27
One big problem is the large variation in the size of marine microorganisms. The very large
cells in particular call for a flow chamber and tubing of larger cross section than is currently
used in commercial instruments. To avoid turbulence and clogging, the smallest cross section
should be at least twice that of the largest organisms passing through the system. Some
phytoplankton may reach 500 ~m (Drebes, 1974), which means that the smallest cross section
of the fluidics system, including the flow chamber, should be no less than 1 mm. That in turn
means that the water consumption will be roughly one order of magnitude higher than in
current instruments. More importantly, it means that the width of the excitation focus must
be several millimeters in order to achieve homogeneous excitation intensity across the cells.
This is hardly compatible with the high sensitivity required to measure the organisms at the
lower end of the size range, which calls for a narrow focus to achieve a sufficiently high
excitation intensity, that is, a focus width of about 100 ~m. One could of course compensate
for a large focus width by means of a very powerful laser. However, that means a large water
cooled device which is so susceptible to mechanical shock and vibration that it is not a true
field instrument. An instrument taking such considerations into account has recently been
developed (Peeters et aI., 1989; Dubelaar et al., 1989). In conclusion, it appears that based
on the present technology, an instrument which is sufficiently compact and rugged to be used
in the field will not be able to measure simultaneously organisms covering the entire size
range from sub-micron to several hundred microns. However, it is possible to design an
instrument which can be switched between two different focal widths, for example 100 ~m
and 2 mm. Thus, to cover the entire size range, one would have to perform a separate
acquisition cycle with each of the focal widths.
Another problem which is particular to marine organisms is that most of these organisms are
non-spherical and highly asymmetrical, and therefore will produce fluorescence and light
scattering signals which depend on the orientation of the cell in the flow. As noted above, this
type of measuring artifact is most pronounced in laser-based instruments with a near-parallel
beam of excitation light. With such an instrument, the fluorescence signal of flat, disc-formed
cells has been found to vary by more than a factor of two, depending on the orientation of the
cells in the flow (pinkel et al., 1982). It has been demonstrated that a flow exhibiting
asymmetrical convergence can orient flat cells (Kachel et al., 1977; Kay and Wheeless, 1977),
for example, when a cylindrical flow runs into a flow having a rectangular or flattened cross
One big problem is the large variation in the size of marine microorganisms. The very large
cells in particular call for a flow chamber and tubing of larger cross section than is currently
used in commercial instruments. To avoid turbulence and clogging, the smallest cross section
should be at least twice that of the largest organisms passing through the system. Some
phytoplankton may reach 500 ~m (Drebes, 1974), which means that the smallest cross section
of the fluidics system, including the flow chamber, should be no less than 1 mm. That in turn
means that the water consumption will be roughly one order of magnitude higher than in
current instruments. More importantly, it means that the width of the excitation focus must
be several millimeters in order to achieve homogeneous excitation intensity across the cells.
This is hardly compatible with the high sensitivity required to measure the organisms at the
lower end of the size range, which calls for a narrow focus to achieve a sufficiently high
excitation intensity, that is, a focus width of about 100 ~m. One could of course compensate
for a large focus width by means of a very powerful laser. However, that means a large water
cooled device which is so susceptible to mechanical shock and vibration that it is not a true
field instrument. An instrument taking such considerations into account has recently been
developed (Peeters et aI., 1989; Dubelaar et al., 1989). In conclusion, it appears that based
on the present technology, an instrument which is sufficiently compact and rugged to be used
in the field will not be able to measure simultaneously organisms covering the entire size
range from sub-micron to several hundred microns. However, it is possible to design an
instrument which can be switched between two different focal widths, for example 100 ~m
and 2 mm. Thus, to cover the entire size range, one would have to perform a separate
acquisition cycle with each of the focal widths.
Another problem which is particular to marine organisms is that most of these organisms are
non-spherical and highly asymmetrical, and therefore will produce fluorescence and light
scattering signals which depend on the orientation of the cell in the flow. As noted above, this
type of measuring artifact is most pronounced in laser-based instruments with a near-parallel
beam of excitation light. With such an instrument, the fluorescence signal of flat, disc-formed
cells has been found to vary by more than a factor of two, depending on the orientation of the
cells in the flow (pinkel et al., 1982). It has been demonstrated that a flow exhibiting
asymmetrical convergence can orient flat cells (Kachel et al., 1977; Kay and Wheeless, 1977),
for example, when a cylindrical flow runs into a flow having a rectangular or flattened cross