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Introduction Flow cytometry uses the principles of light scattering ,
light excitation, and emission of fluorochrome molecules to generate
specific multi-parameter data from particles and cells in the size range of
0.5um to 40um diameter. Cells are hydro-dynamically focused in a sheath of
PBS before intercepting an optimally focused light source (See Figure 1.1).
Lasers are most often used as a light source in flow cytometry.
Figure 1.1 Flow cytometers use the principle of hydrodynamic focusing for
presenting cells to a laser (or any other light excitation source). The
sample is injected into the center of a sheath flow. The combined flow is
reduced in diameter , forcing the cell into the center of the stream. This
the laser one cell at a time. This schematic of the flow chamber in relation
to the laser beam in the sensing area.(From Current Protocols In Cytometry,
Unit 1.2 , p1.2.2 ).
As your cells or particles of interest intercept the light source they
scatter light and fluorochromes are excited to a higher energy state. This
energy is released as a photon of light with specific spectral properties
unique to different fluorochromes( see Table 1.1 for a listing of commonly
used fluorescent dyes and their excitation and emission spectra. This table
also includes the most common laser light sources with their multiple lines
One unique feature of flow cytometry is that it measures fluorescence per
cell or particle. This contrasts with spectrophotometry in which the percent
absorption and transmission of specific wavelenths of light is measured for
a bulk volume of sample.
Table 1.1 Fluorescence spectra of commonly used fluorochromes. Excitation
spectra is represented by the gray lines while emission spectra is in black.
The bottom part of the table summarizes the emission wavelengths of various
light sources used in flow cytometry. The 488nm line of the argon ion laser
is extended over the spectra. (From Practical Flow Cytometry, Third Edition,
Howard M. Shapiro. P. 245).
Scattered and emitted light from cells and particles are converted to
electrical pulses by optical detectors. Collimated (parallel light
waveforms) light is picked up by confocal lenses focused at the intersection
point of cells and the light source. Light is send to different detectors by
using optical filters. For example , a 525 nm band pass filter placed in the
light path prior to the detector will only allow “green” light into the
detector. The most common type of detector used in flow cytometry is the
photomultiplier tube (PMT). (See Figure 1.2 for a basic layout of the
optical components in flow cytometry).
Figure 1.2 shows the optical system schematic for the XL analyser as well
as the optical configuration for the four FL PMT sensors.
The electrical pulses originating from light detected by the PMTs are
then processed by a series of linear and log amplifiers. Logarithmic
amplification is most often used to measure fluorescence in cells. This type
of amplification expands the scale for weak signals and compresses the scale
for “strong” or specific fluorescence signals.
After the different signals or pulses are amplified they are processed by
an Analog to Digital Converter (ADC) which in turn allows for events to be
plotted on a graphical scale(One Parameter, Two parameter Histograms).
Flow cytometry data outputs are stored in the form of computer files
using the FCS 2.0 or 3.0 standard. Data corresponding to one sample can be
stored as a listmode file and/or histogram file.
Histogram Files Histogram files can be in the form of one-parameter or
two-parameter files.Histogram files consist ofa list of the events
corresponding to the graphical display specified in your acquisition
One-parameter histograms A one-parameter histogram is a graph of cell
count on the y-axis and the measurement parameter on x-axis. All
one-parameter histograms have 1,024 channels. These channels correspond to
the original voltage generated by a specific "light" event detected by the
PMT detector. In other words, the ADC assigns a channel number based on the
pulse height for individual events. Therefore, brighter specific
fluorescence events will yield a higher pulse height and thus a higher
channel number when displayed as a histogram.
Two-Parameter Histograms A graph representing two measurement parameters,
on the x- and y-axes, and cell count height on a density gradient. This is
similar to a topographical map. You can select 64 or 256 channels on each
axis of two-parameter histograms. Particle counts are shown by dot density
or by contour plot.
Two parameter histogram Dot Plot displaying FL1-FITC on the x axis and
FL2-PE on the y axis.
Listmode Data Files Listmode files consist of a complete listing of all
events corresponding to all the parameters collected, as specified by your
acquisition Protocol. This file follows a format specified by the FCS 3.0
standard. Raw listmode data files can be opened or replayed using any
program designed for analysis of flow cytometry data. You should keep in
mind that a Protocol serves as a template. It allows you to collect
specified Parameters (i.e. FLS, FL1, FL2, etc.), and how these parameters
are displayed. Protocols also serve to determine how the data is Gated, and
contains all the Regions from which your statistics will be generated. In
addition, Protocols contain other specific information that serves as direct
interface between the computer workstation and the cytometer. These pertain
to high voltage settings for the PMT detectors, gains for amplification of
linear parameters, sample flow rates, fluorescence compensation,
discrimination settings, etc.
Once your data has been collected and written into a listmode file you
can replay the file either using the specific Protocol used for collection
or any other program specifically designed for analysis of flow cytometry
data. However, you should keep in mind that you can only adjust Regions,
Gating, and Parameters to be displayed. Settings such as amplification,
fluorescence compensation, etc., can not be modified . Therefore, when
collecting data make sure that your instrument settings are correct.
Finally, if you open your listmode files using a programs such as FlowJo,
WINMIDI, and/or ExPO you will have to specify parameter displays, and create
Regions and Gating corresponding to the Protocol used for collecting the
Flow Cytometry Analysis and Sorting Flow cytometry analysis of a single
cell suspension yields multiparameter data corresponding to Forward Light
Scatter (FLS), 90° Light Scatter (90LS), and FL1-FL4.
This information allows researchers to identify and characterize various
subpopulations of cells. The process of separating cells using flow
cytometry multiparameter data, is referred to as sorting. The
Beckman-Coulter XL instruments are bench-top, flow cytometer, analyzers.
They are capable of acquiring multiparameter flow cytometry data but they
can not separate or purify cells. Sorting is a specialized process that
requires sophisticated electronic components not incorporated into most
Schematic of Sorting Components
The Beckman-Coulter ELITE-ESP and Dako-Cyomation MoFli high speed sorters
are representative of a research grade cell sorter. Sorters include the
A tunable transducer which permits the breaking of the fluid sheath into
individual droplets. These individual droplets will encapsulate single
Electric charge delays for charging individual droplets.
Deflection plates for deflecting individually charged droplets into
Software settings for defining sorting criteria, these include regions
defining populations to be sorted.