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 of emission).
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).
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.
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
Protocol.
One-parameter histograms
A one-parameter histogram is a graph of cell count on the y-axis and the
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.
Listmode
Data Files
Listmode files consist of a complete listing
of all events corresponding to
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 data.
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.
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 bench-top instruments.
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 following components:
A tunable transducer which permits the breaking of the fluid sheath into individual droplets. These individual droplets will encapsulate single cells.
Electric charge delays for charging individual droplets.
Deflection plates for deflecting individually charged droplets into collection tubes.
Software settings for defining sorting criteria, these include regions defining populations to be sorted.