35
a few monospecific antibodies. The loss of spatial discrimination in flow cytometry
compounds the limitations for analysis and argues strongly for increased use of DIM. One
approach currently being developed employs specific labelling techniques and oligonucleotide
probes for in situ characterization of microbial ecosystems (Giovannoni et al., 1990; DeLong
et al., 1989) . We have tested a method similar to that of DeLong and colleagues (16) using
direct coupling of fluorophores to synthetic oligonucleotide rDNA probes to identify various
microbiol populations and analyzing the samples with a DIM-CCD camera system in our
laboratory (Suematsu et al., 1987 and N.B. Ramsing, unpublished data). An example of these
data is shown in Figure 2. A mixed culture of Saccharomyces cerevisiae and E. coli was
probed with a eukaryotic oligonucleotide sequence, labeled with rhodamine, and a prokaryotic
sequence, labeled with fluorescein. Both populations are simultaneously visible by DNA
staining or phase contrast but completely distinguished by their respective fluorescent
emissions.
Clearly, the inherent multiparameter nature of digital microscopy affords an integrated
approach to solving the problems of identification and characterization of microscopic and
small macroscopic marine populations. One can summarize the obvious advantages of DIM
over bulk measurements and flow cytometry as follows: (a) precise discrimination between
biological particles and debris, (b) direct assessment of both size and structure, (c) the
mechanism for metabolic determinations, and (d) established procedures for reproducible in
situ hybridization techniques. However, DIM excludes high statistical precision in the
measurement of particle numbers due to the low sample volume that can be measured.
Digital microscopy should be much better able to exploit endogenous fluorescence molecules
than flow cytometry for assessment of the physiological properties of marine organisms since
(a) the range of excitation wavelengths is unlimited, (b) spatial parameters are essential to
correct identification, and (c) very low-light levels can be measured by signal integration
over time without loss of resolution. The sensitivity of digital microscopy has reached the
range where individual bioluminescence organisms can be detected (Suematsu et al., 1987;
Seliger, 1979). The combination of present spectroscopic techniques with microscopy will
certainly provide new understanding of the physiology of these organisms in the very near
future.
a few monospecific antibodies. The loss of spatial discrimination in flow cytometry
compounds the limitations for analysis and argues strongly for increased use of DIM. One
approach currently being developed employs specific labelling techniques and oligonucleotide
probes for in situ characterization of microbial ecosystems (Giovannoni et al., 1990; DeLong
et al., 1989) . We have tested a method similar to that of DeLong and colleagues (16) using
direct coupling of fluorophores to synthetic oligonucleotide rDNA probes to identify various
microbiol populations and analyzing the samples with a DIM-CCD camera system in our
laboratory (Suematsu et al., 1987 and N.B. Ramsing, unpublished data). An example of these
data is shown in Figure 2. A mixed culture of Saccharomyces cerevisiae and E. coli was
probed with a eukaryotic oligonucleotide sequence, labeled with rhodamine, and a prokaryotic
sequence, labeled with fluorescein. Both populations are simultaneously visible by DNA
staining or phase contrast but completely distinguished by their respective fluorescent
emissions.
Clearly, the inherent multiparameter nature of digital microscopy affords an integrated
approach to solving the problems of identification and characterization of microscopic and
small macroscopic marine populations. One can summarize the obvious advantages of DIM
over bulk measurements and flow cytometry as follows: (a) precise discrimination between
biological particles and debris, (b) direct assessment of both size and structure, (c) the
mechanism for metabolic determinations, and (d) established procedures for reproducible in
situ hybridization techniques. However, DIM excludes high statistical precision in the
measurement of particle numbers due to the low sample volume that can be measured.
Digital microscopy should be much better able to exploit endogenous fluorescence molecules
than flow cytometry for assessment of the physiological properties of marine organisms since
(a) the range of excitation wavelengths is unlimited, (b) spatial parameters are essential to
correct identification, and (c) very low-light levels can be measured by signal integration
over time without loss of resolution. The sensitivity of digital microscopy has reached the
range where individual bioluminescence organisms can be detected (Suematsu et al., 1987;
Seliger, 1979). The combination of present spectroscopic techniques with microscopy will
certainly provide new understanding of the physiology of these organisms in the very near
future.