4
In contrast, marine microorganisms span the size range from sub-micron to hundreds of
microns, they have all kinds of intricate shapes, they vary greatly with regard to the chemical
composition, and they may exhibit an endogenous fluorescence which can be much stronger
than that of dyes used to stain specific components of the cell. Many applications of marine
microbiology require that the cells are measured as soon as possible after being harvested, that
is in the often harsh field conditions of a vessel at sea. Hence, this science makes significant
new demands on the technology of flow cytometry. We shall have to develop instruments
which can measure sub-micron organisms which are outside the detection limit of most current
flow cytometers. At the same time these instruments should be able to handle cells several
hundreds of microns in size. That will require new flow chambers and optics which can
illuminate and collect the light from an object of this size. Non-spherical, asymmetrical cells
create measuring artifacts in current instruments, especially the laser-based ones. To be able
to measure organisms reproducibly and reliably, we have to solve this problem. In order to
measure some of the endogenous fluorophores of many marine organisms, such as chlorophyll
and phycocyanins, we have to employ other light sources and develop new optical filters. And
all of this has to be packed into a device which is sufficiently compact and rugged to be
practical for field use. All together, it appears that marine microbiology is a greater technical
challenge to flow cytometry than the mammalian cell biology and immunology for which this
technology was originally developed. But to the extent that this challenge can be met, flow
cytometry represents a unique opportunity to harvest large amounts of scientific information
from a very important and relatively unknown part of the biosphere.
In this presentation I shall discuss some of the principal aspects of flow cytometers and some
of the instrument parameters which determine their performance, especially with regard to
sensitivity and resolution. Various basic designs of flow cytometers are described and design
specifications which are particular to applications in marine microbiology are discussed.
BASIC CONSIDERATIONS
Common to all flow cytometers is a narrow, laminar flow of water which carries the cells,
one by one, through an intense focus of excitation light. The resulting fluorescence, which can
stem either from natural pigments or from dyes used to label specific cell constituents, is
In contrast, marine microorganisms span the size range from sub-micron to hundreds of
microns, they have all kinds of intricate shapes, they vary greatly with regard to the chemical
composition, and they may exhibit an endogenous fluorescence which can be much stronger
than that of dyes used to stain specific components of the cell. Many applications of marine
microbiology require that the cells are measured as soon as possible after being harvested, that
is in the often harsh field conditions of a vessel at sea. Hence, this science makes significant
new demands on the technology of flow cytometry. We shall have to develop instruments
which can measure sub-micron organisms which are outside the detection limit of most current
flow cytometers. At the same time these instruments should be able to handle cells several
hundreds of microns in size. That will require new flow chambers and optics which can
illuminate and collect the light from an object of this size. Non-spherical, asymmetrical cells
create measuring artifacts in current instruments, especially the laser-based ones. To be able
to measure organisms reproducibly and reliably, we have to solve this problem. In order to
measure some of the endogenous fluorophores of many marine organisms, such as chlorophyll
and phycocyanins, we have to employ other light sources and develop new optical filters. And
all of this has to be packed into a device which is sufficiently compact and rugged to be
practical for field use. All together, it appears that marine microbiology is a greater technical
challenge to flow cytometry than the mammalian cell biology and immunology for which this
technology was originally developed. But to the extent that this challenge can be met, flow
cytometry represents a unique opportunity to harvest large amounts of scientific information
from a very important and relatively unknown part of the biosphere.
In this presentation I shall discuss some of the principal aspects of flow cytometers and some
of the instrument parameters which determine their performance, especially with regard to
sensitivity and resolution. Various basic designs of flow cytometers are described and design
specifications which are particular to applications in marine microbiology are discussed.
BASIC CONSIDERATIONS
Common to all flow cytometers is a narrow, laminar flow of water which carries the cells,
one by one, through an intense focus of excitation light. The resulting fluorescence, which can
stem either from natural pigments or from dyes used to label specific cell constituents, is