Flow Cytometry: Introduction Flashcards
What is flow cytometry?
A technique that simultaneously measures several physical characteristics of a cell in suspension. This is done by light scatter and fluorescence.
What is Fluorescence-Activated Cell Sorting (FACS)?
Sorting (separating) cells based on properties measured in flow
What can a flow cytometer tell us about a cell?
- Relative size
- Relative granularity
- Relative fluorescence intensity
Describe the key differences between flow cytometry and fluorescence microscopy.
- Microscopy can only visualise small numbers of cells (in a single field) at a time compared to flow cytometry.
- Flow cytometry can compare characteristics of multiple cell populations. This would be much harder to do with microscopy.
- In microscopy quantitation of fluorescence intensity is subjective and therefore imprecise compared to flow cytometry.
Describe the 3 basic components of a flow cytometer.
- Fluidics - cells in suspension flow in single-file
- Optics - illuminated cells scatter light and emit fluorescence
- Electronics - data is collected, filtered and converted to digital values which are stored on the computer
How is a single-file suspension of cells achieved?
- Inject cells into a sheath fluid as it passes through a narrow orifice (50-300uM)
- Sample fluid flows in a central core that does not mix with the sheath fluid - laminar flow
- Introduction of a large volume into a small volume - hydrodynamic focusing - ensures cells flow in single-file
What does light scatter tell us about size and granularity?
- Forward light scatter is proportional to size
- 90 degree light scatter is proportional to granularity
- We can measure size and granularity without fluorescence
What does a dot plot show?
- Different populations of cells cluster together based on size and granularity
- Cells appear as dots on graph
- Can identify different cell populations and their characteristics
Why does flow cytometry require filters and mirrors?
- The 4 different colours of light used have overlapping emission spectra
- Diverting light enables us to cut-out overlap and only collect light from the specific fluorochrome that is desired
- The 4 colours can then be quantified separately
What do the electronics do in the flow cytometer?
Change light into digital information - analog-digital conversion
Label a diagram of a flow cytometer.
![](https://s3.amazonaws.com/brainscape-prod/system/cm/229/450/602/a_image_thumb.png?1516012161)
Label a diagram of the channel layout in flow cytometry.
![](https://s3.amazonaws.com/brainscape-prod/system/cm/229/450/669/a_image_thumb.png?1516012193)
What is Stokes shift?
The energy difference between the lowest energy peak of absorbance and the highest energy of emission, measured in wavelength (nm) or frequency (hz) units.
Name 3 fluorochromes - immunofluorescent dye molecules.
- Fluorescein isothiocyanate (FITC) - green
- Phycoerythrin (PE) - orange
- Peridinin Chlorophyll Protein (PerCP) - red
Explain how and why combinations of fluorochromes are used together.
- The more fluorochromes expressed, the greater the number of cell populations possible.
- Gating can then be used to visualise different cell populations separately.
- Using combinations of fluorochromes allows us to compare the characteristics of more cell populations at the same time.