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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a flow cytometry apparatus, and more
particularly, concerns a flow cytometry apparatus for determining one or
more characteristics of cells or the like which includes improved optics
for wide angle light scatter and fluorescence detection.
2. Description of the Prior Art
Flow cytometry apparatuses rely upon the flow of cells or other particles
in a liquid flow stream in order to determine one or more characteristics
of the cells under investigation. For example, a liquid sample containing
cells is directed through the flow cytometry apparatus in a rapidly moving
liquid stream so that each cell passes serially, and substantially one at
a time, through a sensing region. Cell volume may be determined by changes
in electrical impedance as each cell passes through the sensing region.
Similarly, if an incident beam of light is directed at the sensing region,
the passing cells scatter such light as they pass therethrough. This
scattered light has served as a function of cell shape, index of
refraction, opacity, roughness and the like. Further, fluorescence emitted
by labeled cells which have been excited as a result of passing through
the excitation energy of the incident light beam is detectable for
identification of specifically labeled cells. Not only is cell analysis
performed on the flow cytometry apparatuses, but sorting of cells may also
be achieved. Lasers have been used as the source of the incident beam of
illumination in flow cytometry apparatuses, as well as sources of
incoherent and non-collimated light, such as mercury or xenon arc lamps.
In most of the presently known and available flow cytometry apparatuses,
fluorescence emitted by the cells is typically collected at an angle whose
viewing axis is 90.degree. relative to the axis of light excitation. In
addition, wide angle light scatter, typically at 90.degree., has been
found to obtain information about cells relating to shape and internal
morphology. Inasmuch as both light scatter and fluorescence at 90.degree.
may be collected with the same optical collection system, filters or light
beam separators have been utilized to split the 90.degree. fluorescence
and light scatter so that each may be detected separately. Furthermore,
for most efficient collection, and if the incident light beam is provided
by a laser, the light beam is typically polarized with the electric vector
oriented normal to the plane containing the 90.degree. angle defined by
the excitation and collection axes.
It is a property of scattering, that light scattered at right angles from
the incident beam retains its polarization. On the other hand, the
polarization of fluorescence depends upon the molecules themselves, and
not upon the incident light beam. In practice, however, fluorescence from
most cells is usually modestly unpolarized.
At present, dichroic filters are employed in separating a light beam having
both fluorescence and scatter components. Such filters frequently do not
transmit fluorescence as efficiently as would be desirable. Additionally,
dichroic filters are typically wavelength dependent. For instance,
changing the excitation wavelength from one frequency to another would
require a different dichroic filter. It is also expensive to fabricate
these types of dichroic filters.
In some instances, dielectric broadband filters have been employed to
separate the light scatter and fluorescence components of a light beam.
Such broadband filters normally do not have polarization benefits. In
other words, and for example, reflecting 25% of the scatter would imply
transmitting only 75% of the fluorescence.
It has also been known to employ uncoated glass or quartz as a beam
splitter plate in a flow cytometry apparatus. Such a glass plate has
typically been positioned at a 45.degree. angle relative to the incident
light beam comprising both light scatter and fluorescence components. At
the 45.degree. angle, fluorescence is transmitted efficiently, but light
scatter is reflected rather inefficiently.
Many different flow cytometry apparatuses and techniques have been
described in a review article by John A. Steinkamp, entitled "Flow
Cytometry," Review of Scientific Instruments 55 (9), September 1984. While
there are a variety of different approaches for optically sensing the
characteristics of cells, there is still a need for further improvements
in such optics so that cellular information may be obtained more
efficiently and reliably.
SUMMARY OF THE INVENTION
The flow cytometry apparatus of the present invention for determining one
or more characteristics of particles or the like flowing in a liquid
stream comprises means for moving particles, substantially one at a time,
in a liquid flow stream. Means provides a plane polarized beam of light to
illuminate the particles moving in the stream. Beam splitter means is
positioned in the collected beam light path so that light scattered and
fluorescence emitted by each moving particle are directed thereto. The
splitter means is oriented at an incidence angle greater than 45.degree.,
preferably approaching Brewster's angle, so as to transmit fluorescence
and to reflect light scatter. Means for detecting the transmitted
fluorescence and the reflected light scatter related to each particle is
included, which also associates the detected scattered light and
fluorescence with one or more characteristics of such particle.
In a preferred embodiment of the present invention, the source for
providing an incident beam of linearly polarized light is a laser, which
also provides excitation energy to cause fluorescently labeled cells in
the flow stream to emit fluorescence. The preferred apparatus includes a
light scatter detector and a fluorescence detector which respectively
detects light scattered and fluorescence emitted by each moving cell at
substantially 90.degree. relative to the axis of the laser beam. A beam
splitter is positioned in the light path between the cells and the
detectors so that both light scatter and fluorescence at substantially
90.degree. are directed to the beam splitter. It is preferred that the
beam splitter be oriented at its Brewster's angle relative to the incident
light scatter and fluorescence to transmit fluorescence and to reflect
light scatter.
In accordance with the principles of the present invention, improved optics
are provided to split the light scatter/fluorescence beam, particularly
for wide angle detection of such light-related information about the cells
moving through the flow cytometry apparatus. The present invention is
particularly advantageous if both light scatter and fluorescence are to be
collected with the same optical collection system. Use of a preferably
uncoated flat plate of glass at a wide incidence angle, such as at
Brewster's angle, with the plane of incidence normal to the electric
vector of the laser beam, serves as an ideal beam splitter. A very high
percentage of unpolarized fluorescence is transmitted through the beam
splitter of the present invention, while an effective percentage of light
scatter is reflected off the beam splitter so that such light scatter may
be detected. Furthermore, the present invention employs such a beam
splitter without the reliance upon dichroic filters or dielectric
broadband filters.
Accordingly, the present beam splitter is straightforward and economical to
fabricate, and is modestly independent of excitation wavelength. In
addition, the beam splitter may be oriented at an optimum angle of
incidence so as to balance the loss in fluorescence with the gain in
scatter to thereby obtain the best efficiency for both light scatter and
fluorescence detection. A number of other advantages, in addition to the
aforementioned significant advantages, are offered by the present
invention as will become more apparent from a reading the the detailed
description set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of a preferred embodiment of the optical
elements and light paths of a flow cytometry apparatus of the present
invention for determining one or more characteristics of cells or the
like;
FIG. 2 is an enlarged schematic representation of the preferred angularly
oriented, scatter/fluorescence beam splitter of the present invention; and
FIG. 3 is a graphic representation of the efficiency characteristics of the
present beam splitter depicting 90.degree. light scatter and fluorescence
as parameters.
DETAILED DESCRIPTION
While this invention is satisfied by embodiments in many different forms,
there is shown in the drawings and will herein be described in detail a
preferred embodiment of the invention, with the understanding that the
present disclosure is to be considered as exemplary of the principles of
the invention and is not intended to limit the invention to the embodiment
illustrated. The scope of the invention will be measured by the appended
claims and their equivalents.
Adverting to the drawings, and FIG. 1 in particular, the optical and
particle flow elements of a flow cytometry apparatus 10 are illustrated.
The optical and flow elements of FIG. 1 represent the major components of
a flow cytometry apparatus for moving particles, such as cells or the
like, in a liquid stream, substantially one at a time, in order to assess
those particles for specific characteristics thereof. For example, the
elements of the device of FIG. 1 may be included in a FACS.TM.
fluorescence-activated cell sorter, manufactured and sold by Becton
Dickinson Immunocytometry Systems, Mountain View, Calif. The FACS cell
sorter analyzes and sorts cell populations on the basis of light scatter
and fluorescence in a wide variety of research laboratory applications. In
addition to the optical and flow elements to be described in more
particular detail herein, and which may be embodied in an instrument such
as the FACS cell sorter, other details of a cell sorting apparatus useful
in conjunction with the present invention are described in U.S. Pat. No.
3,826,364. It is understood that the present invention is useful in many
different types of flow cytometry or flow fluorometric apparatuses,
whether measuring light scatter, particle volume, fluorescence or other
optical parameters for the identification or quantification of cells or
the like in a sample liquid medium. The optical elements, in particular,
of the present invention represent the essence of the improvement in flow
cytometry apparatuses such as described in the aforementioned patent.
As illustrated in FIG. 1, light energy is provided for the present flow
cytometry apparatus by a light source 12 such as a laser which provides a
linearly polarized collimated beam of light at a singular wavelength or at
a number of discreet wavelengths. Alternatively, light source 12 may be
broad-spectrum arc lamp, such as mercury or xenon, with polarizing means
included in excitation light path 14a produced by light source 12.
Typically, apparatus 10 also includes a lens 15 which focuses the light
beam at a liquid stream 16 containing the particles or cells under
investigation.
A nozzle 18, incorporated within the flow cytometry apparatus of the
present invention, facilitates the flowing of cells or particles within
liquid stream 16. The utilization of a nozzle of this type is well-known
and is described, for example, in U.S. Pat. No. 3,826,364. Nozzle 18
provides a hydrodynamically focused flow of cells within a sheath fluid,
the sheath fluid and cells comprising liquid flow stream 16. As each cell
or particle passes through the focused light region 20, where light beam
14 intersects liquid stream 16, light scattered thereby may be detected.
An appropriate photodetector 21, as illustrated in FIG. 1, is positioned
to receive light scattered forwardly by each cell. Before describing the
elements associated with the detection of fluorescence and wide angle
light scatter, it should be pointed out that the particles in liquid
stream 16 may be collected in an appropriate container 22, or, perhaps,
may be sorted and collected in different containers if the flow cytometry
apparatus employs a sorting capability.
Fluorescence, if emitted by cells energized by the illumination from the
light source, may also be detected. Similarly, light scattered in
different directions, besides the forward direction, may be detected. In
laser excited flow cytometry, both fluorescence and wide angle light
scatter are typically collected at an angle whose viewing axis is
90.degree. relative to the excitation axis of light beam 14. In FIG. 1,
axis 24 represents the 90.degree. viewing access for the collection of
fluorescence and wide angle scatter. Thus, light traveling along axis 24,
for purposes of the ensuing discussion, includes both light scatter and
fluorescence as its components.
In order to collect fluorescence and light scatter at the 90.degree. angle
from the incident laser beam, the light scatter and fluorescence should be
separated or split. To accomplish such splitting without relying upon the
presently used dichroic filters, a beam splitter plate 25 is utilized by
the present invention. Beam splitter 25 functions to receive both
scattered light and fluorescence at the 90.degree. angle and to re-direct
each such component in different directions. Such re-direction of light
scatter and fluorescence then permits each of the light scatter and
fluorescence to be collected separately, even though both originate at the
90.degree. angle.
Specifically, beam splitter 25 is intended to transmit fluorescence and to
reflect light scatter. To this end, the beam splitter is positioned along
axis 24 so that it is angularly oriented relative to axis 24. This angle
normally referred to as the angle of incidence is designated as angle B in
FIG. 2 and is the angle between axis 24 and a vector 26 which is a line
extending normal to the surface 28 of beam splitter 25. For best results
in accordance with the present invention, angle B should be greater than
45.degree.. Preferably, angle B is the Brewster's angle of the beam
splitter plate. As is well-known in the optical field, Brewster's angle
represents the angle of incidence at which the amount of reflectance is
minimized for light polarized parallel to the plane of incidence. Thus, at
such angle of incidence, there would be little or no reflection loses.
Brewster's angle is, moreover, a function of the material out of which
beam splitter 25 is fabricated. When angularly oriented as illustrated in
FIG. 2, light traveling along axis 24, comprised of light scatter and
fluorescence, is split into two beams. Fluorescence, which consists of
both polarization vectors, is efficiently transmitted through beam
splitter 25 and travels along the axis represented by arrows 30. On the
other hand, scattered light consists only of the polarization vector which
has a reflectance component at Brewster's angle, and is thus represented
by arrows 31. Once light scatter and fluorescence have been separated,
each may be collected by appropriate photodetectors.
For example, the reflected light scatter may be collected in photodetector
32 as illustrated in FIG. 1. Before the transmitted fluorescence traveling
along axis 30 is collected, the fluorescence signal may be further
refined. If the transmitted fluorescence includes a number of different
color regions, a dichroic mirror 34 may be utilized to separate the
different color wavelengths. Thus, and for example, fluorescence in the
green color region may be reflected by dichroic mirror 34 along axis 35
and collected in an appropriate photodetector 36. On the other hand,
fluorescence in the red color region may be transmitted through dichroic
mirror 34 along axis 38 and collected in an appropriate photodetector 39.
While not illustrated in FIG. 1, those skilled in the art will appreciate
that various lenses, filters, barriers or the like may be employed in
conjunction with each of the photodetectors to obtain as pure a signal as
possible. Obtaining such optically clean signals is most desirable
particularly when a four-parameter sensing apparatus (two fluorescence
channels and two light scatter channels) is utilized, such as the
apparatus illustrated in FIG. 1.
Referring once again to FIG. 2, it can be seen that both surfaces 28 and 29
of beam splitter 25 have been used to reflect and transmit light scatter
and fluorescence, respectively. In order to effectively use both surfaces
of the beam splitter for the transmission and reflectance properties, it
is preferred that beam splitter 25 be a thin, uncoated flat plate. For
example, a beam splitter having a thickness range of 1 to 2 mm has been
found to perform effectively in the present invention.
While different materials may be chosen for the beam splitter, having the
capability of reflecting light scatter and transmitting fluorescence,
particularly when unpolarized, commonly available low cost glasses are
most desirable. In particular, a borosilicate glass, sold under the trade
name Pyrex, has been found to be most suitable for the present invention.
A thin, flat, polished piece of borosilicate glass provides a beam
splitter that transmits substantially more than 75% of the unpolarized
fluorescence which strikes the splitter. This same borosilicate glass beam
splitter reflects an amount of scattered light so as to obtain a signal
which may be adequately detected. For borosilicate glass, Brewster's angle
occurs at approximately 56.degree.. Thus, in FIG. 2, angle B, when beam
splitter 25 is made from borosilicate glass, would be at approximately
56.degree. to obtain the results in accordance with the present invention.
It should be pointed out that the angle at which beam splitter 25 is
oriented along axis 24 typically represents a trade-off between the
efficiencies of fluorescence transmission and light scatter reflectance.
Assuming the fluorescence to be unpolarized, reference to FIG. 3
illustrates the efficiencies of fluorescence transmission and light
scatter reflectance at 90.degree. as a function of beam splitter plate
angle. As the incident angle of the plate increases, the fluorescence
efficiency decreases and the scatter efficiency increases. Therefore,
choosing the optimum angle of incidence is a matter of balancing the loss
in fluorescence with gain in scatter. Since scattering is a far more
efficient process than fluorescence, the angle should be increased only to
the point where the loss in fluorescence is substantially undetectable in
terms of signal-to-noise performance. For example, by increasing the
incidence angle from 45.degree. to Brewster's angle (seen in FIG. 3 as
approximately 56.degree.), the fluorescence efficiency only decreases by
about 4%, an amount so small as to be substantially undetectable in terms
of signal-to-noise. At the same time, the scatter efficiency is increased
nearly 60%. If the plate angle is further increased to 65.degree., for
example, the fluorescence efficiency is decreased by about 11%, which
starts to become noticeable in terms of fluorescence sensitivity.
Therefore, at the Brewster's angle for borosilicate glass, the beam
splitter of the present invention transmits approximately 88% of the
unpolarized fluorescence and reflects approximately 25% of the available
light scatter at 90.degree.. These efficiencies are quite satisfactory in
collecting the respective fluorescence and light scatter signals.
Once the above-described photodetectors receive the scatter and
fluorescence signals, the information gained thereby may be further
utilized. The various photodetectors may be well-known photomultiplier
tubes or similar devices which convert light signals into electrical
impulses so that the light thereby detected may be associated with the
cells flowing through the apparatus. The electrical signals from the
photodetectors are typically fed to the electronics (not shown) of the
apparatus for purposes of display, storage or further processing so that
one or more characteristics of the cells under analysis may be determined.
Thus, the present invention provides a scatter/fluorescence beam splitter
for a flow cytometry apparatus which is particularly useful for wide angle
light scatter and fluorescence detection. Particularly when the present
beam splitter is oriented at a wide incidence angle, such as its
Brewster's angle, a simple, economical light filter is provided.
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