Flow Cytometry - Introduction Flashcards
What is flow cytometry?
A technique that simultaneously measures several physical characteristics belonging to a single cell in suspension. This is done by light scatter and fluorescence.
What are the advantages of flow cytometry?
- Integrate cells and get information about many different characteristics all at the same time
- Very quick
Define flow cytometry
Measuring properties of cells in flow for example, how many CD4 cells are there in a sample of blood.
What is flow sorting?
Sorting (separating) cells based on properties measured in flow also called fluorescence-activated cell sorting (FACS) for example, separate the CD4 cells from the rest.
What can a flow cytometry tell us about a cell?
- About the relative sound
- About the relative granularity/internal complexity
- About the relative fluorescence intensity
What can be measured using flow cytometry?
- Adhesion
- DNA
- Cytokines
- Enzymes
- Surface Receptors
- Also apoptosis
What are the methods of visualisation for cells?
- Fluorescence Microscopy
- Flow Cytometry
How does a fluorescence microscopy work?
- Fluorescence is attached to the CD4
- Limited no. of cells in each field (20 cells per field)
- Not quantitative compared to flow cytometry that looks at thousands of cells per second
Difference between fluorescence microscopy and flow cytometry
Flow Cytometry:
- Lots of cells at once
- Quantitative
- An accurate view of the intensity
Fluorescence Microscopy:
- Limited No. Cells in each field
- Not quantitative
- Varied intensity subjective
What are the 3 components of flow cytometry?
- Fluidics: cells in a single cell suspension
- Optics: an illuminated volume where they scatter light and emit fluorescence that is collected and filtered
- Electronics: The fluorescence is then converted to digital values that are stored on a computer.
Describe the flow cytometry analyser
- The light source then to,
- Flow Chamber then to the,
- The optical system then to the,
- Light detectors and lastly the computer.
What are the fluidics of flow cytometry?
- Need to have cells in suspension flow in single file
- Accomplished by injecting the sample into a sheath fluid as it passes through a small (50-300 um) orifice.
- Sample fluid flows in a central core that does not mix with the sheath fluid; this is laminar flow
- Introduction of a large volume into a small volume caused by hydrodynamic focusing.
How does the nozel tip work in the flow cytometry?
The cells are forced to flow in a single file through an orifice because of the sheath fluid by hydrodynamic focusing.
Describe how flow cytometry works
- Single file cell
- The laser hits cells and light is scattered
- The light is picked up by detectors
What are the light sources of optics in flow cytometry?
Lasers
What lasers are used in flow cytometry?
- The single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
- Can provide from milliwatts to watts of light
- Can be inexpensive, air-cooled units or expensive, water-cooled units
- Provide coherent light (single frequency)
- Generally, FC lasers are a single wavelength of light or a 488nm laser.
Describe how a laser scatters light
- The laser hits the cell at 488 nm.
- The light is scattered in the forward direction which is proportional to the size of the cell
- Side scatter; 90o Light Scatter proportional to the granularity or the internal complexity
What does a white cell population dot plot show? When is a white cell population dot plot used?
- X-axis: increase in forward scatter
- Y-axis: increase in size scatter
Every dot represents a cell forward scatter and side scatter shows a distinct correlation.
This technique is used to quantitative different populations of cells. This is done on peripheral blood.
When is a channel layout for laser-based flow cytometry used?
It is used for antibodies and fluorescence.
- The laser hits cells and cells are labelled with (4) different antibodies and colours.
- Light is emitted and picked up by photomultiplier tubes (PMT)
- This happens after the light has gone through filters and mirrors. This is needed because of the emission change.
- It converts analogue into digital
Why are electronics needed?
To process signals from detectors
- Analog-Digital Conversion
What is the Stokes shift?
The energy difference between the lowest energy peak of absorbance and the highest energy of emission.
What is FITC?
The most commonly emitted fluorochrome Fluorescein isothiocyanate (FITC) at the green energy peak
How does fluorescence occur?
Fluorescence occurs when a fluorochrome is excited by a laser and goes back to the unexcited state. This is the difference between the two peaks is the stoke shift.
What causes fluorescent energy peaks to overlap in a histogram?
Mirror and filters cause overlapping histograms
