Apparatus for counting particles suspended in a fluid

Abstract: An apparatus for counting and analyzing sample particles like cells suspended in a fluid by applying an irradiation beam such as a laser beam to the sample particles in a flow cell. The apparatus has a segment for supplying a flow cell with the suspension of sample particles, an optical one for applying a laser beam to sample particles in the above-mentioned suspension, a light gathering one for forming the image of the above-mentioned sample particles, a light sensing one for sensing light coming from the above-mentioned formed image of the sample particles and an analysis one for analyzing the above-mentioned sample particles on the basis of the above-mentioned sensed light. A polarizing beam splitter and such convergent lenses as have magnification different between the two directions perpendicular to the optical axis of the laser are placed in the course of the above-mentioned laser beam between the above-mentioned optical segment and flow cell.

Description: BACKGROUND OF THE INVENTION The present invention relates to an apparatus for analyzing the nature of particles by causing them to flow, and is particularly concerned with an apparatus efficient for an analysis of flowing cells such as the classification of leukocytes. An example in which the above-noted particles are cells will be taken in the description below. One of the conventional methods of distinguishing sample particles in a suspension is flow cytometry (US-A-4 636 075; US-A-4 548 499). In this method, cells in a suspension, typically a blood sample are run through the minute passage of a flow cell, where each particle in the blood sample is shone on by one or more beams of light in the sensing zone. Then, the result of the interaction of a light beam or a group of light beams on each particle is sensed by one or more light sensors. Interaction here means such a phenomenon as the occurrence of fluorescence from particles caused by light beams scattered by particles. Conventionally, light sensors are designed so that they can measure scattered light at a particular scattering angle or fluorescence having a particular wavelength. In this way, each particle flowing through the flow cell is characterized by scattered light, fluorescence or one or more other optical or electrical characteristics. According to these characteristics, each particle is mapped in a specific space based on radiation intensity or another characteristics such as red or green which is measured by the sensor. It has been desired to map different kinds of particles in a sample in correspondingly different areas in the specific space so that the type of each particle (e.g. cell) can be estimated from their mapping in the specific space. Therefore, to improve accuracy of the analysis of particles, the sheath flow method has been employed for causing flow in the flow cell. In this method, a blood sample flows only in the center of the minute passage. Furthermore, a light beam of a substantially elliptical shape whose major axis is perpendicular to the direction of the stream in the flow cell is used so that the quantity of light each particle receives can be uniform. Under Provisional Publication No. 47635/88, further, the Official Gazette made public a means to scan with a laser beam perpendicularly to the direction of the stream in the flow cell in order to expand the area in which the quantity of light is uniform. As the rate of the flow of a sample increases, the position of each cell flowing through the flow cell varies more greatly. Unless the quantity of light of laser is uniform in the area where particles pass, the analysis will be less accurate, because the intensity of scattered light or fluorescence from particles depends on the quantity of light of laser. The quantity of light of laser in the area where particles pass needs to be uniform. The laser for use in flow cytometry is called a gauss beam whose quantity of light is large in the center and small in the periphery. The above-mentioned conventional techniques has enlarged the width of a laser beam into an elliptical shape to use the center where the quantity of the light is uniform. Such a laser beam has caused energy loss, so a large-scale laser has been necessary for the techniques. Then, a large laser leads to the large size of entire equipment which costs much more. Moreover, these techniques have such a drawback as the heat and vibration from lasers which lower their reliability and shorten their life. Besides, the section of the sample flow in the flow cell must be small because of the narrow range where the quantity of light is uniform. This has prevented the flow of the sample from being enlarged, necessitating a long period of time for analysis. When a laser beam is operated for scanning perpendicular to the direction of the stream in the flow cell, it is necessary to apply a uniform radiation intensity of the laser to the range where cells move during laser scanning. The laser beam must be expanded in the direction of the stream in the flow cell, which leads to a large loss of laser energy. SUMMARY OF THE INVENTION The present invention relates to an apparatus as defined in claim 1. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic composition of an embodiment of the present invention. FIG. 2 is a sectional view of the flow in a flow cell. FIG. 3 is a schematic view illustrating the working of the above first embodiment. FIG. 4 is a graphical representation of the distribution of the radiation intensity of a laser beam at the observation point of conventional apparatus. FIG. 5 is a graphical representation of the distribution of the radiation intensity of a laser beam at the observation point of the above first embodiment. FIG. 6 is a schematic composition of another embodiment of the present invention. FIG. 7 is a schematic composition of the principal part of a third embodiment of the present invention. FIG. 8 is a graphical representation of the distribution of the radiation intensity of a laser beam at the observation point of the above third embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT In a flow cytometry apparatus in accordance with the present invention, a laser beam is broken up by a polarizing beam splitter into two orthogonal polarizing components at an angle of a minute degree with each other. Each component is focused in a flow cell by light gathering lenses whose magnification is different between the two directions perpendicular to the optical axis of the laser, but the two components have their spots at a minute distance from each other. The distance depends on the divided angle the polarizing beam splitter has and on the focal length of the light gathering lenses, but as long as the distance is specified to overlap the spots, the quantity of the light will be uniform in the range between the adjacent spots. Therefore, the rate of the flow of the sample can be increased and then can be lessen the time for the analysis of cells. In addition, the range of uniform quantity of light can be provided without enlarging the width of the laser beam at its light source, so the laser beam has smaller energy loss. Therefore, a small output laser can be used as the light source with the result that the entire apparatus can be small-sized and highly reliable. More reduction of energy loss is possible by using more than one polarizing beam splitter to provide many spots at an equal interval in a line for more enlargement of the range of uniform quantity of light. In this case, polarizing beam splitters are placed on the optical axis with a polarization rotator between adjacent polarizing beam splitters. When the laser beam for use is one whose two orthogonal polarizing components for a polarizing beam splitter are the same percentage, the laser beam is broken up by the first polarizing beam splitter for the beam to pass into two laser beams having the same intensity. The two laser beams each have only one half of the orthogonal polarizing component, but they are formed by the polarization rotator so that their two orthogonal polarizing components for a polarizing beam splitter can be the same percentage again. Then, the two laser beams are broken up by the second polarizing beam splitter for them to pass into four laser beams having the same intensity. Repeating the above-mentioned process obtains many laser beams having the same intensity. On the other hand, given that the smallest divided angle is  d , the divided angle that the orthogonal polarizing components provided by each polarizing beam splitter has will be 2 d , 4 d and so on. Then, in the case where the first polarizing beam splitter has the divided angle 2 d and the second one the divided angle  d , the laser beam is broken up by an angle of 2 d by the first polarizing beam splitter and broken up further by an angle of  d by the second polarizing beam splitter, resulting in four laser beams separated by the same angle  d . Such many laser beams with the same intensity at the same angle with adjacent others are focused in the flow cell by convergent lenses, resulting in many spots lined at the same interval. Laser beams at the same angle with adjacent others can be obtained in whatever order more than one polarizing beam splitter may be placed. However, placing the polarizing beam splitter having the smallest divided angle nearest to the flow cell allows adjacent spots to have mutually orthogonal polarizing components. Since mutually orthogonal polarizing components do not interfere with each other, there is no possibility of quantity of light lacking uniformity due to the interference of adjacent spots with each other. Referring first to FIG. 1, there is shown the composition of the principal part of a flow cytometry apparatus which is the first embodiment of the present invention. On the extension of a laser beam 3 emitted from the light source 15 of laser are a 1/2 wave plate 7, a Wollaston prism 8, convergent lenses 2, a flow cell 1, a beam trap 11, light gathering lenses 6 and light sensor 10. The flow cell 1 is supplied with a sheath fluid by the means 19 for providing a sheath fluid and with a suspension of sample particles by a sample provision means 20. The light sensor 10 is connected to an analyzer 17, which is connected to a display unit 18. A controller 16 is connected to the analyzer 17, the sheath fluid provision means 19 and the sample provision means 20. The laser beam 3 travels though the light beam forming means composed of the 1/2 wave plate 7, Wollaston prism 8 and convergent lenses 2, comes to the observation point 5 on the axis of the flow cell 1 and is absorbed by the beam trap 11 provided ahead of the observation point 5. When a suspension of cells is run through the flow cell 1 in the form of sheath flow, light is emitted from cells at the observation point 5. The abovementioned light emitted at the observation point 5 is focused on the sensing surface of the light sensor 10 and is then sensed in respect of intensity. The light sensed by the light sensor 10 is scattered light or fluorescence emitted from cells, so it is closely related to the type and nature of the cells. Therefore, by sensing and analyzing the scattered light and fluorescence from each cell it is possible to find out the number or ratio of each of various kinds of cells in a blood sample. FIG. 2 is a sectional side view of the flow cell 1. In the portion measuring d in diameter flows a sample 21 which is a suspension of sample particles, and around it a sheath fluid 22, both forming sheath flow. To shorten the time for the analysis, it is necessary to increase the rate of the sample flow. However, increasing the rate leads to the enlargement of the diameter d of the sample 21. For highly accurate analysis, the radiation intensity of a laser beam needs to be uniform and also higher than a certain level in the area of the sample 21. FIG. 3 is an illustration of the operation of the light beam forming means in the embodiment shown in FIG. 1. Generally, before the light beam forming means (left of the means in the figure), the plane of polarization for the laser beam 3 is vertical or horizontal. The 1/2 wave plate 7 changes the plane of polarization into a plane tilted 45° from the horizontal to provide the laser beam with both vertical and horizontal polarizing components. Then, the laser beam 3 is broken up by the Wollaston prism 8 into two orthogonal polarizing components. The convergent lenses 2 are composed of cylindrical lenses which converge the two components of the laser beam on two points adjacent to each other on the convergence plane 13, each of them forming an elliptical spot. FIG. 4 is a graph showing the distribution of the radiation intensity of a laser beam at the observation point of conventional apparatuses. The spot of the laser beam is shaped like a horizontally long ellipse so that the radiation intensity can be uniform inside the sectional area of the sample 21 measuring 6 in diameter. FIG. 5 is a graph showing the distribution of the radiation intensity of a laser beam at the observation point 5 of the embodiment shown in FIG. 1. The radiation intensity for one of the two spots is denoted by the broken line I 1 and that for another by I 2 . The synthesized radiation intensity is denoted by the solid line I 3 . The radiation intensity is almost uniformly distributed in the range where the two spots overlap each other. Therefore, since the uniformity of the radiation intensity distribution can be achieved despite the narrowness of the width of a spot, the greater part of the energy of the laser beam is concentrated inside the sectional area measuring d in diameter, so this allows a higher energy density than the conventional apparatuses do as shown in FIG. 4. Therefore, only a small output laser light source is sufficient for obtaining energy density required for the analysis of particles. In addition to the above, the wide range of uniform radiation intensity provides the ability to enlarge the sectional area of the flow of the sample suspension of cells and thus to increase the rate of the flow of the suspension, which then leads to the shortening of the time for the analysis. Furthermore, since in this embodiment each of the two spot beams converged by the convergent lenses 2 is of a substantially elliptical shape whose major axis is perpendicular to the direction of the stream, the observation point is of a vertically short shape, which lessens the time for a cell to pass the observation point and then shortens the time for the analysis again. The length of a spot in the above-mentioned direction of the stream can be changed by adjusting the focal length of one of the cylindrical lenses composing the convergent lenses 2 without altering the distribution of the radiation intensity perpendicular to the above-mentioned direction of the stream. FIG. 6 is a schematic composition of the principal part of a flow cytometry apparatus which is another embodiment of the present invention. The difference from the embodiment shown in FIG. 1 lies in the use of spherical lenses 2' for the convergent lenses 2 and in the mounting of forming prisms 12 on the optical axis of the laser beam 3. The laser beam 3 is formed into a beam of a vertically long elliptical shape by the forming prisms 12. Then, the beam travels through the 1/2 wave plate 7 and the Wollaston prism 8 and is converged on the observation point 10 by the convergent lens 2, so that it becomes a spot of a substantially elliptical shape whose major axis is perpendicular to the direction of the stream as in the embodiment in FIG. 1. In contrast to the embodiment in FIG.1, this second embodiment does not use cylindrical lenses, so the whole apparatus can be less expensive, and the use of a spherical lens for the convergent lenses 2 facilitates checking and adjustment. FIG. 7 is a view showing the operation of the means of forming a light beam in a third embodiment of the present invention. This embodiment uses Wollaston prisms 8a and 8b and a 1/4 wave plate 14 between them. Before entering the means of forming a light beam, the laser beam 3 generally has a vertical or horizontal plane of polarization. The 1/2 wave plate 7 changes the plane of polarization into one tilted 45° from the horizontal in order for the laser beam to have both vertical and horizontal polarizing components. Then, the laser beam 3 is divided by the Wollaston prism 8a into two orthogonal polarizing components. Each of the two polarizing components is formed by the 1/4 wave plate 14 into circularly polarized light. The two components are then broken up by the Wollaston prism 8b into four orthogonal polarizing components. The four laser beams 3' are converged by convergent lenses 2" on the convergence plane 13 in line with each other, each forming a circular spot. In this case, the Wollaston prism 8a has a divided angle specified as 2 d in response to the divided angle  d the Wollaston prism 8b has, so the four beams from the Wollaston prism b are directed with the angle  d among them, resulting in the equal distance among their spots on the convergence plane 13. Since the four beams produced by breaking up by the Wollaston prism have orthogonal polarizing components, the polarizing component of each beam intersects that of their adjacent one, so no interference occurs. FIG. 8 is a graph showing the distribution I 9 of the radiation intensity of a laser beam on the convergence plane 13 in the embodiment shown in FIG. 7. Since the range where the spots I 5 to I 8 overlap has almost uniform radiation intensity, the intensity of the scattered light and the fluorescence from cells does not vary no matter where they may pass within the range, which allows accurate analysis. In this embodiment, the range of uniform radiation intensity is wider than the other embodiments, so the embodiment is capable of providing higher energy efficiency. In addition, the capability of providing a radiation range whose vertical size is smaller than its horizontal without the use of a cylindrical lens or forming prism facilitates checking and adjustment. It is possible to increase the number of Wollaston prisms in order to have more overlapping spots. In this case, as long as a polarizing beam splitter having the smallest splitting angle is placed nearest to the flow cell, other polarizing beam splitters can be placed in any order. On the other hand, a 1/4 wave plate is used for the polarization rotator in the third embodiment, but a 1/2 wave plate or a polarizing plate can be used to obtain the same effect. Wollaston prisms are used as polarizing beam splitters in the above embodiments, but Rochon prisms or Senarmont prisms can be used for the same purpose. The above description has related to the use of fluid as the carrier of sample particles. However, it will be obvious to those skilled in the art that not only fluid but other forms of substance like gas can be used as the above-mentioned carrier if they are suitable for the above purpose. According to the present invention, it is possible for a laser light source in a flow cytometry apparatus to provide a range of uniform radiation intensity without enlarging the width of the laser beam because the beam can be broken up into more than one beams by polarizing beam splitters and converged as more than one spot in line with each other on the observation point in the flow cell. Therefore, embodiments in accordance with the present invention allow the use of a small output laser as their light source, thus as a whole being low-cost and high-reliability flow cytometry apparatuses.

Claims: An apparatus for flow cytometry of particles suspended in a fluid, comprising fluid supplying means for supplying a flow cell with a fluid containing sample particles, optical means for applying a laser beam to said sample particles in said fluid, a polarizing beam splitter dividing the irradiation laser beam into a plurality of beam components, convergent lenses placed in the course of said beam components between said beam splitter and said flow cell, light gathering means for forming an image of the sample paticles at the observation point, light sensing means for sensing light coming from the formed image of the sample particles and analysis means for analyzing said sample particles on the basis of the sensed light, characterized in that at the observation point said at least two beam components form at least two spots centered at a such a minute but nonzero distance from each other, that said at least two spots overlap, but only partly, such that the radiation intensity is almost uniformly distributed in the range where the two spots overlap. An apparatus according to claim 1 characterized in that the magnification of said convergent lenses is different between the two directions perpendicular to the optical axis of said irradiation beam. An apparatus according to claim 2, characterized in that said polarizing beam splitter is either a Wollaston prism or a Senarmont prism. An apparatus according to claim 2, characterized in that said polarizing beam splitter placed on the optical axis is more than one in number. An apparatus according to claim 2, characterized in that said polarizing beam splitter placed on the optical axis is more than one in number and that a polarization rotator is placed between them so that the divided angle that each polarizing beam splitter gives to orthogonal polarized light can be specified as two, four, eight or 2n times as large as the smallest divided angle. An apparatus according to claim 5, characterized in that one of said more than one polarizing beam splitter which is placed nearest to said flow cell has the smallest divided angle. An apparatus according to claim 2, characterized in that said irradiation beam is beam of a small output laser. An apparatus according to claim 2, characterized in that said fluid is a suspension. An apparatus according to claim 2, characterized in that said irradiation beam is a laser beam and that said fluid is a suspension. An apparatus according to claim 7, characterized in that said polarizing beam splitter is a Wollaston prism, a Rochon prism or a Senarmont prism. An apparatus according to claim 7, characterized in that said polarizing beam splitter placed on the optical axis is more than one in number. An apparatus according to claim 7, characterized in that said polarizing beam splitter placed on the optical axis is more than one in number and that a polarization rotator is placed between them so that the divided angle that each polarizing beam splitter gives to orthogonal polarized light can be two, four, eight or 2n times as large as the smallest divided angle. An apparatus according to claim 7, characterized in that the incident beam to said flow cell is formed by a laser light source, 1/2 wave plate, Wollaston prism and light gathering lens. An apparatus according to claim 7, characterized in that the light output is entered into an analyzer by way of a light gathering lens and a light sensor. An apparatus according to claim 7, characterized in that the incident beam to said flow cell is formed by a laser light source, 1/2 wave plate, Wollaston prism and light gathering lens and that the light output from said flow cell is entered into an analyzer by way of a light gathering lens and a light sensor. An apparatus according to claim 7, characterized in that said convergent lenses are composed of spherical lenses and that forming prisms are placed in the course of the laser beam from said laser light source. An apparatus according to claim 7, characterized in that the incident beam to said flow cell is formed by forming prisms, a 1/2 wave plate, a Wollaston prism and spherical lenses placed in this order between a laser light source and a flow cell. An apparatus according to claim 7, characterized in that the incident beam to a flow cell is passed through a 1/2 wave plate, a Wollaston prism, 1/4 wave late, a Wollaston prism and convergent lenses in this order.

Inventors: KANEKO TOSHIO, KATAOKA KEIJI, OHKI HIROSHI, YAMAZAKI ISAO, KANEKO, TOSHIO, KATAOKA, KEIJI, OHKI, HIROSHI, YAMAZAKI, ISAO
Applicants: HITACHI LTD, HITACHI, LTD.
Application year: 1990
Main classification: G01N15
IPC codes: G01N21, G01N15