0022- 1554/79/2712THE
JOURNAL
1649$02.OO/O HISTOcHEMISTRY
OF
AND
© 1979 by The Histochemical
Copyright
Vol. 27, No. 12, pp. 1649-1651, 1979 Printedin U.S.A.
CYTOCHEMISTRY Inc.
Society,
Future
Directions LOUIS Ortho
During the
most
of the
development
concern
was
biologists
to
cathode
ray
last
of not
with
bridge
its
the
tube
needs,
and
of acceptability
to develop
take?
and,
future
relate
for
research
measure?
Once as are
be taken
get on
the
a
major
I believe
it is
as: What
good
these
to expand
I define
an
and
obtained
analytic
flow
displaying
from
a
cell
taking way
such
cells
to
it, diluting
a cytometric
system;
of individual
measurements
and
biologist.
inception, since, valuable to somehow
Analytic
flow
on
these
will be future
making
3) a means has
been
one
and
or
difficult
flow
techniques
results. for
that
each
it is not
I will
of the
discuss
three
what
elements
appropriate
for
sorting
I believe
issues completely
with new
a discussion directions
for
flow
Sample
plates
to automate based
to discuss
the
although
cytometry. Handling
Second,
with
energy
rate,
consistency
artefacts commercial
and
of the
measurements,
capability
research
flow
sion into the measuring pressure. Some clinical (Coulter
Electronics,
cytometry the
of
studying
cytometers
Fla.)
reduction viable
aperture
light
measurement of
cells.
slide-related All
!nstruments’
HEMAC
There
has
been
interest
in developing
Systems
separating
two
can
be measured
cause
into
of a cell
the
(Or-
light
tively
tho Instruments, Westwood, Mass.) and ELT-8 (Ortho Instruments) and Technicon’s HEMALOG (Techmcon Corporation, Tarrytown, N. Y.) have been designed as fast automatic screening instruments to perform multiple tests on a sample. In these instruments, blood samples are automatically-multiply diluted, and differentially lysed, and techniques are used for dilution, mixing and transport that allow replicate cell counting precisions of better than 1%. The Technicon HEMALOG D (Technicon Corporation) additionally automatically stains leukocytes to perform differential counts by a subsequent
focused
large
focused
into
times,
down
irradiated
pendent
measure actions
the state
volume,
to
any
multiple useful determined of polarization
by a lens
frequency
of small
with
measurement
adequate
use of sheath liquid
flow,
stream,
measurement
the
rates
volumes since
can
energy,
the of
using
a rela-
light
can
be
for shorter without
many
or
signal
flow through
be illuminated obtainable
high
suspension
caused by cell coincidences. flow through the image of a light electromagnetic
the
particles in which
the
as they
Third,
cell can
to focus
to achieve
measurements
aperture.
measurement
interactions
are
of the
sensitive and precise without using a small In an optical system,
to a few microns
to small
the
by
the larger
physical
a small
increasing
volume
to make constituent
minimum
cisions of measurement As a cell is made fact,
of which
it is difficult
to make currents problems.
clogging
over electrical noise. Through cells are injected into a second cells can be confined in position
present
techniques,
volume electric
a small
required
amounts
each
as a function
understood
may
be focused
densities
chambers,
circuit. I believe that the number by this principle are limited in for the following reasons. First,
current source to yield useful multiple parameters, could be related to cell size, perhaps electrical properties, but probably not to cell constituents.
into a small with reasonable
which
can
small
transport a cell suspeninstrument by differential air such as the Coulter Model S
Ortho
aperture
presently
field
measurements
simply
part of the flow cytometers,
Hialeah,
in a rapid
sample.
impedance
provides
for flow
an
of an alternating these measurements and morphologic an electric
advantages
sample
future
on cytotoxicity.
through
The handling of liquid suspension samples is the feature that distinguishes the flow cytometer from other cell photometric methods in which cells are transported to a measuring system on a microscope slide, continuous tape, or string. Maintaining cells in suspension major
reagent
Another
handling
contains one electrode of an electric of useful parameters measurable number, resolution and sensitivity
at this time, since the techniques is complex. I application needs may lead to
of how
disposable
of tests.
samples as small as bA, using microtiter various immunologic profile tests, such as tissue
for
typing
flow
of future directions for cell sorting of how it relates to other separation
will conclude
a large
sets
Present flow cytometry instruments are based on either, or cornbisations, of two measurement principles: electrical or optical. Electrical measurements, first introduced to the biologist as the Coulter Counter (Coulter Electronics, Inc.), depend on a change in electrical impedance as a cell in an electrically conducting medium is made to
to
of analytic
me
to use
specific
Measurement
these
way
to flow
to get
more
relevant
coupled
developed for
the
of processing
in a useful
cytometry
measurement I feel
and
them
for
transporting
be
appropriate
cells
1) a means of sample in some
it, and of
doing
will
direction for flow cytometry sample handling systems will be measuring small samples, either because a reagent, such as an antibody, is expensive or difficult to obtain, few cells are available, or it is clinically
if individual cells could be measured, it was divert this flow into different vessels de-
directions
cytometry.
subject
for
basic elements: processing this
a means
cells;
its
pending
2)
system
02090
instruments
containers
their
on individual
instrument
it, or staining
displaying
from
instrument
Any
as mixing
the
an
suspension.
measurements
flow
as
measurements
measurements has three a suspension of cells, perhaps
cytometry
felt
cytometer
one or more
79-185)
Massachusetts
and
instru-
instruments
applicability?
making
(BR
photometric
research
issues
What should
to
own
and such
should
was
Cytometry
system. As flow cytometric techniques are developed so they are routinely used in research or clinical laboratories, some sample handling aspects of these tests will be automated to minimize the manual labor involved, and, more importantly, to make these tests consistent from laboratory to laboratory so they can be properly calibrated and controlled. This automation will probably follow two parallel development paths in which cytometric tests will be incorporated into screening instruments for the large clinical laboratories,
in
major
dots
to their
directions
directions
my it
is overcome,
configurations
involved
electronic
its application.
cytometers
What
been
Rather,
concept
KAMENTSKY Westwood,
cytometry,
between
concept
on future
flow
What
this
A.
Instruments,
I have
flow
directions. gap
of this
should
specifications?
for
techniques
now, we can concentrate ment
in which
conceptual
to cells,
hurdle parameters
15 years
instrumentation
for Flow
source, different
impre-
or, in inde-
be measured, leading to the capability to properties of each cell. These different interby the angles of collection, the wavelengths, and the phase of the energy emitted from the
1649
Downloaded from jhc.sagepub.com at University of Southern Queensland on April 28, 2015
1650 cell
KAMENTSKY and/or
the
chromophores
a function
of time
interactions
can
as the be
cell
bound
to it. These
interactions
passes
through
energy
processed
and
the
combined
vary as source. The
in a variety
measurement parameters. that each cell is illuminated mated energy, it will absorb some of that of it at the incident wavelength (scatter),
of manners
resting
to yield
the
with monochromatic cothenergy and reradiate some or at a longer wavelength
Assuming
(fluorescence). collected over
!f
the a large
remaining numerical
incident aperture,
to measure absorption. My own were to study the simultaneous nucleic
acids
and
system.
This
type
the difficulties tional error, receive
by
again
aperture
If the
angular
with
a high
with
microscope
energy
reemitted
ranges,
these
parameters
the the
in the
aperture
not been
flow cytometry
cell
in
high
over
or
more
measurements can be used to derive the size, shape, granularity or absorption of cells. There has been extensive work during the last calculations of the scatter from cell models as a scatter
in
10 years
refining
function
of cell
morphology
and
composition.
There
have
been
many
empirical studies of flow cytometry scattering to estimate cell constituents, characterize cell morphology and differentiate subpopulations in heterogeneous populations. Distributional error and nonspecific
scattering
obtaining this
effects high
presently
resolution
technique
has
make
cell
been
it unacceptable
constituent
successfully
as
measurements.
applied
both based on stains for various cellular enzymes light scatter of unstained cells, to counting live versus trypan
blue
staining,
determinantslabeled red
cells
to
with
and
platelets.
differentiating
peroxidase, Scatter
continue
to be developed
a simple
light
source,
and
dye,
wavelengths
generally
fluorescent
in
constituents
such
1%. Since
than
are
measured
stained at
of the incident major
cells
with
one
or
energy.
advantages
These
can be measured emitted
detection
Through
the
into
the
use
of
system
a
more
over
specific to precisions now
cytometry. energy
cells
three
will
other
dyes, better can
be
separated from the incident energy by virtue of its longer wavelength, measurements can be obtained at high signal-to-noise ratio to provide sensitive
estimates
fluorescent
found
that
different can
of
tagged
are
small
amounts
surface
specific
of
for
different
since
constituents
and
be
used
as
or metachromatic dyes a stain and emit at different
two
or more
cell
constituents
can
simultaneously. Still another attribute begun to be explored. If a fluorescent molecule, is excited by polarized light the
constituents
wavelengths,
constituents,
laser),
cell
Third,
determinants.
the
polarization
fluorescence
to which
of the
lifetime
it is bound
is able
fluorescence
of the
dye,
to rotate
emit
like acridine colors for be precisely
and
to the
within
this
will
degree
to of the
of the
image,
or
to the
distribution
the
the
is normally
pulse of cell
fluorescence
which
is related
through this
pulse
can
a
passage
i.e.,
additional
There
are
instruments.
to per to the
the
energy
which
may
be
be used
as other
measurement
mo-
energy
now
two
different
types
of
ical
at
energy
at
lines
in
the
measurements along
a path
aperture
energy
analytic
flow
cytometry
In the first, a laser, often a Helium Neon laser emitting 6300 A for scatter measurements, or an Argon ion laser ultraviolet
as
at
a right
as mechanically possible
fluorescent
to
energy
to
is imaged
by
at
differ-
at
dalities provide morphologic information useful for such purposes as distinguishing single cells from cell clusters. By narrowing the energy source image, in principle, a profile of the chromophore distribution can be obtained as a set of parameters to more completely characterize morphology. It should be noted that with each additional parameter obtained from a flow cytometer, more data processing is required. The complexity of this data processing in terms of the amount of equipment, programs or time required increases in proportion to the number of parameters used to characterize a cell.
light
molecule
will, in each
This
of chromophores,
light is generally measured by sensors and fluorescence is collected by a lens to the plane of the laser beam and the imaging the stream onto an aperture is
to
most
will produce
pulse
These
resolution it the
in intensity
chromophores
cytometer.
ampli-
in intensity
duration
of
better
particles.
source
quantity,
value
this
to
make
a change
voltage
cell
maximum
a flow
energy
the
moving
lifetime.
small
width
related
angle
feasible
increase
from
both
the
to
blue
a lens the
optical
the
for cell
axis.
flow
Scattered
placed opposite to the laser, whose axis is at a right angle cell flow. This condenser lens generally of as high a numer-
to collect
as much
resolution
aperture
wavelengths onto
is
and
fluorescent
sensitivity.
imaged
The
through
dichroic
and/or optical filters appropriate to the experiment onto photomultiplier optical sensors. In some instruments, the cell stream is jetted into air, where it intersects the light beam; in others, the mirrors
measurement
is
made
in
a quartz
flow
cuvette
with
optically
flat
Although the air stream measurement is less susceptible to defects on the quartz surface, I favor measurement in a cuvette because I believe the superior optical design possible with optical flats over the air stream leads to superior sensitivity and resolution. The second type of instrument utilizes an epi-illumination micro-
which
detect
The
of passage
will
of time.
during
related
parameter
in
as a function to time
present
staining
sensitivity,
change
the
paid
specificity,
This
pulse
respect
be
fluorescent
cause
utilize
I believe
coated
systems,
to
a horsepower to
will
of fluorescence
measurement
been
been
of fluorescent
the great
as a photomultiplier.
a
attention
deter-
determinant.
has
indeed
for flow cytometry. of a cell through a focused
source
applications
is, to reproducibly
that
voltage
to the
leading
antibody
as
capability
measured.
walls.
that
more
of flow molecules
instruments.
development
such
with
produce
Thus, by measuring the polarization of the emission, it is possible to characterize the structure of the dye’s environment. There has been a considerable effort to develop fluorescent staining methods for use specifically in flow cytometers to measure cell constituents, such as DNA, RNA, total protein and enzymes, in order to study cell kinetics or to characterize various cells as a function of their environment. The key requirement of a flow cytometer in these is resolution;
to the
of
resolution.
sensitivity
of per cell surface
manufacturers that
one
of fluorescent
laser-based
as
related
the
such
the
has
be
orange different
fluorescence
sensor
cytornetery
sensitivity
there
can
measured
be
above
fluorescence
of fluorescence has recently dye, bound to a biologic (normally available from a emission
and
and
useful technique The passage
emitting
such
dyes
cytometer
multiwatt,
is no question
of the
this and
the
enabling
Perhaps
antibody
improve
sources,
multiconstituent
parameters
can use
denatured
and
to
light
is wrong
duration
sizing
technique
employed
minimally
is
that
provide
as DNA the
and
emission
longer
flow
system
numbers
a fluoresceinated
techniques,
cell,
surface and
cytometry
measurements, their
measurements
techniques than
and
a flow
of
and wide angle dead cells based
containing
fewer
to detect with
flow
integrated
leuko-
to differentiating
the optical
unstained
can be characterized. To perform fluorescence fluorescence
as
since
cells and
numbers
an electronic
However,
to differentiating
cytes on
a means
small
designs
There
of flow
to detect
expensive,
fication
describing
chromophores
among
factors
in order
used
race
differences.
to improve
approach
approach
limiting
a percent,
cell
developers has been to improve most certainly continue, as will
made
tagged powerful
than
chromosomal
by instrument This will
the
less
has been
primarily
more
of
effort
minants
optical
one
single
resolution.
per cell,
large,
differences
efforts
to defme
The
systems.
is measured
of some
cytometers
of
DNA
major
A parallel
is
because
interest
detection
research
and distribustains. It may
renewed
have
measurement
optical
pursued
light scatter of avoiding
current
based
by
energy in principle,
cells
the most
flow cytometry ultraviolet by
to use
numerical
has
of dealing with nonspecific although it has the advantage
attention
numerical
proteins
and reradiated it is possible,
first attempts absorptions
of measurement
ences in a constituent such as the DNA of each cell of different clones. !t is now possible to resolve cell populations in which
scope
system
is imaged
in which by
can
an
the
objective
be flowing
condenser lens
is the
Downloaded from jhc.sagepub.com at University of Southern Queensland on April 28, 2015
of high
direction
lens
of an
numerical
of the
arc
lamp
aperture
optical
illuminator onto
axis,
each
cells
cell
FUTURE
crossing
the focal
by the
objective
plane. lens
The
and
fluorescent
directed
DIRECTIONS
energy
emitted
by a dichroic
mirror
FOR
is collected to one
ments, or
or more
optical filters and photomultipliers. This system can use standard high quality microscope lenses with Kohler illumination to yield the uniform fields and high numerical apertures required to achieve good measurement resolutions, and this type of instrument has, to date, achieved the best resolution. It has the additional advantages of freeing the cytochemist from the restriction of finding dyes that will work
with
system
excitation
has
been
not
at
been
concerned
specific
achieved,
with
high
ever, that this approach antibody tagging as well experiments. a less
will yield
based
simpler
flow cytometry
partially
High
on these
this
its developers
have
I believe,
how-
useful
for
constituent
with for
data
will offer
doing
fluorescent
most
various
the biologist
made
to present
display
or
stereo
meters,
the
more
data
and
other
The
measurements
Handling
obtained
in a useful manner to interpret the test. If one parameter such as amount of a DNA specific dye, is are best displayed as a population In this case, the data handling analyzer used in liquid scintillation present
flow
cytometry
cytometer
must
be displayed
results of an experiment or clinical fluorescence, proportional to the measured, the measurement results histogram of per cell fluorescence. electronics can be a multichannel spectrometry.
experiments,
For
particularly
cell
many
of the
cycle
versatile,
distributions
relative
from
numbers
experiments,
the
of cells
counts
raw
single
parameter
at each
stage
of the
of cells
whose
data
complex
systems
parameters
cell
cycle.
For
are
within
ranges
Data involving either uncorrelated
more than one or as correlated
as uncorrelated, parameter
each can
measurement parameters.
measurement
may be handled as If the data is treated
is treated
be independently
displayed
independently
and
as a population
each
histogram.
to
tinely
4) cell
and
All
of the
with
on
one
For
parameter example,
of per cell fluorescence
for
some
it is useful
to
of one
know
the
more
time
energy,
original
antibody
determinant. a mode is within
stored
per
cell
In these
in which a certain
one
different distribution
cases, parameter
range,
fluorochrome of cells
a multichannel gates it (turns
so the
value
to with
analyzer it on),
of a second
determine a second
the surface
parameter
can
be
for subsequent display, or cells counted based on its value. In certain experiments, particularly is scatter or some dual fluorescence measurements, the parameter measurements are correlated so that subpopulations of cells cannot be distinguished based on parameter ranges, and functions of two or more parameters must be used instead. Analog circuitry to define these functions has been built into the earliest flow cytometers, and current computerized systems allow for methods to define them manually. We have employed techniques for computerized definition of population clusters, and I believe these techniques will be available in future computerized flow cytometers. In some experiments, it is convenient to display correlated measure-
disk
be
or
capable
for
program
of different
histochemical
methods
rou-
for
to
of various
various
agents
are
based
cells.
and
individual cell
on
to additionally
constituent
cytometers
as
or fluorescence
of both
undenatured
beginnings
and the energy
Other
structure forms
as
its
resonance
their
retic
techniques
I
treated
being
made
of different
could
based measurements,
with
fluorescence
chromophores
measurements impedance
of biologic
be possible using
probes
cells,
bound conceivably
on ultrasonic or nuclear
measurements.
It will also be of interest cells,
the
amount.
at wavelengths
and
should
energy
including of electrical
membrane, are
transfer
constituents.
new
cell
used based
characterize
well
scatter,
in the
constit-
will use forms of energy other and other constituents. We should
these
range
cell
quenching
light
Some
characterize
chro-
measurements
absorption,
particularly
different
as
populations
measuring
scatter
Fluorescence
developed
being
visible
polarization
such
of cells;
of light
some
to measure
the
of cells heterogeneous
exception
flow
light
DNA
enzymes
of a specific
future
that
agents;
analysis.
now
cytometry.
to
have
are now
of applications
response
applications
synthesis
magnetic
can be used in ifthat parameter
useful
probably beginning with the ultraviolet and infrared. Measurement of cell structure and the relative positions
antibody
a
be
microprocessor-
determinants;
and some
count
visible
flow
with
what
are programmed.
of various
surface
analysis
viability
are
of
molecules,
from
as a function of cellular
above
techniques
believe
distribution
of
as
or drugs;
differentially
a fluoresceinated antibody to a cell determinant for cells 1) within a specific size range to distinguish one group of cells from another, or from cellular debris based on size, or 2) having fluorescence at a second wavelength related to a second tagged
resulting
ranges to
and
the possible
or
based
parameters.
will
small
will
they
types
following
counting
cell
count
such
a floppy
they
how
the
for RNA
cell
the
other
that on
of cell
5) differential including
outside
a computer
be
inserts
development
constituent
mosomes
to study
or
but
will
user
exception
analysis
antigens
begin
analyzers
expensive,
second
the
3) characterization
than
multichannel
and
depending
2) characterization
which
multiple
of ranges
questions
done:
print or experof, or
requires
will
the
where
store the sets of measurements of each parameter and display histograms for each. In multiparameter measurement iments, the user is often interested in knowing the distribution
This
answer
cyto-
multiparameter
as a function
thus
The
cytometric
uents
ments.
parameters
use
which
with
advanced
some
are needed rather than distributions. For other experwe have found it useful to characterize populations of cells by derived statistically from the distributions of their measure-
to
of operation
mitogens, of
flow
to handle
Applications
measurements
iments, indices
in
1) cell cycle
estimate
have a color
computerize
be able
contour
using
simultaneously
will
two
dot,
attempts
and the instrument is set up to perform the user’s specific store his data, and generate the appropriate report. Such will be run and controlled as present clinical hematology
Flow
analysis,
to
Some
manufacturers
could
methodology.
modes
this is all that is required, especially if the inherent resolution of the instrument is good. If not, a computer has been used to reconstruct model
least
using
data.
and RNA per cell of cells: 1) develop the techniques with a certain enzyme or surface determinant. I believe there will be two types of data handling systems developed in the future. The first will be used on the large multiparameter flow cytometer systems, and will
instruments
in a flow
As users
They
developed
parameters
viewer. at
been
parameter
three
affluent
display
instruments Data
have two
are the correlated distributions of DNA of a certain size, or 2) when cytochemists
based
experiments.
of
parameters.
cartridge, experiment,
analytic
techniques
displays
developing
handling
routine
and
been
1651
CYTOMETRY
isometric
be
measurement
appropriate
principles
instrument
for
because
instruments
that
sensitivity
measurements.
resolution
believe
a system
expensive,
lines.
resolution as high
I further
electronics,
laser
FLOW
surface
charges combined
to characterize and
the electrical
transmembrane
with
optical
potential. measurements
properties
of
Electrophocould
be
one
approach to characterizing the electrical properties of cells as a function of various agents and their relationship to other cells. Although a major impetus to flow cytometry has been to characterize large cell populations rapidly, the fact that some cell characteristics are heterogeneous, no matter how precisely we perform measurements, will make it of interest to study one or more properties of an individual cell as a function of changes in its environment. Thus, last
in
my
list
of
future
directions,
is the
belief
that
techniques developed for flow cytometry instrumentation applied in this opposite direction to determine the kinetics ual cells within large populations.
Downloaded from jhc.sagepub.com at University of Southern Queensland on April 28, 2015
some
of
the
will now be of individ-
JOURNAL
1649$02.OO/O HISTOcHEMISTRY
OF
AND
© 1979 by The Histochemical
Copyright
Vol. 27, No. 12, pp. 1649-1651, 1979 Printedin U.S.A.
CYTOCHEMISTRY Inc.
Society,
Future
Directions LOUIS Ortho
During the
most
of the
development
concern
was
biologists
to
cathode
ray
last
of not
with
bridge
its
the
tube
needs,
and
of acceptability
to develop
take?
and,
future
relate
for
research
measure?
Once as are
be taken
get on
the
a
major
I believe
it is
as: What
good
these
to expand
I define
an
and
obtained
analytic
flow
displaying
from
a
cell
taking way
such
cells
to
it, diluting
a cytometric
system;
of individual
measurements
and
biologist.
inception, since, valuable to somehow
Analytic
flow
on
these
will be future
making
3) a means has
been
one
and
or
difficult
flow
techniques
results. for
that
each
it is not
I will
of the
discuss
three
what
elements
appropriate
for
sorting
I believe
issues completely
with new
a discussion directions
for
flow
Sample
plates
to automate based
to discuss
the
although
cytometry. Handling
Second,
with
energy
rate,
consistency
artefacts commercial
and
of the
measurements,
capability
research
flow
sion into the measuring pressure. Some clinical (Coulter
Electronics,
cytometry the
of
studying
cytometers
Fla.)
reduction viable
aperture
light
measurement of
cells.
slide-related All
!nstruments’
HEMAC
There
has
been
interest
in developing
Systems
separating
two
can
be measured
cause
into
of a cell
the
(Or-
light
tively
tho Instruments, Westwood, Mass.) and ELT-8 (Ortho Instruments) and Technicon’s HEMALOG (Techmcon Corporation, Tarrytown, N. Y.) have been designed as fast automatic screening instruments to perform multiple tests on a sample. In these instruments, blood samples are automatically-multiply diluted, and differentially lysed, and techniques are used for dilution, mixing and transport that allow replicate cell counting precisions of better than 1%. The Technicon HEMALOG D (Technicon Corporation) additionally automatically stains leukocytes to perform differential counts by a subsequent
focused
large
focused
into
times,
down
irradiated
pendent
measure actions
the state
volume,
to
any
multiple useful determined of polarization
by a lens
frequency
of small
with
measurement
adequate
use of sheath liquid
flow,
stream,
measurement
the
rates
volumes since
can
energy,
the of
using
a rela-
light
can
be
for shorter without
many
or
signal
flow through
be illuminated obtainable
high
suspension
caused by cell coincidences. flow through the image of a light electromagnetic
the
particles in which
the
as they
Third,
cell can
to focus
to achieve
measurements
aperture.
measurement
interactions
are
of the
sensitive and precise without using a small In an optical system,
to a few microns
to small
the
by
the larger
physical
a small
increasing
volume
to make constituent
minimum
cisions of measurement As a cell is made fact,
of which
it is difficult
to make currents problems.
clogging
over electrical noise. Through cells are injected into a second cells can be confined in position
present
techniques,
volume electric
a small
required
amounts
each
as a function
understood
may
be focused
densities
chambers,
circuit. I believe that the number by this principle are limited in for the following reasons. First,
current source to yield useful multiple parameters, could be related to cell size, perhaps electrical properties, but probably not to cell constituents.
into a small with reasonable
which
can
small
transport a cell suspeninstrument by differential air such as the Coulter Model S
Ortho
aperture
presently
field
measurements
simply
part of the flow cytometers,
Hialeah,
in a rapid
sample.
impedance
provides
for flow
an
of an alternating these measurements and morphologic an electric
advantages
sample
future
on cytotoxicity.
through
The handling of liquid suspension samples is the feature that distinguishes the flow cytometer from other cell photometric methods in which cells are transported to a measuring system on a microscope slide, continuous tape, or string. Maintaining cells in suspension major
reagent
Another
handling
contains one electrode of an electric of useful parameters measurable number, resolution and sensitivity
at this time, since the techniques is complex. I application needs may lead to
of how
disposable
of tests.
samples as small as bA, using microtiter various immunologic profile tests, such as tissue
for
typing
flow
of future directions for cell sorting of how it relates to other separation
will conclude
a large
sets
Present flow cytometry instruments are based on either, or cornbisations, of two measurement principles: electrical or optical. Electrical measurements, first introduced to the biologist as the Coulter Counter (Coulter Electronics, Inc.), depend on a change in electrical impedance as a cell in an electrically conducting medium is made to
to
of analytic
me
to use
specific
Measurement
these
way
to flow
to get
more
relevant
coupled
developed for
the
of processing
in a useful
cytometry
measurement I feel
and
them
for
transporting
be
appropriate
cells
1) a means of sample in some
it, and of
doing
will
direction for flow cytometry sample handling systems will be measuring small samples, either because a reagent, such as an antibody, is expensive or difficult to obtain, few cells are available, or it is clinically
if individual cells could be measured, it was divert this flow into different vessels de-
directions
cytometry.
subject
for
basic elements: processing this
a means
cells;
its
pending
2)
system
02090
instruments
containers
their
on individual
instrument
it, or staining
displaying
from
instrument
Any
as mixing
the
an
suspension.
measurements
flow
as
measurements
measurements has three a suspension of cells, perhaps
cytometry
felt
cytometer
one or more
79-185)
Massachusetts
and
instru-
instruments
applicability?
making
(BR
photometric
research
issues
What should
to
own
and such
should
was
Cytometry
system. As flow cytometric techniques are developed so they are routinely used in research or clinical laboratories, some sample handling aspects of these tests will be automated to minimize the manual labor involved, and, more importantly, to make these tests consistent from laboratory to laboratory so they can be properly calibrated and controlled. This automation will probably follow two parallel development paths in which cytometric tests will be incorporated into screening instruments for the large clinical laboratories,
in
major
dots
to their
directions
directions
my it
is overcome,
configurations
involved
electronic
its application.
cytometers
What
been
Rather,
concept
KAMENTSKY Westwood,
cytometry,
between
concept
on future
flow
What
this
A.
Instruments,
I have
flow
directions. gap
of this
should
specifications?
for
techniques
now, we can concentrate ment
in which
conceptual
to cells,
hurdle parameters
15 years
instrumentation
for Flow
source, different
impre-
or, in inde-
be measured, leading to the capability to properties of each cell. These different interby the angles of collection, the wavelengths, and the phase of the energy emitted from the
1649
Downloaded from jhc.sagepub.com at University of Southern Queensland on April 28, 2015
1650 cell
KAMENTSKY and/or
the
chromophores
a function
of time
interactions
can
as the be
cell
bound
to it. These
interactions
passes
through
energy
processed
and
the
combined
vary as source. The
in a variety
measurement parameters. that each cell is illuminated mated energy, it will absorb some of that of it at the incident wavelength (scatter),
of manners
resting
to yield
the
with monochromatic cothenergy and reradiate some or at a longer wavelength
Assuming
(fluorescence). collected over
!f
the a large
remaining numerical
incident aperture,
to measure absorption. My own were to study the simultaneous nucleic
acids
and
system.
This
type
the difficulties tional error, receive
by
again
aperture
If the
angular
with
a high
with
microscope
energy
reemitted
ranges,
these
parameters
the the
in the
aperture
not been
flow cytometry
cell
in
high
over
or
more
measurements can be used to derive the size, shape, granularity or absorption of cells. There has been extensive work during the last calculations of the scatter from cell models as a scatter
in
10 years
refining
function
of cell
morphology
and
composition.
There
have
been
many
empirical studies of flow cytometry scattering to estimate cell constituents, characterize cell morphology and differentiate subpopulations in heterogeneous populations. Distributional error and nonspecific
scattering
obtaining this
effects high
presently
resolution
technique
has
make
cell
been
it unacceptable
constituent
successfully
as
measurements.
applied
both based on stains for various cellular enzymes light scatter of unstained cells, to counting live versus trypan
blue
staining,
determinantslabeled red
cells
to
with
and
platelets.
differentiating
peroxidase, Scatter
continue
to be developed
a simple
light
source,
and
dye,
wavelengths
generally
fluorescent
in
constituents
such
1%. Since
than
are
measured
stained at
of the incident major
cells
with
one
or
energy.
advantages
These
can be measured emitted
detection
Through
the
into
the
use
of
system
a
more
over
specific to precisions now
cytometry. energy
cells
three
will
other
dyes, better can
be
separated from the incident energy by virtue of its longer wavelength, measurements can be obtained at high signal-to-noise ratio to provide sensitive
estimates
fluorescent
found
that
different can
of
tagged
are
small
amounts
surface
specific
of
for
different
since
constituents
and
be
used
as
or metachromatic dyes a stain and emit at different
two
or more
cell
constituents
can
simultaneously. Still another attribute begun to be explored. If a fluorescent molecule, is excited by polarized light the
constituents
wavelengths,
constituents,
laser),
cell
Third,
determinants.
the
polarization
fluorescence
to which
of the
lifetime
it is bound
is able
fluorescence
of the
dye,
to rotate
emit
like acridine colors for be precisely
and
to the
within
this
will
degree
to of the
of the
image,
or
to the
distribution
the
the
is normally
pulse of cell
fluorescence
which
is related
through this
pulse
can
a
passage
i.e.,
additional
There
are
instruments.
to per to the
the
energy
which
may
be
be used
as other
measurement
mo-
energy
now
two
different
types
of
ical
at
energy
at
lines
in
the
measurements along
a path
aperture
energy
analytic
flow
cytometry
In the first, a laser, often a Helium Neon laser emitting 6300 A for scatter measurements, or an Argon ion laser ultraviolet
as
at
a right
as mechanically possible
fluorescent
to
energy
to
is imaged
by
at
differ-
at
dalities provide morphologic information useful for such purposes as distinguishing single cells from cell clusters. By narrowing the energy source image, in principle, a profile of the chromophore distribution can be obtained as a set of parameters to more completely characterize morphology. It should be noted that with each additional parameter obtained from a flow cytometer, more data processing is required. The complexity of this data processing in terms of the amount of equipment, programs or time required increases in proportion to the number of parameters used to characterize a cell.
light
molecule
will, in each
This
of chromophores,
light is generally measured by sensors and fluorescence is collected by a lens to the plane of the laser beam and the imaging the stream onto an aperture is
to
most
will produce
pulse
These
resolution it the
in intensity
chromophores
cytometer.
ampli-
in intensity
duration
of
better
particles.
source
quantity,
value
this
to
make
a change
voltage
cell
maximum
a flow
energy
the
moving
lifetime.
small
width
related
angle
feasible
increase
from
both
the
to
blue
a lens the
optical
the
for cell
axis.
flow
Scattered
placed opposite to the laser, whose axis is at a right angle cell flow. This condenser lens generally of as high a numer-
to collect
as much
resolution
aperture
wavelengths onto
is
and
fluorescent
sensitivity.
imaged
The
through
dichroic
and/or optical filters appropriate to the experiment onto photomultiplier optical sensors. In some instruments, the cell stream is jetted into air, where it intersects the light beam; in others, the mirrors
measurement
is
made
in
a quartz
flow
cuvette
with
optically
flat
Although the air stream measurement is less susceptible to defects on the quartz surface, I favor measurement in a cuvette because I believe the superior optical design possible with optical flats over the air stream leads to superior sensitivity and resolution. The second type of instrument utilizes an epi-illumination micro-
which
detect
The
of passage
will
of time.
during
related
parameter
in
as a function to time
present
staining
sensitivity,
change
the
paid
specificity,
This
pulse
respect
be
fluorescent
cause
utilize
I believe
coated
systems,
to
a horsepower to
will
of fluorescence
measurement
been
been
of fluorescent
the great
as a photomultiplier.
a
attention
deter-
determinant.
has
indeed
for flow cytometry. of a cell through a focused
source
applications
is, to reproducibly
that
voltage
to the
leading
antibody
as
capability
measured.
walls.
that
more
of flow molecules
instruments.
development
such
with
produce
Thus, by measuring the polarization of the emission, it is possible to characterize the structure of the dye’s environment. There has been a considerable effort to develop fluorescent staining methods for use specifically in flow cytometers to measure cell constituents, such as DNA, RNA, total protein and enzymes, in order to study cell kinetics or to characterize various cells as a function of their environment. The key requirement of a flow cytometer in these is resolution;
to the
of
resolution.
sensitivity
of per cell surface
manufacturers that
one
of fluorescent
laser-based
as
related
the
such
the
has
be
orange different
fluorescence
sensor
cytornetery
sensitivity
there
can
measured
be
above
fluorescence
of fluorescence has recently dye, bound to a biologic (normally available from a emission
and
and
useful technique The passage
emitting
such
dyes
cytometer
multiwatt,
is no question
of the
this and
the
enabling
Perhaps
antibody
improve
sources,
multiconstituent
parameters
can use
denatured
and
to
light
is wrong
duration
sizing
technique
employed
minimally
is
that
provide
as DNA the
and
emission
longer
flow
system
numbers
a fluoresceinated
techniques,
cell,
surface and
cytometry
measurements, their
measurements
techniques than
and
a flow
of
and wide angle dead cells based
containing
fewer
to detect with
flow
integrated
leuko-
to differentiating
the optical
unstained
can be characterized. To perform fluorescence fluorescence
as
since
cells and
numbers
an electronic
However,
to differentiating
cytes on
a means
small
designs
There
of flow
to detect
expensive,
fication
describing
chromophores
among
factors
in order
used
race
differences.
to improve
approach
approach
limiting
a percent,
cell
developers has been to improve most certainly continue, as will
made
tagged powerful
than
chromosomal
by instrument This will
the
less
has been
primarily
more
of
effort
minants
optical
one
single
resolution.
per cell,
large,
differences
efforts
to defme
The
systems.
is measured
of some
cytometers
of
DNA
major
A parallel
is
because
interest
detection
research
and distribustains. It may
renewed
have
measurement
optical
pursued
light scatter of avoiding
current
based
by
energy in principle,
cells
the most
flow cytometry ultraviolet by
to use
numerical
has
of dealing with nonspecific although it has the advantage
attention
numerical
proteins
and reradiated it is possible,
first attempts absorptions
of measurement
ences in a constituent such as the DNA of each cell of different clones. !t is now possible to resolve cell populations in which
scope
system
is imaged
in which by
can
an
the
objective
be flowing
condenser lens
is the
Downloaded from jhc.sagepub.com at University of Southern Queensland on April 28, 2015
of high
direction
lens
of an
numerical
of the
arc
lamp
aperture
optical
illuminator onto
axis,
each
cells
cell
FUTURE
crossing
the focal
by the
objective
plane. lens
The
and
fluorescent
directed
DIRECTIONS
energy
emitted
by a dichroic
mirror
FOR
is collected to one
ments, or
or more
optical filters and photomultipliers. This system can use standard high quality microscope lenses with Kohler illumination to yield the uniform fields and high numerical apertures required to achieve good measurement resolutions, and this type of instrument has, to date, achieved the best resolution. It has the additional advantages of freeing the cytochemist from the restriction of finding dyes that will work
with
system
excitation
has
been
not
at
been
concerned
specific
achieved,
with
high
ever, that this approach antibody tagging as well experiments. a less
will yield
based
simpler
flow cytometry
partially
High
on these
this
its developers
have
I believe,
how-
useful
for
constituent
with for
data
will offer
doing
fluorescent
most
various
the biologist
made
to present
display
or
stereo
meters,
the
more
data
and
other
The
measurements
Handling
obtained
in a useful manner to interpret the test. If one parameter such as amount of a DNA specific dye, is are best displayed as a population In this case, the data handling analyzer used in liquid scintillation present
flow
cytometry
cytometer
must
be displayed
results of an experiment or clinical fluorescence, proportional to the measured, the measurement results histogram of per cell fluorescence. electronics can be a multichannel spectrometry.
experiments,
For
particularly
cell
many
of the
cycle
versatile,
distributions
relative
from
numbers
experiments,
the
of cells
counts
raw
single
parameter
at each
stage
of the
of cells
whose
data
complex
systems
parameters
cell
cycle.
For
are
within
ranges
Data involving either uncorrelated
more than one or as correlated
as uncorrelated, parameter
each can
measurement parameters.
measurement
may be handled as If the data is treated
is treated
be independently
displayed
independently
and
as a population
each
histogram.
to
tinely
4) cell
and
All
of the
with
on
one
For
parameter example,
of per cell fluorescence
for
some
it is useful
to
of one
know
the
more
time
energy,
original
antibody
determinant. a mode is within
stored
per
cell
In these
in which a certain
one
different distribution
cases, parameter
range,
fluorochrome of cells
a multichannel gates it (turns
so the
value
to with
analyzer it on),
of a second
determine a second
the surface
parameter
can
be
for subsequent display, or cells counted based on its value. In certain experiments, particularly is scatter or some dual fluorescence measurements, the parameter measurements are correlated so that subpopulations of cells cannot be distinguished based on parameter ranges, and functions of two or more parameters must be used instead. Analog circuitry to define these functions has been built into the earliest flow cytometers, and current computerized systems allow for methods to define them manually. We have employed techniques for computerized definition of population clusters, and I believe these techniques will be available in future computerized flow cytometers. In some experiments, it is convenient to display correlated measure-
disk
be
or
capable
for
program
of different
histochemical
methods
rou-
for
to
of various
various
agents
are
based
cells.
and
individual cell
on
to additionally
constituent
cytometers
as
or fluorescence
of both
undenatured
beginnings
and the energy
Other
structure forms
as
its
resonance
their
retic
techniques
I
treated
being
made
of different
could
based measurements,
with
fluorescence
chromophores
measurements impedance
of biologic
be possible using
probes
cells,
bound conceivably
on ultrasonic or nuclear
measurements.
It will also be of interest cells,
the
amount.
at wavelengths
and
should
energy
including of electrical
membrane, are
transfer
constituents.
new
cell
used based
characterize
well
scatter,
in the
constit-
will use forms of energy other and other constituents. We should
these
range
cell
quenching
light
Some
characterize
chro-
measurements
absorption,
particularly
different
as
populations
measuring
scatter
Fluorescence
developed
being
visible
polarization
such
of cells;
of light
some
to measure
the
of cells heterogeneous
exception
flow
light
DNA
enzymes
of a specific
future
that
agents;
analysis.
now
cytometry.
to
have
are now
of applications
response
applications
synthesis
magnetic
can be used in ifthat parameter
useful
probably beginning with the ultraviolet and infrared. Measurement of cell structure and the relative positions
antibody
a
be
microprocessor-
determinants;
and some
count
visible
flow
with
what
are programmed.
of various
surface
analysis
viability
are
of
molecules,
from
as a function of cellular
above
techniques
believe
distribution
of
as
or drugs;
differentially
a fluoresceinated antibody to a cell determinant for cells 1) within a specific size range to distinguish one group of cells from another, or from cellular debris based on size, or 2) having fluorescence at a second wavelength related to a second tagged
resulting
ranges to
and
the possible
or
based
parameters.
will
small
will
they
types
following
counting
cell
count
such
a floppy
they
how
the
for RNA
cell
the
other
that on
of cell
5) differential including
outside
a computer
be
inserts
development
constituent
mosomes
to study
or
but
will
user
exception
analysis
antigens
begin
analyzers
expensive,
second
the
3) characterization
than
multichannel
and
depending
2) characterization
which
multiple
of ranges
questions
done:
print or experof, or
requires
will
the
where
store the sets of measurements of each parameter and display histograms for each. In multiparameter measurement iments, the user is often interested in knowing the distribution
This
answer
cyto-
multiparameter
as a function
thus
The
cytometric
uents
ments.
parameters
use
which
with
advanced
some
are needed rather than distributions. For other experwe have found it useful to characterize populations of cells by derived statistically from the distributions of their measure-
to
of operation
mitogens, of
flow
to handle
Applications
measurements
iments, indices
in
1) cell cycle
estimate
have a color
computerize
be able
contour
using
simultaneously
will
two
dot,
attempts
and the instrument is set up to perform the user’s specific store his data, and generate the appropriate report. Such will be run and controlled as present clinical hematology
Flow
analysis,
to
Some
manufacturers
could
methodology.
modes
this is all that is required, especially if the inherent resolution of the instrument is good. If not, a computer has been used to reconstruct model
least
using
data.
and RNA per cell of cells: 1) develop the techniques with a certain enzyme or surface determinant. I believe there will be two types of data handling systems developed in the future. The first will be used on the large multiparameter flow cytometer systems, and will
instruments
in a flow
As users
They
developed
parameters
viewer. at
been
parameter
three
affluent
display
instruments Data
have two
are the correlated distributions of DNA of a certain size, or 2) when cytochemists
based
experiments.
of
parameters.
cartridge, experiment,
analytic
techniques
displays
developing
handling
routine
and
been
1651
CYTOMETRY
isometric
be
measurement
appropriate
principles
instrument
for
because
instruments
that
sensitivity
measurements.
resolution
believe
a system
expensive,
lines.
resolution as high
I further
electronics,
laser
FLOW
surface
charges combined
to characterize and
the electrical
transmembrane
with
optical
potential. measurements
properties
of
Electrophocould
be
one
approach to characterizing the electrical properties of cells as a function of various agents and their relationship to other cells. Although a major impetus to flow cytometry has been to characterize large cell populations rapidly, the fact that some cell characteristics are heterogeneous, no matter how precisely we perform measurements, will make it of interest to study one or more properties of an individual cell as a function of changes in its environment. Thus, last
in
my
list
of
future
directions,
is the
belief
that
techniques developed for flow cytometry instrumentation applied in this opposite direction to determine the kinetics ual cells within large populations.
Downloaded from jhc.sagepub.com at University of Southern Queensland on April 28, 2015
some
of
the
will now be of individ-
