Method and vessel for imaging fluorescent particles

Abstract: A vessel for containing fluorescent particles that are to be imaged is proposed. The fluorescent particles are stained with a fluorescent dye, collected in the bottom portion (3') of the vessel (3) and illuminated from below by a beam of excitation light (1). Part of the exterior surface of the bottom portion is an excitation light entry surface (3e). Thus, with only the bottom portion (3') or the vicinity thereof being illuminated, background light is reduced, thereby improving the contrast of the images of the particles.

Description: This invention relates to a method for imaging fluorescent particles, and more particularly to a vessel for holding fluorescent particles such as leukocytes or the like stained with a fluorescent dye for imaging the fluorescent particles. In the medical field, platelet preparation and erythrocyto preparation are produced by extracting platelets and erythrocytes from whole blood. These platelet and erythrocyte preparation are each used for blood transfusions, and it is undesirable for either preparation to contain leukocytes. It is therefore important to be able to know how many leukocytes the preparations contain. Conventionally this is done by placing a sample platelet preparation in a NAGEOTTE chamber, staining with a fluorescent dye, projecting an excitation light onto the sample and counting leukocytes via a microscope. Specifically, a 50 microliter sample is taken from a 200 or 400 milliliter bag of platelet preparation, the leukocytes in the sample are counted and converted to a leukocytes count for the whole bag. This is a tiring, inefficient, time-consuming task that has to be done by skilled personnel. An apparatus has been proposed to enable leukocytes to be counted, instead, by staining the leukocytes with a fluorescent dye, illuminating the sample with an excitation light having predetermined wavelengths, using a CCD camera or the like to image the sample and then analysing the images to obtain a count of the leukocytes. However, the solution containing the stained leukocytes also contains fluorescent dye that also emits fluorescent light. Thus, since not only the stained leukocytes but also the fluorescent dye itself is excited by the excitation light, there is a marked decrease in the contrast of the leukocytes that it is desired to observe or image. In some cases, the contrast may worsen to the point that the leukocyte images become so buried in the background that they cannot be picked out, making it impossible to count the leukocytes. US 5242837 discloses a method and vessel and for the detection of analytes using light attenuating magnetic particles in which labelled analytes are excited by means of a beam directed onto an optically transparent surface. Signal radiation produced by the excitation returns through the surface and is detected along the same axis of excitation by conventional spectroscopic means. This document discloses all the features of the preamble to claim 1. EP 661532A discloses a transparent cuvette for measuring constituents by applying a beam of light through a first flat surface of the cuvette, through the constituents contained in the cuvette and through an opposite, second surface of the cuvette so that measurement is uniaxial. EP 671622A discloses an optical measurement instrument and method therefor in which a prism for introducing an excitation light is formed as a unitary body at one of a slab-type waveguide and a reaction vessel is formed on one side of the waveguide. Labelled antibodies are constrained in the reaction vessel near the waveguide and an excitation light is directed into the prism along the waveguide. Signal radiation produced by the excitation returns along the waveguide and exits the prism for measurements. A similar arrangement is known from US5677196. An object of the present invention is to provide a vessel for containing fluorescent particles that enables the fluorescent particles to be well imaged by reducing the effect of background light. According to the present invention, there is provided a method of imaging fluorescent particles that are stained with a fluorescent dye, the particles being contained in in a fluid and the fluid also containing the fluorescent dye, the method comprising: disposing the fluid in a vessel; and collecting the fluorescent particles in a bottom portion of a volume enclosed by the vessel, characterised by further comprising: guiding an excitation radiation for exciting the fluorescent particles through the vessel, wherein of the volume enclosed by the vessel said radiation is guided in a direction from the side of the vessel directly and only through said bottom portion, such that the fluorescent dye disposed in any part of the fluid other than the bottom portion is not substantially excited, and measuring radiation from the fluorescent particles from below the bottom portion. To attain the above object, the present invention provides a vessel for imaging fluorescent particles that are stained with fluorescent dye and illuminated by excitation light, the fluorescent particles being contained in a bottom portion of a volume enclosed by the vessel and imaged from below the bottom portion, wherein a side part of the exterior surface of the vessel enclosing the bottom portion is formed to be an excitation light entry surface. In accordance with this arrangement, as the fluorescent particles that are the object of interest are accumulated in the bottom part of the imaging vessel and the bottom portion of the vessel has an external side surface that is an entry surface for the excitation light, only the bottom portion of the vessel is illuminated by the excitation light, so background light can be reduced, thereby making it possible to obtain high-contrast images of the fluorescent particles in the vessel. Background light can be further reduced by a considerable amount by using a shield member to cover parts of the imaging vessel other than the vicinity of the bottom portion. It is preferable to form the bottom portion so that it has a square or rectangular horizontal cross-section with one side thereof forming an excitation light entry surface. The bottom portion may also be given a horizontal cross-section having a round shape, and a negative cylindrical lens disposed on the side from which the bottom portion is illuminated by the excitation beam. Further features of the invention, its nature and various advantages will become more apparent from the accompanying drawings and following detailed description of the invention. Figure 1 is a block diagram showing the general configuration of an apparatus for imaging fluorescent particles according to the present invention. Figure 2 is a front view of the apparatus used for analyzing and displaying obtained fluorescent particle images. Figure 3 is a diagram illustrating an arrangement of a cover used to shield the upper part of the imaging vessel. Figure 4 is a diagram illustrating another arrangement for shielding the upper part of the imaging vessel from illuminating light. Figure 5 is a diagram of an optical system used to form a strip-shaped excitation light beam. Figure 6 is a diagram of another optical system used to form a strip-shaped excitation light beam. Figure 7 is a diagram showing the configuration of optical elements used to form a strip-shaped excitation light beam. Figure 8a is an exterior perspective view of the imaging vessel. Figure 8b is a cross-sectional view of the vessel. Figure 8c is a bottom view of the vessel. Figure 9 is a cross-sectional horizontal view of an imaging vessel according to another configuration. Figure 10 is a cross-sectional view of an imaging vessel according to yet another configuration. Figures 1 and 2 show the arrangement of a first embodiment of the present invention. In the drawings, reference numeral 1 denotes a laser light source, such as, for example, a YAG laser that produces a green laser beam. The laser beam from the laser light source 1 impinges upon, and is diffused by, a diffusion plate 2 comprised of ground glass or other such member that is able to diffuse light. The light thus diffused is projected at a bottom portion 3' of an imaging vessel 3, the upper part of which is covered by a cover 4. Fluorescent particles are accumulated in the bottom portion of the imaging vessel 3, and these fluorescent particles fluoresce when illuminated by the laser beam. The images of the fluorescent particles illuminated by the laser beam pass via a cover-glass 5 and objective lens 6 to a mirror 7 that reflects the images to a barrier filter 8 that transmits light in a prescribed frequency band, and are then picked up by a CCD camera 9. The images of the fluorescent particles picked up by the CCD camera 9 are passed via a signal line 10 to a video capture device 11 of a computer 12, where they are processed by an image processing circuit 13 (Figure 2) to enable the fluorescent particles to be recognized. There is a change in brightness where there is a fluorescent particle, so the fluorescent particles can be recognized by, for example, using the differentiation of signal values to detect the positional coordinates of the particles. The fluorescent particles thus recognized are displayed on a monitor 14. Figure 2 depicts the image 15 of the bottom portion of the vessel together with a plurality of fluorescent particles 15a therein, displayed on the monitor 14. The fluorescent particles 15a are counted and the count is also displayed at the lower part 16 of the monitor 14. The imaging vessel 3 is molded in one piece from transparent polystyrene resin, glass, or acrylic resin, preferably polystyrene resin. Inserted into the imaging vessel 3 are a platelet preparation sample (100 microliters, for example), a chemical (Triton X) that dissolves platelet and leukocyte cytoplasm, and a fluorescent dye (propidium iodide) for staining leukocyte nuclei. The imaging vessel 3 is then subjected to centrifugal separation in a centrifuge (not shown), causing the leukocyte nuclei to collect in the bottom portion of the imaging vessel 3. All of the leukocyte nuclei can be collected in the bottom portion 3' of the imaging vessel 3 by applying a prescribed centrifugal force. The cover 4 is then used to cover the imaging vessel 3 in which the leukocyte nuclei stained with a fluorescent dye are collected in the bottom portion 3' thereof, and the imaging vessel 3 is mounted on the imaging apparatus. For imaging, the laser light source 1 is activated, producing a laser beam which is diffused by the diffusion plate 2 and projected onto the bottom portion 3' of the imaging vessel 3. As the nuclei of the leukocytes in the bottom portion 3' of the imaging vessel 3 have been stained with a fluorescent dye, when they are illuminated by the beam of excitation light, they emit fluorescent light having a frequency of around 600 nm. This is picked up via the cover-glass 5, objective lens 6, mirror 7 and barrier filter 8 below the imaging vessel 3. The barrier filter 8 only transmits light having the frequency of fluorescent light, allowing light of harmful frequencies to be blocked at this point. The laser beam is projected only at the bottom portion of the vessel, effectively illuminating the leukocytes collected there. Therefore, even if there is fluorescent dye floating in the solution in the imaging vessel 3, it is possible to prevent the fluorescent dye from forming harmful background light, thereby enabling the images to be obtained with improved contrast. With reference to Figure 2, the images of fluorescent particles thus obtained by the CCD camera 9 are passed via a signal line 10 to a video capture device 11 of a computer 12, where they are processed by an image processing circuit 13 to count the number of leukocytes 15a. In accordance with the arrangement described above, the laser beam is projected only onto the bottom portion of the imaging vessel and does not illuminate the upper part of the vessel. As shown by Figure 3, the effect of only illuminating the bottom portion can be enhanced by providing a cover 20 that shields all parts other than the bottom portion from the illuminating light beam. Instead of the cover arrangement of Figure 3, an arrangement such as that shown in Figure 4 may be used. In this arrangement, a mask 21 having a central slit-shaped aperture 21a is used. The laser beam from the laser light source 1 passes through the aperture 21a, ensuring that only the bottom portion 3' of the imaging vessel 3 is illuminated. A linear or line-shaped light beam would illuminate more of the bottom portion of the imaging vessel than a spot-shaped beam. Figure 5 shows the type of arrangement that could be used in such a case, with laser beam 30 being shaped to a flat beam 33 by passage through cylindrical lenses 31 and 32, and the flat beam 33 being used to illuminate the bottom portion 3' of the imaging vessel 3. Figure 6 shows another arrangement, in which a scanning mirror 40 is used to deflect the laser beam 30, which passes through a lens 41 to scan the bottom portion of the imaging vessel. Figure 7 shows another arrangement, that uses a bundle of optical fibers. In this arrangement, the fibers at the entrance end 50 from which the laser beam enters are arranged in a round configuration, while at the exit end 51 the fibers are arranged in a straight line, with the exit end 51 being disposed in the vicinity of the bottom portion of the imaging vessel so as to illuminate the bottom portion thereof. In each of these embodiments, only the bottom portion of the vessel, or the vicinity thereof, is illuminated, by a strip-shaped beam, thereby making it possible to obtain images of the fluorescent particles with good contrast. Moreover, if a diffusion plate 2 is used to diffuse the laser beam, it enables the bottom portion of the imaging vessel to be illuminated uniformly. Figure 8 shows a preferred embodiment of the imaging vessel 3, preferably formed as a one-piece molding of polystyrene resin. The vessel has a ringshaped upper portion 3a having a notch 3f for positioning purposes. The imaging vessel 3 comprises a cylindrical portion 3b that extends vertically downward from the upper portion 3a to a small-diameter portion 3d, via a sloping portion 3c. A substantially square or rectangular block portion 3e is formed at the lower end. One side of the block portion 3e is arranged to be illuminated by a laser beam, indicated by the arrow. When fluorescent particles are to be imaged, the imaging vessel 3 is attached to the apparatus, with the notch 3f being used to position the imaging vessel 3. When the vessel 3 has been fitted into position, a flat face of the block portion 3e is perpendicular to the direction of laser beam illumination, forming the entry surface for the incident beam. Thus, the laser beam illuminates only the bottom portion 3g of the imaging vessel 3. The bottom portion may also be illuminated by an arrangement such as the one shown in Figure 9, in which the block portion 3e has a round cross-section, and a negative cylindrical lens 60 is disposed on the side from which the bottom portion is illuminated by the excitation beam. In the arrangement shown in Figure 10, a cover 70 is used to shield parts of the imaging vessel 3 other than the bottom portion from the laser beam. In this case, the cover 20, shown in Figure 3, on the imaging apparatus side may be omitted. The cover or shielding function may be realized by applying a light shield coating to the vessel, or by painting the vessel in a shielding color. It is to be understood that while in the foregoing the invention has been described with reference to leukocytes as the fluorescent particles, the invention is not limited thereto but can be applied to other fluorescent particles. As described in the foregoing, in accordance with the present invention, fluorescent particles to be imaged are collected in the bottom portion of the imaging vessel. Part of the exterior surface of the bottom portion is an excitation light entry surface, so that only the bottom portion of the vessel is illuminated by the excitation light beam. Background light is thereby reduced, making it possible to obtain high-contrast images of the fluorescent particles contained in the vessel.

Claims: A method of imaging fluorescent particles that are stained with a fluorescent dye, the particles being contained in a fluid and the fluid also containing the fluorescent dye, the method comprising: disposing the fluid in a vessel (3); and collecting the fluorescent particles in a bottom portion (3') of a volume enclosed by the vessel, characterised by further comprising: guiding an excitation radiation for exciting the fluorescent particles through the vessel, wherein of the volume enclosed by the vessel said radiation is guided in a direction from the side of the vessel directly and only through said bottom portion, such that the fluorescent dye disposed in any part of the fluid other than the bottom portion is not substantially excited, and measuring radiation from the fluorescent particles from below the bottom portion. A method according to claim 1, wherein the excitation radiation is guided through the bottom portion of the volume enclosed by the vessel in a direction substantially perpendicular to a longitudinal axis of the vessel. A method according to claim 1 or claim 2, wherein a shielding member (20, 70) is provided for shielding all parts of the vessel other than the bottom portion from the excitation radiation. A method according to any one of the preceding claims, wherein the excitation radiation is a laser (1). A method according to any one of the preceding claims, wherein the excitation radiation is a beam having a rectangular cross-section. A method according to anyone of claims 1 to 4, wherein the excitation radiation is guided through a plurality of optical fibres (50) before being guided through the bottom portion, the ends of the optical fibres (51) at which the excitation radiation exits being arranged in a straight line. A method according to any one of the preceding claims, wherein the excitation radiation is diffused before being guided through the bottom portion. A method according to any one of the preceding claims, wherein a cross-section along a longitudinal axis of the vessel of a region of the vessel enclosing the bottom portion comprises a bottom surface and two side surfaces; and wherein the excitation radiation is guided through the bottom portion such that it enters the bottom portion through a first one of the side surfaces and exits the bottom portion through a second one of the side surfaces. A vessel (3) for use in a method according to any one of the preceding claims, the fluorescent particles being imaged from below the bottom portion (3'), wherein a side part of the exterior surface of the vessel enclosing the bottom portion is formed to be an excitation light entry surface. A vessel according to claim 9, wherein parts of the vessel other than in a vicinity of the bottom portion are covered by a shielding member (20, 70). A vessel according to-claim 9, wherein a region of the vessel enclosing the bottom portion has a square or rectangular horizontal cross-section and one side thereof forms the excitation light entry surface. A vessel according to claim 9, wherein the bottom portion has a round horizontal cross-section.

Inventors: KOMATSU AKIO, MATSUI HIROKI, NIINO MASAO, KOMATSU, AKIO, MATSUI, HIROKI, NIINO, MASAO, Komatsu, Akio,c/o Kowa Co. Ltd., Matsui, Hiroki,c/o Kowa Co. Ltd., Niino, Masao, c/o Kowa Co. Ltd.
Applicants: KOWA CO, KOWA CO., LTD.
Application year: 1998
Main classification: G01N21
IPC codes: G01N15, G01N21, G01N33