Flow Cytometry - Introduction and applications Flashcards
What is flow cytometry?
Flow cytometry = A technique used to simultaneously measure several physical characteristics of a single cell in suspension.
Done by light scatter and fluorescence.
What is the definition of flow cytometry?
Measuring properties of cells in flow
What is the definition of flow sorting ?
Sorting (separating ) cells based on properties measured in flow.
aka Fluorescence-Activated Cell Sorting (FACS)
What information can flow cytometer tell us about a cell?
- Relative size
- Relative Granularity/Internal Complexity
- Relative fluorescence intensity (using markers)
What markers can be measured ?
- Cell surface receptors
- Intercellular cytokines
- DNA-Apoptosis/cell cycle/viability
What are two methods of visualisation?
- Flow cytometry
- Fluorescence Microscopy
What technique can we use to quantitate cells?
Flow cytometry - quantifies fluorescence
How does a flow cytometry work?
Fluidics :
-Cell in suspension flow through in a single file.
Optics:
- Cells are hit by laser
- An illuminated volume where they scatter light and emit fluorescence that is collected and filtered.
Electronics:
Light signals are converted into digital values that are stored on a computer
(Then analysed later)
Outline the different processes involved in flow cytometry
Light source Flow chamber Optical system Light detectors Computer
Describe the Fluidics phase
- Cells must be in suspension and flow in single file. Achieved by:
- The sample is injected into sheath fluid through a small orifice (opening)
- Sample fluid flows in a central core that does not mix with the sheath fluid (Laminar flow)
- Introducing a large volume into a small volume (Hydrodynamic Focusing)
slides 12 + 13
Describe the Light source for the Optic stage.
Light source = laser:
- Single wavelength of light (laser) /mix of wavelengths (rare)
- Can provide milliwatts-watts of light
- Inexpensive air-cooled units/Expensive water cooled units
- emit coherent light (single frequency)
Describe light scatter
Light scatter occurs when light hits the cell.
- Most common wavelength of laser is 488nm.
- The light becomes scattered in a forward direction (FSC) and this is proportional to cell size
- Light is also emitted at a 90 degree angle to cell
- 90 degrees light scatter proportional to granularity of cell = SSC Side scatter
no fluorochromes, just laser hitting cell
Describe dot plot
X axis = forward scatter (size)
Y axis = side scatter (granularity )
Each dot is a cell.
We can identify different type of cells based on their size and granularity.
The scatter shows this information - blood - characteristic scatter
What is Granularity ?
Granularity = A cell’s internal complexity
Describe the Passage of Laser-Based Flow Cytometry
1.Flow cell - cells leaving the nozzle tip are hit by the laser
- The cells have been labelled with fluorochromes that emit different-coloured light.
- There is an overlap in emission spectrum of each fluorochrome.
3.Mirrors and filters restrict the amount of light hitting the PMT (photomultiplier tube) = we can differentiate the fluorescence from each fluorochrome separately
see ppt slides 13 + 17
What is the electronic stage of flow-cytometry?
Electronic stage:
- Final stage
- PMT + computer converts light(analogue) signals → digital signals.
What are the key differences between flow cytometry and fluorescence microscopy?
- Fluorescence microscopy (FM) has many fields which means its not quantitative as you would need to look at many fields to quantify cells accurately.
- Flow cytometer (FC) can analyse 1000s of cells every second = more accurate
- FC quantifies rare cells accurately
- FM = difficult by eye (brightness of cells) using FM but FC quantifies the fluorescence of each cell
slide 8
How does fluorescence happen?
What is this process called?
Fluorescence:
-A laser hits fluorochrome, exciting it at a specific wavelength.
-Fluorochrome then returns to its unexcited state, emitting fluorescence at a higher wavelength.
This is called excitation-emission spectrum
What is stokes shift ?
Stoke’s shift = The energy difference between the lowest energy peak of absorbance and the highest energy peak of emission
What are some different fluorochromes and dyes ?
FITC (Fluorescein isothiocyanate)- Green
PE (Phycoerythrin)-Orange
PerCP (Peridinin Chlorophyll Protein)-Red
What are the wavelengths at which each of these fluorochromes emit/are excited?
FITC - Excited at 488nm and emits at 520nm (green)
PE-Excited at 488nm and emits 580nm (orange)
PerCP- Excited at 488nm and emits at 620nm (red)
What is fluorescence ?
Fluorescence = the emission of light by a substance that has absorbed light/other electromagnetic radiation
Why can different fluorochromes be used together and what is a benefit of this ?
- We can use 3 fluorochomes together, all excited by the same laser but they emit at different wavelengths.
- Emission of diff. fluorochromes always overlaps therefore the overlapping light is filtered out by mirrors + filters so we can analyse data from all 3 fluorochromes together
What are some cells which are in single cell suspension ?
Peripheral blood Bone Marrow Fine Needle aspirate CSF (+ other fluids) Cell lines in single suspension Fresh tissue