Flow Cytometry

M S Motswaledi
 Components of a Flow Cytometer
• Fluidics
• Optics
• Electronics (Detection)
The flow cell
Fluidics - 1
 Flow cell
• Sheath fluid at constant pressure
• Sample pressure can be increased or decreased
• High pressure, more cells pass
• Less pressure, single file passage
• Laser interception results in light scatter and
fluorescence
• Cell interacts with laser light
• Forward and side scatter measured
Optics
 Optics Components
• Lenses to concentrate light
• Mirrors to reflect and filter desired wave
lengths
 Laser-cell interaction
• Laser intercepts (interrogates) cell
• Cell scatters light FSC and SSC
• Fluorochrome, if present, is activated
• Fluorochrome emits light
• Emitted light and scattered light collected by
optics
• Collected light converted to digital signal
Application in Microbiology
Filters
 Light Detectors
• Photodiode (FSC and SSC)
• Photomultiplier tube (Fluorescence)
• Conversion of light to digital signal
Differential Count with Light Scatter
Fluorescence
 Fluorochromes
• Also called Fluorophores
• fluoresce only when energized with correct
wavelength
• Choose a flow cytometer that has the correct
laser light wavelength
• Absorb at shorter λ and emit at longer λ
Specific Wavelength is Selected for each Fluorophore
 Emission of longer
Fluorophore in Excitation with Specific shorter wavelength
ground state wavelength
Flow Cytometry

M S Motswaledi
Fluorescence Compensation
 Fluorescence Compensation
• Occurs when a cell is labelled with two or
more fluorochromes (Multicolour Flow
cytometry)

517 576
 Fluorescence compensation
• Subtracting the amount of stray fluorescence
coming from a different fluorochrome
• Done by altering the PMT voltage
Dot Plot
Fluorescence Compensation
Many cell characteristics measured
 Channel
• The measured value of a parameter,
representing the signal intensity of an event
after amplification. To appear on a plot, data
for an event must fall into one of either 256
channels (0-255) or one of 1024 channels (0-
1023) depending on the resolution of the plot.
Scatterplot Interpretation
Immunophenotyping
Scatter Plot, Histogram, Dot Plot