361
Cell sorting also enabled us to determine something about what made the strains different: by
sorting several million cells of each kind from a natural sample, we were able to obtain
fluorescence excitation spectra (Fig. 6D) that indicated that the pigments from the two kinds
of cells were strikingly different. The fluorescence of the "bright" cell type was excited by
blue light much more effectively than that of the "dim" type, while both cell types were also
excited by green light. These results explain why we observed bright and dim fluorescence
from the two populations: since we were using a blue laser beam (488 nm) to excite
fluorescence, the cells with blue-absorbing pigment absorbed more light and hence emitted
brighter fluorescence than the "dim" cells (Fig. 6D).
These fluorescence excitation patterns, it turns out, reflect differing proportions of two
chromophores in the phycoerythrin of these cells: phycoerythrobilin (PEB) absorbs maximally
at 550 nm, and a second kind of chromophore, phycourobilin (PUB), absorbs at about 490
nm (Waterbury et aZ. 1986). It is noteworthy that PUB, the blue-absorbing chromophore, was
described in 1985 as a new and unusual chromophore (Ong 1984), only rarely encountered
in cultured strains. Since many oceanic organisms are not easily cultured, we wondered how
prevalent these cells were in the oceans as opposed to in laboratory collections; we used
another flow cytometric technique to address this question.
Because the fluorescence intensity of each cell depends not only on its pigment type but also
the amount of pigment (which can change dramatically depending on photoacclimation or
nutrient status), we can't simply use "brightness" as an index for pigment type (except perhaps
in the relatively small number of cases where we see both bright and dim cells together).
However, by using dual beam excitation in the flow cytometer, one can a obtain crude,
2-point excitation "spectrum" from each cell. If we pass the cell through first a blue beam and
then a green beam, we can measure two sets of fluorescence signals, and their ratio will tell
us the relative absorption properties of the cell's fluorescent pigment. In this case, we use the
488 and 515 nm laser lines from the argon ion laser: 488 nm is absorbed much more
effectively by PUB than by PEB, and 515 nm is absorbed about equally by both pigments
(Fig.6D).
Cell sorting also enabled us to determine something about what made the strains different: by
sorting several million cells of each kind from a natural sample, we were able to obtain
fluorescence excitation spectra (Fig. 6D) that indicated that the pigments from the two kinds
of cells were strikingly different. The fluorescence of the "bright" cell type was excited by
blue light much more effectively than that of the "dim" type, while both cell types were also
excited by green light. These results explain why we observed bright and dim fluorescence
from the two populations: since we were using a blue laser beam (488 nm) to excite
fluorescence, the cells with blue-absorbing pigment absorbed more light and hence emitted
brighter fluorescence than the "dim" cells (Fig. 6D).
These fluorescence excitation patterns, it turns out, reflect differing proportions of two
chromophores in the phycoerythrin of these cells: phycoerythrobilin (PEB) absorbs maximally
at 550 nm, and a second kind of chromophore, phycourobilin (PUB), absorbs at about 490
nm (Waterbury et aZ. 1986). It is noteworthy that PUB, the blue-absorbing chromophore, was
described in 1985 as a new and unusual chromophore (Ong 1984), only rarely encountered
in cultured strains. Since many oceanic organisms are not easily cultured, we wondered how
prevalent these cells were in the oceans as opposed to in laboratory collections; we used
another flow cytometric technique to address this question.
Because the fluorescence intensity of each cell depends not only on its pigment type but also
the amount of pigment (which can change dramatically depending on photoacclimation or
nutrient status), we can't simply use "brightness" as an index for pigment type (except perhaps
in the relatively small number of cases where we see both bright and dim cells together).
However, by using dual beam excitation in the flow cytometer, one can a obtain crude,
2-point excitation "spectrum" from each cell. If we pass the cell through first a blue beam and
then a green beam, we can measure two sets of fluorescence signals, and their ratio will tell
us the relative absorption properties of the cell's fluorescent pigment. In this case, we use the
488 and 515 nm laser lines from the argon ion laser: 488 nm is absorbed much more
effectively by PUB than by PEB, and 515 nm is absorbed about equally by both pigments
(Fig.6D).