U.S. patent application number 11/094723 was filed with the patent office on 2006-10-05 for flow cytometer for differentiating small particles in suspension.
This patent application is currently assigned to Beckman Coulter, Inc.. Invention is credited to Mark A. Wells.
Application Number | 20060221325 11/094723 |
Document ID | / |
Family ID | 37018928 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060221325 |
Kind Code |
A1 |
Wells; Mark A. |
October 5, 2006 |
FLOW CYTOMETER FOR DIFFERENTIATING SMALL PARTICLES IN
SUSPENSION
Abstract
A flow cytometer includes an optical flow cell through which
particles to be characterized on the basis of at least their
respective side-scatter characteristics are caused to flow
seriatim. A plane-polarized laser beam produced by a laser diode is
used to irradiate the particles as they pass through a focused
elliptical spot having its minor axis oriented parallel to the
particle flow path. Initially, the plane of polarization of the
laser beam extends perpendicular to the path of particles through
the flow cell. A half-wave plate or the like is positioned in the
laser beam path to rotate the plane of polarization of the laser
beam so that it is aligned with the path of particles before it
irradiated particles moving along such path.
Inventors: |
Wells; Mark A.; (Davie,
FL) |
Correspondence
Address: |
BECKMAN COULTER, INC.
P.O. BOX 169015
MAIL CODE 32-A02
MIAMI
FL
33116-9015
US
|
Assignee: |
Beckman Coulter, Inc.
Fullerton
CA
|
Family ID: |
37018928 |
Appl. No.: |
11/094723 |
Filed: |
March 30, 2005 |
Current U.S.
Class: |
356/73 ; 356/318;
356/336; 356/442 |
Current CPC
Class: |
G01N 15/1434 20130101;
G01N 15/1404 20130101 |
Class at
Publication: |
356/073 ;
356/336; 356/442; 356/318 |
International
Class: |
G01N 21/00 20060101
G01N021/00; G01N 15/02 20060101 G01N015/02 |
Claims
1. A flow cytometer comprising: (a) an optical flow cell having a
particle-interrogation zone through which particles to be
characterized can be made to pass, one-at-a-time, along a
substantially linear particle path; (b) a laser for producing a
plane-polarized laser beam, said laser being oriented so that the
plane of polarization of said laser beam is perpendicular to the
particle path; (c) a beam-shaping lens system for focusing said
laser beam as an elliptical spot centered on said particle path
within said particle-interrogation zone, the minor axis of said
elliptical spot being arranged parallel to said particle path; (d)
a side-scatter photodetector positioned to detect a portion of said
laser beam upon being scattered by particles irradiated by said
focused elliptical spot in a direction substantially perpendicular
to said particle path and to the direction from which said laser
beam irradiates a particle at said particle-interrogation zone; and
(e) a polarization-rotating optical element positioned in said
laser beam between said laser and said optical flow cell, said
optical element serving to rotate the plane of polarization of said
laser beam before said laser beam irradiates particles at said
particle-interrogation zone.
2. The apparatus as defined by claim 1 wherein said
polarization-rotating optical element serves to rotate the plane of
polarization of said laser beam by an amount sufficient to render
the plane of polarization of the particle-irradiating laser beam
substantially parallel to said particle path.
3. The apparatus as defined by claim 2 wherein said
polarization-rotating optical element operates to rotate the plane
of polarization of said laser beam by 90 degrees.
4. The apparatus as defined by claim 3 wherein said
polarization-rotating optical element comprises a half-wave
plate.
5. The apparatus as defined by claim 3 wherein said
polarization-rotating optical element comprises a pair of
quarter-wave plates.
6. A flow cytometer comprising: (a) an optical flow cell having a
linear particle path along which particles to be characterized can
be made to pass seriatim; (b) a laser diode for producing a
plane-polarized laser beam of elliptical cross-section having
mutually perpendicular major and minor axes, said laser diode being
arranged so that the major axis of said elliptical cross-section
extends perpendicular to said particle path; (c) a lens system for
focusing said laser beam to an elliptical spot having mutually
perpendicular major and minor axes on said particle path, said spot
being oriented so that said minor axis of the focused spot is
parallel to said particle path; (d) a side-scatter photodetector
positioned to detect laser radiation scattered in a direction
substantially perpendicular to the direction of particle flow and
to the direction at which said laser beam irradiates a particle
moving along said particle path; and (e) an optical element
positioned in said laser beam and functioning to rotate the plane
of polarization of said laser beam by 90 degrees before said laser
beam irradiates particles on said particle path.
7. The apparatus as defined by claim 6 wherein said optical element
comprises a half-wave plate.
8. The apparatus as defined by claim 6 wherein said optical element
comprises a pair of quarter-wave plates arranged in tandem.
9. A flow cytometer comprising: (a) an optical flow cell having a
particle-interrogation zone through which particles to be
characterized can be made to pass, one-at-a-time, along a
substantially linear particle path; (b) a laser that produces a
plane-polarized laser beam of expanding elliptical cross-section
having mutually perpendicular major and minor axes, said laser beam
being plane-polarized in a plane parallel to the major axis of the
expanding elliptical cross-section, said being oriented with
respect to said optical flow cell so that said plane of
polarization is substantially perpendicular to said linear particle
path; (c) a collimating lens for collecting and collimating a major
portion of said laser beam to provide a collimated laser beam, said
collimating lens further operating to truncate a portion of said
expanding elliptical cross-section and thereby give rise to
spurious light sources appearing at opposite sides of said
collimating lens; (d) a beam-shaping lens system for (i) focusing
the collimated laser beam as an elliptical spot centered on the
linear particle path within the particle-interrogation zone, and
(ii) condensing light from said spurious light sources as spurious
light spots on opposite sides of, and along the major axis of, the
focused elliptical spot, said beam-shaping lens system being
effective to orient said focused elliptical spot so that its minor
axis extends parallel to said particle path, thereby positioning
the spurious light spots on opposite sides of and spaced from said
particle path; (e) a side-scatter photodetector positioned to
detect laser radiation scattered from a particle irradiated by said
focused elliptical spot in a direction substantially perpendicular
to the particle path and to the direction at which said laser beam
irradiates a particle at said particle-interrogation zone; and (f)
a polarization-rotating optical element, positioned in said laser
beam and adapted to rotate the plane of polarization of said laser
beam by 90 degrees before said laser beam irradiates particles at
said particle-interrogation zone.
10. The apparatus as defined by claim 9 wherein said
polarization-rotating optical element is positioned in the
collimated portion of said laser beam.
11. The apparatus as defined by claim 9 wherein said
polarization-rotating optical element comprises a half-wave
plate.
12. The apparatus as defined by claim 9 wherein said
polarization-rotating optical element comprises a pair of
quarter-wave plates arranged in tandem.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to improvements in flow
cytometers of the type commonly used to differentiate small
particles, e.g., various types of blood cells, in a liquid
suspension. More particularly, this invention relates to
improvements in flow cytometers of the type that use
plane-polarized radiation, e.g., that emitted from laser diodes, to
irradiate individual particles passing through an optical flow cell
in order to detect the light-scattering characteristics of such
particles and thereby characterize each particle as being a member
of a particular class or type.
[0003] 2. The Prior Art
[0004] Flow cytometers are commonly used to differentiate
individual small particles of different types in a particle
suspension on the basis of the light-scattering and/or fluorescence
characteristics of each particle. Such instruments generally
include an optically-transparent flow cell having a
particle-interrogation zone through which particles from the sample
are made to pass in single file; a laser light source for
irradiating such particles, one-at-a-time, as they pass through the
particle-interrogation zone; and a plurality of optical detectors
that are strategically located about the flow cell to receive both
scattered radiation from the irradiated particles, and fluorescence
radiation emitted by fluorochromes that have been previously
attached to certain particles in a class or of a type of interest.
Typically, the photodetectors are positioned to detect both
forwardly-scattered radiation within angular ranges determined by
the geometry of the light-sensitive elements of the photodetector,
and side-scattered radiation that is scattered in direction
substantially perpendicular to the directions of the irradiating
laser beam and of the particle path. The respective outputs of the
photodetectors are then processed in a known manner to identify
each of the irradiated particles as a member of a particular class
or type. Usually, the flow cytometer provides a histogram or
scattergram indicating the number of particles in each class or of
each type.
[0005] In flow cytometers of the above type, it is becoming
increasingly common to employ laser diodes as the
particle-irradiating laser light source. Such solid-state devices
are often preferred over the more conventional gas lasers, e.g.,
helium-neon and argon lasers, on the basis of size and economic
considerations. Being of relatively small size, these devices can
be easily positioned and oriented within the instrument housing to
achieve any of various design objectives. While laser diodes may be
considered advantageous in many respects, they are not without
disadvantages. For example, in addition to being relatively
low-power devices, laser diodes typically emit non-collimated
radiation that must be collimated for practical use. In most
devices, the output radiation emitted from the active
semi-conductive element or "die" tends to diverge relatively
quickly and, since the die is usually rectangular in shape, the
emitted radiation diverges differentially in mutually perpendicular
planes. Thus, the radiant output from a laser diode is commonly in
the form of an expanding ellipse that typically expands in one
plane at a relatively large angle of divergence of, say, 30
degrees, while expanding in a perpendicular plane at a much smaller
angle of divergence of, say, 10 degrees. To capture and collimate
the laser energy in this expanding beam, it is common to position a
collimating lens of relatively high numerical aperture in close
proximity to the laser source. While this collimating lens readily
collects all of the energy diverging from the source at the smaller
angle, it often truncates a portion of the beam diverging at the
larger angle. In many laser diodes, the result of this truncation
is that a pair of extraneous or spurious light sources (or
far-field diffraction patterns) composed of diffracted and/or
reflected light appear at the opposing sides of the collimating
lens where the beam-truncation occurs. While these spurious light
sources are usually of relatively low intensity compared to the
collimated main beam, they can be problematic to the performance of
a flow cytometer. For example, when focusing the output beam from a
laser diode to an elliptical spot adapted to irradiate particles
moving through a flow cell, the focused elliptical spot will be
accompanied by a pair of relatively faint and ill-defined lobes of
radiation or "light-lobes" representing the focused spurious beams
of radiation emanating from opposite sides of the collimating lens.
These light-lobes appear on opposite sides and outside the boundary
of the focused elliptical spot. In the event these light-lobes are
positioned in the particle path, they will give rise to low-level
light-scatter and fluorescence signals that act to complicate the
signal processing of the flow cytometer. Specifically, such
low-level signals appear to emanate from small particles that, in
fact, are not present in the particle sample.
[0006] In U.S. Pat. No. 6,713,019 to Ozasa et al., the above-noted
light-lobe problem is addressed by simply adjusting the orientation
of a laser diode in a flow cytometer so that the above-noted
light-lobes are located outside the particle path through the flow
cell, i.e., to position the lobes in a plane that is perpendicular
to the particle path. Ozasa et al. note that it is conventional to
orient a laser diode in a flow cytometer so that the major axis of
the expanding ellipse is parallel to the direction of particle flow
through the flow cell (which is normally vertical). This
orientation of the laser diode enables the beam to be focused, upon
passing through the combination of a cylindrical lens and a
condensing lens, to a particle-irradiating ellipse that is (a)
diffraction-limited in a plane parallel to its minor axis, and (b)
centered on the nominal particle path within the flow cell with it
minor axis extending parallel to such path. Because the focused
ellipse is diffraction-limited in a plane parallel to the particle
path, the flux density of the focused beam is maximized which, in
turn, enhances the light-scatter and fluorescence detection
sensitivity of the instrument. Also, being diffraction-limited in a
plane parallel to the particle path, the focused ellipse prevents
the simultaneous irradiation (and detection) of multiple particles
traveling relatively close together in the time domain, i.e., in
the direction of particle flow. But, as noted by Ozasa et al., a
significant drawback of this conventional orientation of a laser
diode in a flow cytometer is that it acts to position the
above-noted light-lobes directly in the particle path, causing the
detectors to mistakenly detect and count small particles that do
not, in fact, exist. Ozasa et al.'s solution to this problem, as
indicated above, is simply to rotate the laser diode by 90 degrees
relative to its support housing so that the major axis of the
expanding ellipse is now horizontal, i.e., perpendicular to the
(normally vertical) particle path through the flow cell. This has
the effect of shifting the lobe radiation outside the particle
path, on opposite sides thereof. As a result of this orientation,
the particles passing through flow cell are not irradiated by the
light lobes and, hence, cannot scatter radiation or emit
fluorescent radiation as a result of such irradiation. Thus, there
is no need to compensate for the presence of such lobe radiation in
the respective outputs of the photodetectors. Note, while such an
orientation of the laser diode would result in a 90 degree rotation
of the focused elliptical spot, causing its major axis to be
undesirably aligned with the particle path, Ozasa et al. avoids
this situation by adding an additional lens to the beam-shaping
optical system. The additional lens operates to restore the shape
of the focused ellipse to that occurring before the rotation of the
laser diode, i.e., to an ellipse in which the minor axis is
parallel to the particle path.
[0007] While the orientation of the laser diode taught in the Ozasa
et al. patent may solve the light-lobe problem identified, it has
been observed to create another optical problem affecting the
detection of radiation scattered by the irradiated particles. More
specifically, when it is desirable to detect side-scatter radiation
from the irradiated particles (i.e., radiation scattered at 90
degrees relative to both the optical axis of the irradiating beam
and to the particle path through the flow cell), it has been
observed that the suggested (horizontal) orientation of the laser
diode has the effect of dramatically reducing the signal-to noise
ratio (SNR) of the side-scatter signal. The extent of this SNR
reduction is such that the side-scatter parameter cannot be used as
part of the particle characterization process.
SUMMARY OF THE INVENTION
[0008] In view of the foregoing discussion, an object of this
invention is to provide a solution to the problem identified, i.e.,
the problem of detecting side-scattered radiation when a laser
diode is so oriented that the major axis of its expanding
elliptical output beam is perpendicular to the particle path
through the flow cell.
[0009] In accordance with one aspect of the invention, it is
recognized that the output beam from a laser diode is
plane-polarized in the plane in which the beam is diverging the
fastest. Further, it is recognized that measurement of
side-scattered radiation in a flow cytometer requires that the
plane of polarization of the irradiating laser beam must be
parallel to the direction of the particle path through the flow
cell. Still further, it is recognized that in a flow cytometer of
the type described above, i.e., one in which the orientation of a
laser diode has been rotated by 90 degrees so as to eliminate the
"light lobe" problem identified, the plane of polarization of the
particle-irradiating laser beam is no longer parallel to the
particle path and, hence, the ability to detect side-scattered
radiation is compromised. Thus, in order to make side-scatter
measurements with such an instrument, it is necessary to
re-establish the requisite relationship between the plane of
polarization of the irradiating beam and the particle path.
[0010] In view of the above, a flow cytometer structured in
accordance with the present invent comprises the following
elements: (a) an optical flow cell having a particle-interrogation
zone through which particles to be characterized can be made to
pass, one-at-a-time, along a substantially linear particle path;
(b) a laser source for producing a plane-polarized laser beam, such
laser being oriented so that the plane of polarization of its laser
beam is perpendicular to the particle path; (c) a beam-shaping lens
system for focusing the laser beam as an elliptical spot centered
on the particle path within the particle-interrogation zone, the
minor axis of such elliptical spot being arranged parallel to the
particle path; (d) a side-scatter photodetector positioned to
detect a portion of the laser beam upon being scattered by
particles irradiated by the focused elliptical spot in a direction
substantially perpendicular to the particle path and to the
direction from which the laser beam irradiates a particle at the
particle-interrogation zone; and (e) a polarization-rotating
optical element positioned in the laser beam path between the laser
source and the optical flow cell, such optical element serving to
rotate the plane of polarization of the laser beam to such an
extent that the plane of polarization is arranged substantially
parallel to the particle path, rather than perpendicular to such
path, as in the prior art system. Preferably, the
polarization-rotating optical element comprises either a half-wave
plate, or the combination of two quarter-wave plates, which serves
to rotate the plane of polarization of the laser beam by 90
degrees.
[0011] The invention and its advantages will be better understood
from the ensuing detailed description of preferred embodiments,
reference being made to the accompanying drawings in which like
reference characters denote like parts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates the diverging output beam from a
conventional laser diode;
[0013] FIGS. 2 and 3 are schematic illustrations of prior art
optical systems for focusing a diverging laser beam at a desired
location within an optical flow cell of a flow cytometer;
[0014] FIGS. 4A and 4B illustrate the effect on light scatter of a
change in the plane of polarization of a plane-polarized light
source; and
[0015] FIG. 5 is a perspective schematic illustration of a flow
cytometer embodying the present invention
DETAILED DECRIPTION OF PREFERRED EMBODIMENTS
[0016] Referring now to the drawings, FIG. 1 schematically
illustrates a conventional laser diode LD of the type that emits a
diverging laser beam LB having an elliptical cross-section that
expands in size in accordance with two different angles of
divergence, .theta.1 and .theta.2. As illustrated, these angles are
measured in mutually perpendicular planes, and one angle, in this
case angle .theta.1, is usually substantially larger than the
other. As is characteristic of laser diodes, the emitted laser beam
LB will be plane-polarized in a plane P that is parallel to the
major axis A' of the expanding elliptical cross-section of the
beam. The minor axis A'' of the elliptical cross-section is, of
course, perpendicular to the major axis A'.
[0017] In FIG. 2, the laser diode LD of FIG. 1 is shown as being
embodied in a flow cytometer of the earlier type described above. A
particle sample PS containing particles to be analyzed, e.g., blood
cells, is introduced by a nozzle 7 into an optically-transparent
flow cell 6 in the direction of arrow A. A sheath liquid S is
supplied to the flow cell in such a manner as to surround the
particle stream provided by nozzle 7, and thereby serves to
hydrodynamically constrain the flow of the particle stream to the
central axis of the flow cell, which is commonly vertically
disposed. The rate of flow of the sheath fluid, in combination with
the rate at which the particle sample is introduced by the nozzle
7, causes the particles to pass, one-at-a-time, through a particle
interrogation zone Z' located at the center of the flow cell. The
diverging laser beam LB emitted by the laser diode is collimated by
a collimating lens, 3 and the resulting collimated main beam MB is
passed through the combination of a cylindrical lens 4 and a
condensing lens 5 to bring the main beam to an elliptical spot
focus at the particle-interrogation zone. For the reasons noted
above, it was conventional in the art to orient the laser diode so
that major axis A' of its elliptical output beam extended parallel
to the (normally vertical) particle path through the flow cell.
(Note, in this orientation, the plane of polarization P of the
laser beam also extends parallel to the particle path.)
Notwithstanding this vertical orientation of the elliptical beam,
the beam is brought to an elliptical spot focus in which the minor
axis of the focused spot extends parallel to the particle path.
This provides the technical advantages noted above.
[0018] With the laser diode orientation shown in FIG. 2, the
collimating lens 3 will usually truncate a portion of the diverging
laser beam in the plane in which the beam is expanding the faster,
i.e., in the vertical plane in which angle .theta.1 is measured.
Assuming the collimating lens is centered with respect to the
diverging laser beam, this truncation gives rise to a pair of
spurious beams SB1 and SB2 of relatively low intensity which appear
to emanate from opposite sides 3a and 3b of the collimating lens 3
where the truncation takes place. In addition to focusing the main
beam as an elliptical spot as described above, lenses 4 and 5 act
to focus these spurious beams as beam spots (or light lobes) BS1
and BS2 on opposite sides of the focused beam spot BS of the main
beam MB. As shown, the respective intensities S1 and S2 of these
spurious beam spots are substantially lower than the intensity S0
of the main beam; nevertheless, these spurious beam spots are
located directly on the particle path through the flow cell. Thus,
each particle passing through the flow cell will be irradiated at
three locations, only one of which (the middle location) is
desirable. The light-scatter and fluorescence detectors, discussed
below, will then receive signals from the spurious beams, thereby
necessitating a scheme for ignoring these signals in the
particle-differentiation process.
[0019] In the flow cytometer schematically illustrated in FIG. 3,
it will be appreciated that the laser diode LD of FIGS. 1 and 2 has
been rotated by 90 degrees so that the diverging laser beam LB now
expands in the plane of the drawing and in the plane of the
particle path at its smaller angle .theta.2. In this orientation of
the laser diode, the major axis A' of its expanding elliptical
output beam now extends in a horizontal plane, perpendicular to the
particle path and to the plane of the drawing. Thus, while portions
of the laser beam are still truncated by the collimating lens, the
resulting spurious beam spots BS1 and BS2 (shown in FIG. 2) are now
located on opposite sides of the particle path, rather than in
alignment therewith. Note, this orientation of the laser diode also
has the effect of orienting the plane of polarization P of the
laser beam in a horizontal plane, perpendicular to the particle
path. Thus, while the beam spots BS1 and BS2 are no longer located
in a position to irradiate the particles passing through the flow
cell, the plane of polarization of the beam is now problematic.
[0020] Referring to FIGS. 4A and 4B, it will be seen in FIG. 4A
that a small particle P' irradiated by a plane-polarized light beam
B that is polarized in the vertical X/Z plane, as indicated by the
vector E, will act to scatter light in the horizontal Y/Z plane as
shown. The scattered light in the horizontal plane, including the
forward scatter FSH, side-scatter SSH, and the forward scatter FSH'
at all intermediate angles, will retain its vertical polarization,
and its intensity, measured at any angle in the horizontal plane,
will be determined only by the physical and optical properties of
the irradiated particle. Note, however, that the intensity of light
scattered forwardly in the vertical X/Z plane will depend on the
angle of measurement .differential. and, as angle .differential.
approaches 90 degrees (measured with respect to the horizontal Y/Z
plane) to a position in which the forwardly scattered light is
directed vertically upwards (or downwards) and thereby becomes
side-scattered light SSV in the vertical direction, the intensity
of such scattered light will approach zero, as indicated. In
effect, the wave oscillation in the vertical plane of polarization
acts to cancel out the scattered beam intensity in the vertical
direction. Similarly, as shown in FIG. 4B, if the beam source is
rotated by 90 degrees so that its plane of polarization is now
horizontal, i.e., in the Y/Z plane, the intensity of the light
scattered in the vertical (X/Z) plane will be unaffected by this
beam source orientation, but the light scattered in the horizontal
(Y/Z) plane will depend on the angle .beta. at which the
measurement is made. Notice, the intensity of the side-scattered
light SSH in the horizontal plane will become zero as the angle
.beta. approaches 90 degrees. Applying this information to the flow
cytometer environment described above, it will be understood why
the side-scatter signal is undetectable with the diode laser
orientation shown in FIG. 3. As so oriented, the scattering
conditions of FIG. 4B apply, and there is no side-scatter signal to
detect.
[0021] In view of the foregoing discussion, FIG. 5 schematically
illustrates a flow cytometer 10 of the type in which the invention
has utility and in which the present invention is embodied. As
shown, the flow cytometer of the invention includes an optical flow
cell FC through which a particle sample PS containing different
particles P' to be characterized is caused to flow. The sample may
comprise, for example, a whole blood sample in which the particles
to be characterized are in the form of various types of blood cells
(e.g., monocytes, lymphocytes, basophils, etc.). The flow cell
comprises an optically transparent housing 12 that defines a narrow
central channel through which the particles flow, one-at-a-time,
while being irradiated by a focused laser beam B' provided by a
laser diode LD. As shown, the laser diode is oriented so that its
diverging elliptical output beam expands in a horizontal plane
faster than in the vertical plane, whereby the aforementioned
spurious beam spots are located on opposite sides of and outside
the vertical particle through the flow cell, as discussed above
with reference to FIG. 3. Upon passing through the
particle-interrogation zone Z and being irradiated by the focused
laser beam, each individual particle will scatter beam light and
emit fluorescent radiation according to the type of particle
irradiated. Forwardly scattered light 14 will be collected by a
lens 16 and focused onto a suitable photodetector 18, and the
output FS of the latter is applied to a microprocessor MP. Axial
beam light is absorbed by a suitable light stop 20. Side-scattered
light 22, i.e., beam light scattered at 90 degrees in the
horizontal (Y/Z) plane, together with multicolor fluorescent light
24 emitted by fluorochromes carried by the selected particles, is
collected and collimated by a lens 26, and the resulting collimated
beam 28 is passed or reflected by a pair of dichroic mirrors 30, 32
arranged at 45 degrees to the collimated beam path. Side-scattered
light 33 is reflected by mirror 30 to a suitable photodetector 34,
typically a photomultiplier tube. Fluorescent light 35 of a first
wavelength transmitted by mirror 30 and reflected by mirror 32 is
directed to a second photodetector 36 that, again, is preferably a
photomultiplier tube. Fluorescent light of a second wavelength that
passes through both mirrors 30 and 32 is detected by a third
photodetector 40, also preferably of the photomultiplier tube type.
The respective outputs SS, FL1 and FL2 of photodetectors 34, 36 and
40 are fed to the microprocessor for processing. In addition to the
light-scatter and fluorescence signals obtained from each particle
passing through the flow cell, it is common to detect an impedance
signal Z representing the Coulter volume of each particle
interrogated. Such signal is derived by passing a constant-current
through the interrogation zone of the flow cell and monitoring
changes in current as occasioned by the electrical impedance of a
particle in the zone; the larger the particle, the larger the
change in current. A constant-current circuit 42, connected between
a pair of electrodes 44, 46 arranged on opposite sides of the
particle-interrogation, provides the requisite current through the
zone and operates to detect particle-induced changes in such
current. The output Z'' of circuit 42 is also fed to the
microprocessor. Upon simultaneously processing the FL1, FL2, SS, FS
and Z'' input signals in accordance with one or more known
algorithms, the microprocessor provides an output, typically in the
form of a plurality of scattergrams SG, indicating the relative
numbers and different types of particles in the particle sample
PS.
[0022] As indicated above, a laser diode oriented as discussed with
reference to FIG. 3 is problematic in that the plane of
polarization P of the laser beam is perpendicular to the direction
of particle flow. As shown in the flow cytometer of FIG. 5, the
major axis of the expanding ellipse E' is in the horizontal plane,
as is the major axis of the collimated beam that has passed through
the collimating lens 3. Since the plane of polarization P of the
beam will be parallel to the major axis, FIG. 4A shows that
side-scattered radiation in the horizontal plane will be canceled
out by wave vibrations in the horizontal plane. Thus, in accordance
with the invention, an optical element 50 is positioned in the
laser beam B', preferably in the collimated portion of the beam
between the collimating lens 3 and the cylindrical lens 4. Element
50 has the effect of rotating the plane of polarization of the beam
by an amount sufficient to orient the plane of polarization of the
laser beam so that it is now substantially parallel to the
(vertical) particle path through the flow cell. Accordingly, the
benefit of rotating the laser diode to eliminate the undesirable
spurious beam spots is combined with the benefit of being able to
detect side-scattered light since the plane of polarization is as
required to detect such side scattered light. Preferably, element
50 operates to rotate the plane of polarization of the laser beam
by 90 degrees, and it takes the form of a conventional half-wave
plate. Alternatively, element 50 may be in the form of two
quarter-wave plates arranged in tandem.
[0023] While the invention has been described with reference to a
preferred embodiment, it will be appreciated that various changes
can be made without departing from the spirit of the invention.
Such changes are intended to fall within the scope of the appended
claims.
* * * * *