The summary is based on the lessons given in class and on a guest lecture from BD Science. It contains information about fluorochromes, detection of apoptosis, immunophenotyping, etc.
Flowcytometry is a technology that simultaneously measures and analyses multiple
characteristics of single cells or particles as they pass through a beam of light. The
instrument focuses light on cells and measures fluorescence and the laser light, which is
scattered by the cells. The cells are measured one-by-one, in a single cell suspension.
Flowcytometry is used to measure qualities of cells, yeast, bacteria, isolated nuclei and beads
in a fluidic system. Multiple parameters can be studied at the same time. Also large amounts
of particles/cells can be measured, compared to microscopy. Flowcytometry is a relative
objective measurement.
A fluidic current passes the cells
one-by-one through a laser. The
lasers, mirrors and filters (optics)
generate and collect light. The laser
hits particles and excites (prikkelt)
fluorochromes and leads light to
flowcell . The flowcell needs to be
quartz (kwarts), because it transmits
UV-light. Collection optics guide
signals to detectors. Dichroïc mirrors
separate light and filters select
signals with specific wavelengths.
Optical signals (photons) are
converted to electronic signals
(electrons) and are digitalized for computer analysis. It measures height, width (time) and
area of surface below pulse (pulse area) above threshold.
Filters and dichroïc mirrors
Longpass (LP) filters transmit light that is equal to or longer than the specified wavelength. It
reflects light wavelengths shorter than 530 nm and transmit light wavelengths that are 530
nm or longer. Bandpass (BP) filters transmit light centered around the first value, within the
range specified by the second value. It reflects light wavelengths shorter than 460nm and
transmits light till 560nm. Shortpass (SP) filters transmit light that is equal to or shorter than
the specified wavelength. It reflects light wavelengths longer than 530nm. Dichroïc mirrors
are like longpass filters.
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