lycopodium and pecan, the diameters differ by ~1.73-fold
(28 μm vs. 48.5 μm, respectively). Sorting of individual pollen/
spore types based on the appropriate windows is highly effective, as
indicated by re-analysis of the sorted samples (Fig. 4a). Ragweed
and mulberry pollen are smaller, and differ in size by a factor of
about 1.56 (diameters of 12–13 μm versus 19–20 μm, respectively).
Their FS signals are almost identical in magnitude, whereas their SS
signals are clearly separated and can be used for sorting (Fig. 4b).
The observation of FS signals of similar magnitudes for particles of
different sizes emphasizes the unreliability of FS as a direct measure
of cell size.
3.3.1 Analysis and
Sorting of Arabidopsis
Protoplasts and Pollen
using the S3
1. In the three laser configuration, for detection of fluorescence
arising from chlorophyll autofluorescence, from pollen, and
from GFP, trigger events on forward-angle light scatter
(FALS) or 90
o side-scatter (SS), and visualize the protoplasts
using bivariate analysis of green fluorescence (the FL2 detector:
[525/30 nm]) versus red fluorescence (the FL3 or FL4 detector). Fluorescence measurements are conveniently done using a
log scale. While most flow cytometers are routinely configured
to trigger using the FALS signal, for plant samples, triggering
based on SS is a better option, since it is less noisy. The
threshold should be optimized to allow visualization of the
particles of interest, while excluding debris to the greatest
extent possible.
2. Draw an amorphous or polygonal sort window to include the
population of protoplasts or pollen. Perform sorting in Purity
Mode. Define the position of the sort region by first analyzing
the negative control, and setting a lower boundary for a positive GFP signal. Next, adjust the boundaries of the sort region
to exclude non-GFP positive protoplasts; determine the effects
of sort window placement by sorting a few protoplasts onto a
slide and examining them under a fluorescence microscope.
3. Protoplast and pollen sizes can be most conveniently measured
by determination of the time-of-flight of the particles through
the laser bean as reflected by the pulse-width value measured
either for the scatter or the fluorescence signals.
Figure 5 illustrates schematically the passage of particles (pollen, protoplasts) of progressively larger sizes through the focused
laser in the cytometer. Since the flow stream moves at a constant
velocity, the pulse widths of the scatter and fluorescence signals
produced by the particles as they traverse the beam are a convolution of the sizes of the particles and the width of the laser beam itself
(Fig. 5a). As the particle sizes decrease, the pulse widths asymptotically trends to the width of the beam itself. This means that the
pulse width parameter can be used to accurately define particle size
(Fig. 5b).
282
David W. Galbraith and Guiling Sun
(28 μm vs. 48.5 μm, respectively). Sorting of individual pollen/
spore types based on the appropriate windows is highly effective, as
indicated by re-analysis of the sorted samples (Fig. 4a). Ragweed
and mulberry pollen are smaller, and differ in size by a factor of
about 1.56 (diameters of 12–13 μm versus 19–20 μm, respectively).
Their FS signals are almost identical in magnitude, whereas their SS
signals are clearly separated and can be used for sorting (Fig. 4b).
The observation of FS signals of similar magnitudes for particles of
different sizes emphasizes the unreliability of FS as a direct measure
of cell size.
3.3.1 Analysis and
Sorting of Arabidopsis
Protoplasts and Pollen
using the S3
1. In the three laser configuration, for detection of fluorescence
arising from chlorophyll autofluorescence, from pollen, and
from GFP, trigger events on forward-angle light scatter
(FALS) or 90
o side-scatter (SS), and visualize the protoplasts
using bivariate analysis of green fluorescence (the FL2 detector:
[525/30 nm]) versus red fluorescence (the FL3 or FL4 detector). Fluorescence measurements are conveniently done using a
log scale. While most flow cytometers are routinely configured
to trigger using the FALS signal, for plant samples, triggering
based on SS is a better option, since it is less noisy. The
threshold should be optimized to allow visualization of the
particles of interest, while excluding debris to the greatest
extent possible.
2. Draw an amorphous or polygonal sort window to include the
population of protoplasts or pollen. Perform sorting in Purity
Mode. Define the position of the sort region by first analyzing
the negative control, and setting a lower boundary for a positive GFP signal. Next, adjust the boundaries of the sort region
to exclude non-GFP positive protoplasts; determine the effects
of sort window placement by sorting a few protoplasts onto a
slide and examining them under a fluorescence microscope.
3. Protoplast and pollen sizes can be most conveniently measured
by determination of the time-of-flight of the particles through
the laser bean as reflected by the pulse-width value measured
either for the scatter or the fluorescence signals.
Figure 5 illustrates schematically the passage of particles (pollen, protoplasts) of progressively larger sizes through the focused
laser in the cytometer. Since the flow stream moves at a constant
velocity, the pulse widths of the scatter and fluorescence signals
produced by the particles as they traverse the beam are a convolution of the sizes of the particles and the width of the laser beam itself
(Fig. 5a). As the particle sizes decrease, the pulse widths asymptotically trends to the width of the beam itself. This means that the
pulse width parameter can be used to accurately define particle size
(Fig. 5b).
282
David W. Galbraith and Guiling Sun