What are the main benefits of flow cytometry?
- The simultaneous measurement of multiple physical characteristics of single cells, one at a time, in a cell suspension
- High throughput
- Measurements are made on a per cell basis at rates typically in the order of 500 to 20,000 cells per second in a moving fluid stream
- Alot of data very quickly, allowing you to find very rare cells
What are the 3 requirements for flow cytometry?
- Fluidics
- Optics
- Electronics
What is the order of events in a flow cytometer?
Laser - Fluid - Fluorescence - Filters - Electronics - Quantification
Describe the concept of fluidics in flow cytometry
- Delivery of individual particles/cells to a specific point intersected by a laser beam
What is Sheath Fluid?
Sheath fluid is filtered isotonic saline, or phosphate buffered saline, to allow cells to keep there morphology.
Sheath fluid ensures laminar coaxial flow.
Name some examples of samples that could be used in flow cytometry
- White blood cells
- Processed tissue
- Cells in culture
- Bone Marrow
- Beads
- Bacteria
What is the issue of using whole blood in Flow cytometry?
There is too many red blood cells in a whole blood sample which will affect the result , therefore red blood cells are lysed in a salt solution.
Describe the Bernoulli Effect.
When the velocity of the fluid is slower on the sides of the tube due to drag. The fluid moves towards low pressure. This means that there is no turbulence in the flow and that the cells are single file.
What velocity is needed to ensure laminar flow and hydrodynamic focusing?
Approximately 10 meters/sec. Meaning 10 micron particles will transverse there own diameter in 1 microsecond.
What are the 4 components of the optics in a flow cytometer?
- Excitation Source
- Fluorescence
- Collection optics - filters/mirrors
- Detectors
Why do we use LASERs in flow cytometry?
- Produce a coherent, plane polarized, intense, narrow beam of light
How does a LASER work?
Plasma tube contains a gas under pressure which fluoresces under the amplification of the current. When the photons strike an atoms in an excited state they release another photon of the same wavelength.
What is the physics of fluorescence?
Excitation of a fluorochrome - when it receives the excitation its energy state increases and then very rapidly drops the energy state and at that point emits light at a longer wavelength.
Why use Tandem dyes?
Individual fluorochromes that are bound together can broaden the scope creating different wavelengths.
What are the 3 components of the optics in a flow cytometer?
- Beam mixer - takes lasers from multiple sources into one specific direction
- Filters/mirrors - angle light to the detectors often for specific wavelengths
- Detectors - Photodiodes, Photomultiplier tubes (PMT)
What are the 4 types of Filters/mirrors?
Dichroic mirrors - Allow light of a certain wavelength to be reflected while the remaining wavelength passes through
- Shortpass filters - Allow a light below a specific wavelength through
- Bandpass filters - Only allow a range of wavelengths through
- Longpass filters - Allow a light above a specific wavelength through
What are the positives and negatives of PMTs?
- Most common detector
- Adjustable
- High Sensitivity
- Poor efficiency in red
- Inexpensive
What are the positives and negatives of photodiodes?
- Newer technology
- High efficiency
- No adjustable sensitivity
What are the 3 steps of the electronics system in flow cytometry?
Amplification - Analog to digital converter - Computer system
What is compensation?
Spectral overlap can occur so compensation removes cells fluorescing in the wrong channel
What does Forward Scatter show (FS)
Size of the cells
What does Side Scatter show (SS)
Internal structures of the cells i.e. granularity
Dead cells can also have a high side scatter due to there rough surface
What are the two ways to display the results from flow cytometry?
- Frequency histogram
- Dot Plot
What do the locations on a dot plot show?
Upper left -+
Upper right ++
Lower left –
Lower right +-
