measured
Use photodiodes for forward scatter
Use photomultiplier tubes (PMTs) for fluorescence and side scatter
Steps:
1. As a particle passes through the laser and fluoresces it is detected by the PMT
2. An electrical pulse (or voltage) is generated
3. Voltage pulse height, width and area determined by fluorescence intensity, particle size and speed

Question 7

Photomultiplier Tubes

Answer 7

Amplifies signals for detection
Voltage applied changes the parameter/setup -> increases in log scale
The higher the PMT voltage (user controllable) the greater the output magnitude
However, higher voltages -> level of noise increases

Question 8

Side Scatter vs Forward Scatter

Answer 8

Forward
- detects size
- detector in line with laser path (opposite side of stream)
Side
- detects complexity
- light scattered to side