148
must be evoked in order to assign real numbers to the relative axes
which are the boundaries for flow cytometric measurements.
Coupled with a thrust to develop micro-methods, is the necessity
to search for suitable reaction blockers for photosynthesis incubations,
growth incubations,
respiration incubations and enzyme
activity experimentation.
If the reactions are stopped adequately
with littl~ degradation of the cells and pigments, then appropriate
sorting can be permitted to occur over hours vs. minutes.
Sterile
sorting is also possible and the instrument has a temperature
controlled sampling vial holder which permits use of the vial as a
reaction container.
One new dimension is certain to be the use of fluorescent stains
as probes. Table II is a compilation of some of the stains commonly
used in biomedical research.
Stain and antibody development has
evolved employing mammalian cells (Kruth, 1982), a cell environment
which is spectrally relatively "competition-free."
Phytoplankton
cells, on the other hand, have pigments crowded into the spectrum.
Chlorophyll a is. common to green plants with fluorescence emission
approximately 680 nm.
In addition, various chromatic groups contain
accessory pigments.
In total, these accessory pigments and the
chlorophyll pigments fill up the absorption regions (;;; excitation
wavelengths for fluorescence) in the visible spectrum. Note that the
absorption of water competes with these plant pigments for light in
the aquatic environment (Yentsch and Yentsch, 1983).
When vital staining is desired (a term used to denote that the
cells remain viable and unaltered), stains must be carefully selected
to avoid overlap of excitation and/or emission with algal pigments.
Energy transfer is the phenomenon of concern.
Basically, the light
emitted by one fluorescent reaction can be reabsorbed by another
molecule resulting in additional excitation and emission. A person
unknowledgeable of this might attempt to measure emission in the
spectral region where fluorescence is expected solely from one
molecule and in fact the emission occurs in another spectral region.
In doing so, the fluorescence emission from the first molecule is
"less" than expected. The molecules do not need to touch each other
for this transfer to take place, merely the light emitted by one
molecule excites the second mOlecule.
Another option is to fix the algal cells and extract the
pigments in an organic solvent (such as 50% ethanol or methanol) as
has been used effectively by Olson et al. (1983). Yentsch and
must be evoked in order to assign real numbers to the relative axes
which are the boundaries for flow cytometric measurements.
Coupled with a thrust to develop micro-methods, is the necessity
to search for suitable reaction blockers for photosynthesis incubations,
growth incubations,
respiration incubations and enzyme
activity experimentation.
If the reactions are stopped adequately
with littl~ degradation of the cells and pigments, then appropriate
sorting can be permitted to occur over hours vs. minutes.
Sterile
sorting is also possible and the instrument has a temperature
controlled sampling vial holder which permits use of the vial as a
reaction container.
One new dimension is certain to be the use of fluorescent stains
as probes. Table II is a compilation of some of the stains commonly
used in biomedical research.
Stain and antibody development has
evolved employing mammalian cells (Kruth, 1982), a cell environment
which is spectrally relatively "competition-free."
Phytoplankton
cells, on the other hand, have pigments crowded into the spectrum.
Chlorophyll a is. common to green plants with fluorescence emission
approximately 680 nm.
In addition, various chromatic groups contain
accessory pigments.
In total, these accessory pigments and the
chlorophyll pigments fill up the absorption regions (;;; excitation
wavelengths for fluorescence) in the visible spectrum. Note that the
absorption of water competes with these plant pigments for light in
the aquatic environment (Yentsch and Yentsch, 1983).
When vital staining is desired (a term used to denote that the
cells remain viable and unaltered), stains must be carefully selected
to avoid overlap of excitation and/or emission with algal pigments.
Energy transfer is the phenomenon of concern.
Basically, the light
emitted by one fluorescent reaction can be reabsorbed by another
molecule resulting in additional excitation and emission. A person
unknowledgeable of this might attempt to measure emission in the
spectral region where fluorescence is expected solely from one
molecule and in fact the emission occurs in another spectral region.
In doing so, the fluorescence emission from the first molecule is
"less" than expected. The molecules do not need to touch each other
for this transfer to take place, merely the light emitted by one
molecule excites the second mOlecule.
Another option is to fix the algal cells and extract the
pigments in an organic solvent (such as 50% ethanol or methanol) as
has been used effectively by Olson et al. (1983). Yentsch and