32
health of the ecosystem. The recent advances in DIM hardware and software suggest that
oceanography will experience a bloom in this discipline in the imminent future. The first eight
references in this paper are books or reviews on DIM which cover in detail both
instrumentation and specific applications of the technique in cell biology.
HARDWARE
Microscope systems
Light microscopes can be divided into two types, confocal and non-confocal. The former
require that only light emitted or reflected from the focal point of the objective lens be
collected by the detector thus increasing the resolution of the microscope to the diffraction
limit (super-resolution instrumentation will not be discussed here). Resolution of two point
objects in the optical microscope is limited, as shown by Rayleigh, by a distance at which the
peak intensity of the Airy disc of one object overlaps the first minimum of the Airy disc of
the second, a value of about 0.2 Jim for visible light (Taylor and Wang, 1989; Jovin and
Arndt-Jovin, 1989; Pawley, 1990). The z-axis resolution can be equivalent for phase-contrast,
interference contrast and polarized light microscopy (see Inoue in 6) but is not better than 0.7
Jim for fluorescence (Taylor and Wang, 1989; Jovin and Arndt-Jovin, 1989). Much smaller
objects can be detected if these objects are separated from one another by the requisite
distance indicated above. For example, 5-10 nm gold particles are detectable in the reflection
mode or 50 nm microtubu1es in phase contrast or immunofluorescence modes (see discussion
of Inoue in 3). Thus, resolution of marine microorganisms from the smallest phytoplankton
to the largest algae is realizable with optical microscopes. The range of~onfocal laser
scanning microscopes (CLSM) can be extended to small macroscopic and multicellular species
as well. The optical superiority of the CLSM is, at present, offset somewhat by their image
acquisition systems which are slow and limited to successive single-parameter measurements.
The lessons from flow cytometry of simultaneous, multi-wavelength excitation and emission
will be applied increasingly to CLSM in the future to provide multiparameter measurements
in real time. Such capabilities with the enhanced spatial resolution of these systems will make
them highly competitive to present 2-dimensional array microscopes. The analysis of
multi-cellular or complex unicellular species requires analysis by 3-D image cytometry, a
health of the ecosystem. The recent advances in DIM hardware and software suggest that
oceanography will experience a bloom in this discipline in the imminent future. The first eight
references in this paper are books or reviews on DIM which cover in detail both
instrumentation and specific applications of the technique in cell biology.
HARDWARE
Microscope systems
Light microscopes can be divided into two types, confocal and non-confocal. The former
require that only light emitted or reflected from the focal point of the objective lens be
collected by the detector thus increasing the resolution of the microscope to the diffraction
limit (super-resolution instrumentation will not be discussed here). Resolution of two point
objects in the optical microscope is limited, as shown by Rayleigh, by a distance at which the
peak intensity of the Airy disc of one object overlaps the first minimum of the Airy disc of
the second, a value of about 0.2 Jim for visible light (Taylor and Wang, 1989; Jovin and
Arndt-Jovin, 1989; Pawley, 1990). The z-axis resolution can be equivalent for phase-contrast,
interference contrast and polarized light microscopy (see Inoue in 6) but is not better than 0.7
Jim for fluorescence (Taylor and Wang, 1989; Jovin and Arndt-Jovin, 1989). Much smaller
objects can be detected if these objects are separated from one another by the requisite
distance indicated above. For example, 5-10 nm gold particles are detectable in the reflection
mode or 50 nm microtubu1es in phase contrast or immunofluorescence modes (see discussion
of Inoue in 3). Thus, resolution of marine microorganisms from the smallest phytoplankton
to the largest algae is realizable with optical microscopes. The range of~onfocal laser
scanning microscopes (CLSM) can be extended to small macroscopic and multicellular species
as well. The optical superiority of the CLSM is, at present, offset somewhat by their image
acquisition systems which are slow and limited to successive single-parameter measurements.
The lessons from flow cytometry of simultaneous, multi-wavelength excitation and emission
will be applied increasingly to CLSM in the future to provide multiparameter measurements
in real time. Such capabilities with the enhanced spatial resolution of these systems will make
them highly competitive to present 2-dimensional array microscopes. The analysis of
multi-cellular or complex unicellular species requires analysis by 3-D image cytometry, a