DEVICE FOR COUNTING SMALL PARTICLES AND A SORTING APPARATUS COMPRISING SUCH A DEVICE

Abstract: A device for counting small particles, especially carriers, comprises a capillary (2) for receiving the particles to be counted, a light source (3) for emitting measuring light (M) and a photoelectric detector (5) for receiving the measuring light (M). Also provided are light-delivering means which deliver the measuring light (M) emitted by the light source (3) to the capillary (2) and light-removing means which deliver the measuring light (M) coming from the capillary (2) to the photoelectric detector (5). The internal diameter (ID) of the capillary (2) is such that the individual particles can enter the capillary (2) only one behind the other and can move along inside it only one behind the other. The light-delivering means irradiate the capillary (2) with the measuring light (M) transversely to the longitudinal axis (A) thereof, the measuring light (M) being in the form of a light beam, the width of which is such that only one of the particles can be completely in the light beam at any one time. A sorting apparatus for distributing small particles comprises such a device.

Description: Device for counting small particles and a sorting apparatus comprising such a device The invention relates to a device for counting small particles according to the preamble of independent claim 1. The invention relates especially to a device for counting carriers (beads) as are used in modern research in the synthesis of novel chemical substances. The invention relates also to a sorting apparatus according to the preamble of claim 14 comprising such a device. The search for novel chemical substances is often guided by the objective of synthesizing a substance that has given physical, chemical or bioactive properties. Such chemical substances may be, for example, plastics, colorants, aromatics, flavourings, bioactive materials or other compounds having useful properties. Bioactive materials are, for example, pharmaceutical or therapeutic active ingredients, herbicides, pesticides, fungicides or active ingredients used in veterinary medicine. Recently, combinatory technologies have often been used in the search for such novel chemical substances. These are based on synthesizing a very large number of different chemical compounds by adding together a predetermined number of reagents that serve as "building blocks" in combination to form one substance. For example, the "building blocks" may be amino acids, with which polypeptides of a predetermined length, that is to say a predetermined number of amino acids in a polypeptide, are synthesized. By combining those "building blocks" in every possible manner, a vastly large number of such polypeptides having different, predetermined lengths can be synthesized. A method frequently used in combinatory technology is the synthesis of substances on carriers. The carriers serve as foundations on which the "building blocks" are added together to form the novel substance. Typically, the carriers are small, mostly spherical particles having a diameter of customarily less than 200 μm and are made, for example, of resin or polystyrene. In accordance with the generally customary nomenclature, the carriers are hereinafter referred to as "beads". Given the vastly large number of different substances that can be prepared in the context of combinatory technologies, the task of studying each individual synthesized substance for the desired property or action would be possible, if at all, only by expending a great deal of time. For that reason, a large number of up to several ten thousand beads having on them the generally different substances are customarily placed together in a vessel, for example on a microtitre plate, and are there studied for the desired property or action by means of an indicator medium. If one of the substances on the beads is then found to be active or to have the desired property, the beads must be divided into smaller portions, for example into the various vessels of a further microtitre plate or deep-well plate, and the detection experiment must be repeated. In analogous manner, the beads are divided into smaller and smaller portions. The purpose of that successive procedure is to isolate or detect the bead on which the active substance or substance having the desired property is located. Towards the end of that successive process, the vessel containing the beads to be distributed then contains only a very few beads, for example approximately one hundred beads, which are to be distributed into, for example, one hundred vessels, so that finally only one bead is present in each vessel. It is therefore necessary to divide the beads located in one vessel into a plurality of vessels, for example into the individual positions of a microtitre plate or deep-well plate. For that purpose, the beads are first suspended in a solvent to form a suspension and that suspension is then distributed. The distribution from one vessel into a plurality of other vessels is customarily carried out using syringes or pipettes. Devices are also known in which the principle of a water jet pump is used to divide the beads, the beads being sucked by a conditioned jet of a solvent from a reservoir into the jet. The resulting suspension is then separated into droplets which are then distributed into the various vessels. A disadvantage of such and similar apparatuses is, however, that the beads must first be transferred from the vessel containing them into a reservoir. Individual beads may be lost in the process, the risk being that the very bead that is lost is one that carries an active substance. The known devices prove to be highly problematic especially in cases in which a very small number of beads is to be divided into still smaller portions, where each portion is to contain only from one bead to a few dozen beads. With traditional pipettes or syringes it is possible to determine only the volume of distributed suspension, or the droplets of suspension introduced can be counted. The disadvantage here is that the exact number of distributed beads is unknown. In the case of a volumetric distribution, only the distributed volume is known but not the number of beads distributed, because there is generally no clear relationship between the volume of suspension distributed and the number of beads contained in that volume. Even in the case where droplets are counted, the number of beads introduced is unknown because the number of beads contained in one drop of suspension is, firstly, unknown and, secondly, may vary greatly from one drop to the next. Thus, the exact number of beads introduced into a vessel is neither known nor controllable. If it is known, for example, that in a vessel containing approximately one hundred beads, one bead carries a substance that has the desired property or action, then it is desirable to divide those approximately one hundred beads into vessels in such a manner that each vessel contains a maximum of one bead, because then, in the next detection step, the bead that is of interest can be unambiguously located. If, however, the number of beads in the target vessel either can not be controlled precisely or is unknown, then still further steps must be carried out in order to isolate or detect unambiguously the bead that is of interest. A problem underlying the invention is therefore to provide a device for counting small particles, especially beads. With the device it is to be possible to count beads in the form of a suspension as far as possible without error. The loss of individual beads in the course of the counting process is to be avoided. A further problem underlying the invention is to provide a sorting apparatus with which small particles, for example beads in the form of a suspension, can be removed from one vessel and distributed into other vessels, it being possible to control and determine the number of particles introduced into a vessel. With such a sorting apparatus, it is to be possible, especially, to divide small amounts of, for example, up to a few hundred particles into still smaller portions of from one particle to a few dozen such particles each. The apparatus is also to enable a portion of beads to be distributed, in a controlled manner, into the various positions of a microtitre plate or deep- well plate so that, for example, there is subsequently a maximum of one bead in each position. Furthermore, with such a sorting apparatus, the loss of particles or beads is to be avoided as far as possible. Those problems are solved by a device for counting small particles, especially carriers (beads), that has the features of independent patent claim 1 , and by a sorting apparatus that has the features of patent claim 14. Further advantageous measures and arrangements will be found in the dependent claims. The invention is described hereinafter in greater detail with reference to embodiments and with reference to the drawings. In the schematic drawings, which are not true to scale: Fig. 1 : shows a longitudinal section through a first embodiment of the device according to the invention for counting small particles, Fig. 2: shows a cross-section through a second embodiment of the device according to the invention, Fig. 3: is a diagram showing signal intensities of the measuring light as a function of the wavelength of the measuring light, and Fig. 4: shows a schematic representation of an embodiment of the sorting apparatus according to the invention showing the essential parts. In the following description, the small particles to be counted or sorted are, by way of example, beads in the form of a suspension. In accordance with the generally customary nomenclature, the term "bead" denotes small carriers that, typically, are essentially spherical in shape and have a diameter of customarily up to 200 μm. They are made, for example, of resin or polystyrene. Nowadays, in the context of combinatory technologies, they are often used as solid bodies on which the chemical substances to be synthesized are built up successively from reagents that serve as building blocks. Such beads, which are often used in solid phase synthesis, are sufficiently well known and therefore require no further explanation. It will be understood that the invention is not limited to applications in which beads are counted or sorted. Without departing from the scope of the invention, the particles to be counted or sorted may equally well be other small particles, for example small particles that are not spherical in shape. They may be, for example, relatively large colloids or other small-volume physical structures. The device according to the present invention is also suitable for counting or sorting animal varieties for example small insects, their eggs or larvae, especially in high-throughput- screen (HTS) systems. In said systems plants or plant parts are infested with a predetermined number of such animal varieties. These are then treated with a bioactive compound and the resulting effect is determined by comparision with an untreated standard. In the following description of the preferred embodiments with reference to the drawings, identical parts or parts that have equivalent functions have been given identical reference numerals. The device according to the invention for counting small particles, especially carriers (beads), is based on an optical method. It comprises, typically, a capillary 2 (Fig. 1) for receiving the particles to be counted, a light source 3 for emitting measuring light, which is indicated symbolically in Fig. 1 by the arrows denoted by the reference character M, and also light-delivering means which deliver the measuring light M emitted by the light source 3 to the capillary 2, a photoelectric detector 5 for receiving the measuring light M, and light- removing means which deliver the measuring light M coming from the capillary 2 to the photoelectric detector 5. In particular, in the device according to the invention, firstly the intemal diameter ID of the capillary 2 is such that the individual particles can enter the capillary 2 only one behind the other and can move along inside it only one behind the other. Secondly, the light-delivering means irradiate the capillary 2 with the measuring light M transversely to the longitudinal axis thereof, the measuring light M being in the form of a light beam, the width of which is such that only one of the particles can be completely in the light beam at any one time. In Fig. 1 , the light beam is indicated symbolically in the region of the capillary 2 by the two rays M1 and M2. The term "capillary" is used to mean generally tube-like structures, the cross-section of which is small in comparison with their longitudinal extent, and comprises, for example, also pipettes, micropipettes and syringes. In principle, the device according to the invention operates as follows: the measuring light M emitted by the light source 3 passes through the light-delivering means to the capillary 2 and irradiates the capillary 2 with a light beam transversely to the longitudinal direction of the capillary 2. After passing through the capillary, the measuring light M passes through the light-removing means to the photoelectric detector which converts the measuring light M into an electrical signal which is available for further processing and evaluation. If the capillary 2 is then immersed in a container 20 that contains, for example, a suspension 21 of a solvent and beads 22, a drop of the suspension 21 moves into the capillary 2 and moves along inside it. The drop may contain no beads, one bead or a plurality of beads. Since, according to the invention, the internal diameter of the capillary 2 is such that the beads can move along in the capillary 2 only one behind the other but not next to one another, and furthermore, since the width of the light beam passing through the capillary 2 is such that only one of the beads can be completely in the light beam at any one time, the beads must cross the light beam one after the other. When a bead crosses the light beam, changes occur in the measuring light M received by the photoelectric detector. By reference to those changes, it is possible to count the individual beads that may be present in the drop. Surprisingly, it has been demonstrated that, by reference to the changes in the measuring light M received by the photoelectric detector 5, all the possible interfaces or transitions, namely air/solvent, solvent/bead, bead/solvent and solvent/air can be detected clearly. Those facts are documented further hereinafter. In that manner, it is possible to count the beads contained in a drop without error. Moreover, it is also possible to count the drops moving in the capillary 2. Thus, the number of drops is known and the number of beads contained in each drop can be determined reliably. In addition, the device according to the invention dispenses with additional reservoirs. The drops of the suspension or the beads can be removed directly from the container 20 by means of the capillary 2 so that no beads are lost during the counting process. Fig. 1 shows a longitudinal section of a first preferred embodiment of the device according to the invention for counting small particles, especially beads. That Figure shows the device in its normal position of use. Terms such as "upper" and "lower" used hereinafter relate to that position of use. The device according to the invention is denoted as a whole by the reference numeral 1. The capillary 2 is in the form of a hollow cylinder having a longitudinal axis A and an intemal diameter ID. The light-delivering means preferably comprise a delivering optical waveguide 4 which has a casing 41 and also at least one optical fibre 42, for example a glass fibre. The light-removing means preferably comprise a removing optical waveguide 6 which has a casing 61 and also at least one optical fibre 62, for example a glass fibre. The arrangement of the light-delivering and light-removing means with the optical waveguides 4, 6 is preferred because a simple optical connection for guiding the measuring light M between the light source 3 and the capillary 2, and between the capillary 2 and the photoelectric detector 5, can thus be obtained. In particular, the capillary 2, with the optical waveguides 4, 6 fixed in relation thereto, can be moved independently of the light source 3 and the photoelectric detector 5 without thereby impairing the propagation of the measuring light M along the light path from the light source 3 through the capillary 2 to the detector 5. For reasons of space, it is especially advantageous if the delivering optical waveguide 4 and the removing optical waveguide 6 are arranged - as shown in Fig. 1 - parallel to the longitudinal axis A of the capillary 2. In the region of the light-exit surface 43 of the delivering optical waveguide 4 there is then provided a first reflecting surface 10 which is so arranged that it deflects the emerging measuring light M towards the capillary 2. In similar manner, there is provided in the region of the light-entry surface 63 of the removing optical waveguide 6 a second reflecting surface 11 which is so arranged that it deflects the measuring light M emerging from the capillary 2 into the removing optical waveguide 6. The representation of the light source 3 and of the photoelectric detector 5 in Fig. 1 is to be understood as being symbolic. The light source 3 may comprise, for example, a deuterium lamp, a light-emitting diode or a laser diode. The photoelectric detector may comprise, for example, a photoelement, a photocell, a photomultiplier, a photodiode or a diode array. In the embodiment shown in Fig. 1 , a sleeve 8 which surrounds the capillary 2 closely is also provided. There is also provided a carrier element 9 having a cavity which receives the sleeve 8 with the capillary 2 and also the two optical waveguides 4, 6 arranged parallel to the longitudinal axis A of the capillary 2. The cavity is substantially cylindrical in its upper region. In the region of its lower end, the cavity narrows by an angle of approximately 45° and merges into a substantially circular bore, the diameter of which corresponds substantially to the external diameter of the capillary 2. The capillary 2 extends beyond the lower end of the carrier element 9. The sleeve 8 extends approximately to the start of the narrowing of the cavity in the carrier element 9. The two optical waveguides 4, 6 terminate above the lower end of the sleeve 8. The carrier element 9 may be mounted, for example, in the region of its upper end on a holding device (not shown) which allows the carrier element 9 and also, of course, the capillary 2 to be moved up and down. The sleeve 8 is connected securely to the carrier element 9 by way of the enclosed optical waveguides 4, 6. That can be achieved, for example, by filling the remaining free space of the cavity with an adhesive so that the optical waveguides are cast in between the sleeve 8 and the carrier element 9 in a mechanically stable manner. Suitable adhesives are, for example, epoxy- resin-based adhesives. In the preferred arrangement of the first embodiment, a transparent mounting 7 is fitted into the region of the narrowing portion of the cavity of the carrier element 9. The term "transparent" means that the mounting 7 is substantially transparent at least for the wavelength or the wave range of the measuring light M used for the detection. The transparent mounting 7 also extends into the cylindrical region of the cavity as far as the lower ends of the optical waveguides 4, 6 and substantially completely fills the space between the lower ends of the optical waveguides 4, 6, the sleeve 8 or capillary 2 and the wall that bounds the cavity. The portion of the transparent mounting 7 that is in the cylindrical region of the cavity is, as a result, annular in shape and surrounds the sleeve 8 together with the capillary 2 inside it. The portion of the transparent mounting 7 that is in the narrowing region of the cavity is of substantially annular but in this embodiment conically tapering shape and surrounds the capillary 2 directly so that its bounding face that faces the capillary 2 forms both a light-exit window 71 for the measuring light M coming from the delivering optical waveguide 4 and a light-entry window 72 for the measuring light M coming from the capillary. The bounding face, remote from the capillary 2, of the conical portion of the transparent mounting 7 is preferably in the form of a reflecting surface, so that the first and second reflecting surfaces 10, 11 form part of the transparent mounting 7. The reflecting surface can be produced, for example, by applying a metal layer, for example by vacuum metallisation. Other possibilities are that the inner wall of the carrier element 9 that bounds the narrowing region of the cavity is in the form of a reflecting surface, or the total intemal reflection effect is used. The reflecting surfaces 10, 11 may also be in the form of a curved surface, for example a truncated conical surface and/or a hemispherical surface. As a result, for example, the divergence of the measuring light striking the surfaces 10, 11 can at least be reduced. The transparent mounting 7 can be manufactured, for example, from glass, quartz or sapphire. It forms an optical connection between the optical waveguides 4, 6 and the capillary 2. The lower ends of the optical waveguides 4, 6 are each connected to the transparent mounting 7 in a mechanically stable manner. The connection may be effected, for example, by bonding with a transparent adhesive 13. It is especially advantageous if the adhesive 13 has a refractive index that corresponds substantially to the refractive index of the optical fibres 42, 62 or to the refractive index of the transparent mounting 7. As a result, undesirable reflections and scattered light can be reduced. Suitable adhesives are, for example, two-component epoxy-resin-based adhesives. Of course, it is also possible to form the connection between the optical waveguides 4, 6 and the transparent mounting so that the transparent mounting 7 is provided with bores into which the ends of the optical waveguides are inserted. As shown in Fig. 1 , in the first embodiment of the device according to the invention there is provided in the region of the lower end of the carrier element 9, at the point where the narrowing region of the cavity merges into the circular bore, a seal 14, for example an O- ring, between the outer wall of the capillary 2 and the carrier element 9. The seal 14 prevents solvent or portions of the suspension from penetrating past the capillary 2 into the inside of the carrier element 9 as the capillary 2 is immersed in the container 20 and, for example, contaminating the light-exit window 71 or the light-entry window 72. The described arrangement of the first embodiment with the sleeve 8, the carrier element 9 and the transparent mounting 7 has a number of advantages. As a result of the arrangement of the optical waveguides 4, 6 parallel to the longitudinal axis A of the capillary 2 and of the transparent mounting 7, the form of the device 1 according to the first embodiment is very compact and space-saving. As a result of the mechanically stable mounting of the optical waveguides 4, 6 and of the mounting 7 in the carrier element 9 and also as a result of the robust connection of the optical waveguides 4, 6 to the transparent mounting 7 and also to the carrier element 9 and the sleeve 8, movements or vibrations of the device do not impair the propagation of the measuring light M along the optical light path. In addition, propagation of the measuring light M between the light source 3 and the light-exit window 71 and between the light-entry window 72 and the photoelectric detector 5 always takes place in media that have substantially the same refractive index so that scattered light effects, which impair the intensity of the measuring light M and hence the sensitivity of detection, are reduced considerably. Moreover, the bounding face of the transparent mounting 7 facing the capillary 2, which bounding face contains the light-exit window 71, is curved and thus has the property of at least reducing the divergence with which the measuring light M customarily emerges from the optical fibre 42 of the delivering optical waveguide 4. Furthermore, there is no mechanical connection between the capillary 2 and the transparent mounting 7. The capillary 2 can thus be removed in simple manner from the sleeve 8 that surrounds it closely without it being necessary to manipulate the optical elements of the device for that purpose. That is especially advantageous because, for example in the case of contamination, the capillary 2 can be exchanged for a new capillary rapidly and without great expense. In particular, complicated adjustment of the other parts of the device is not necessary after such an exchange. The upper end of the capillary 2 may be connected by a tube 15 to a syringe or pump device (not shown) in order, on the one hand, actively to suck the suspension out of the container 20 into the capillary 2 or, on the other hand, to eject drops in the capillary in an active and purposeful manner. It is, of course, also possible to arrange the optical waveguides 4, 6 with their longitudinal direction at an angle, especially approximately at right angles, to the longitudinal axis A of the capillary 2. In that case it is possible to dispense with the reflecting surfaces 10 and 11 because the light-exit surface 43 of the delivering optical waveguide 4 is already so oriented that the measuring light M emerging therefrom irradiates the capillary transversely to the longitudinal axis A thereof. Fig. 2 shows a cross-section of a second embodiment of the device according to the invention. The second embodiment differs from the first essentially in that the light- delivering means and the light-removing means each comprise a plurality of delivering optical waveguides 4a, 4b, 4c and removing optical waveguides 6a, 6b, 6c. If that second embodiment were to be shown in longitudinal section, the drawing would be substantially the same as in Fig. 1. The drawing in Fig. 2 corresponds to a section perpendicular to the longitudinal axis A of the capillary 2 and above the transparent mounting 7. The explanations above relating to the first embodiment, especially with regard to the sleeve 8, the carrier element 9 and the transparent mounting 7, apply analogously to the second embodiment also. In the embodiment shown in Fig. 2, each of the delivering optical waveguides 4a, 4b, 4c and each of the removing optical waveguides 6a, 6b, 6c is arranged parallel to the longitudinal axis A of the capillary 2. As can be seen clearly in Fig. 2, the optical waveguides 4a, 4b, 4c, 6a, 6b, 6c are arranged in a ring around the capillary 2 between the outer wall of the sleeve 8 and the wall bounding the cavity in the carrier element 9. The optical waveguides 4a, 4b, 4c, 6a, 6b, 6c all terminate at the same height relative to the capillary 2, as shown, for example, also in Fig. 1. Accordingly, the light-exit surfaces of all the delivering optical waveguides 4a, 4b, 4c and the light-entry surfaces of all the removing optical waveguides 6a, 6b, 6c all lie in one plane, which is substantially perpendicular to the longitudinal axis A of the capillary 2. That arrangement has the advantage that the light path for the measuring light is symmetrical with respect to the longitudinal axis A of the capillary 2. As shown in Fig. 2, the optical waveguides 4a, 4b, 4c, 6a, 6b, 6c are, moreover, so arranged around the capillary 2 that each of the delivering optical waveguides 4a, 4b and 4c and a respective one of the removing optical waveguides 6a, 6b and 6c lie in a pair diametrically opposite one another with respect to the capillary 2. Thus, exactly opposite each light-exit surface of one of the delivering optical waveguides 4a, 4b, 4c, with respect to the longitudinal axis A of the capillary 2, there is located one of the light-entry surfaces of the removing optical waveguides 6a, 6b, 6c. Each delivering optical waveguide thus has an associated removing optical waveguide: the removing optical waveguide 6a is associated with the delivering optical waveguide 4a, the removing optical waveguide 6b is associated with the delivering optical waveguide 4b and the removing optical waveguide 6c is associated with the delivering optical waveguide 4c. The delivering optical waveguides 4a, 4b, 4c may all be connected to the same light source 3 (Fig. 1). In that case, the light source comprises a beam-splitting device, known perse, that splits the measuring light M into the different delivering optical waveguides 4a, 4b, 4c. Alternatively, it is also possible for a separate light source to be associated with each of the delivering optical waveguides 4a, 4b, 4c, in a manner analogous to that shown in Fig. 1 for one optical waveguide. The latter variant with the separate light sources is preferred because a higher intensity of the measuring light M inside the capillary can thus be produced, as a result of which the sensitivity of detection of particles crossing the measuring light beam in the capillary 2 is increased. It is, of course, also possible for a plurality of different light sources to be used as separate light sources. Those light sources may differ, for example, in the wavelength or wave range of the measuring light emitted. It is also advantageous, with a view to as great as possible sensitivity, if a separate photoelectric detector is associated with each of the removing optical waveguides 6a, 6b, 6c, in a manner analogous to that shown in Fig. 1 for one removing optical waveguide. The second embodiment has the advantage that the inside of the capillary 2 can be better and more homogeneously illuminated because the delivering optical waveguides 4a, 4b, 4c irradiate the capillary 2 with the measuring light from different sides. The sensitivity of detection and consequently the reliability with which the individual particles are counted can thus be increased further. It will be understood that the number of the delivering and removing optical waveguides 4a, 4b, 4c and 6a, 6b, 6c shown in Fig. 2 (in each case three) is given by way of example. Arrangements are also possible in which the light-delivering means and the light-removing means each comprise only two or, alternatively, more than three optical waveguides. According to the invention, the internal diameter of the capillary 2 is such that the individual particles to be counted can enter the capillary 2 only one behind the other and can move along inside it only one behind the other and moreover, the width of the measuring light beam that irradiates the capillary 2 is such that only one of the particles can be completely in the light beam at any one time. It is especially preferred that those features be obtained as a result of the diameter of each optical waveguide not exceeding the diameter of the capillary 2. The diameter of the optical waveguide is intended to mean the diameter of the light-guiding fibres or, in the case of a plurality of fibres in an optical waveguide, their total diameter. Especially in view of an application of the device according to the invention in which the particles to be counted are beads, the internal diameter of the capillary 2 is especially less than 300 μm and more especially less than 160 μm. That dimension ensures that, for the beads customarily used today, the beads can move along inside the capillary 2 only one behind the other and not side by side. It will be understood that the invention is not limited to the use of optical waveguides. It is also possible for the light-delivering means and light-removing means to comprise different means for guiding the light, for example graduated index lenses (GRIN lenses). The operation of the device according to the invention for counting small particles will now be explained below with reference to the first embodiment and with reference to Fig. 1. Of course, these descriptions apply analogously to the second embodiment also. In the operating state, the light source 3 (Fig. 1 ) emits the measuring light M, which propagates through the delivering optical waveguide 4 and, via the light-exit surface thereof, enters the transparent mounting 7. The measuring light M is then reflected at the first reflecting surface 10, passes through the light-exit window 71 and irradiates the capillary 2 with the light beam transversely to the longitudinal axis A thereof, the light beam being indicated symbolically in Fig. 1 by the light rays M1 and M2. The distance between the rays M1 and M2 indicates the width of the light beam. The divergence of the measuring light M is at least reduced by the curved light-exit window 71. After passing through the capillary 2, the measuring light passes through the light-entry window 72 into the transparent mounting 7, is reflected once again at the second reflecting surface 11 and, via the removing optical waveguide 6, reaches the photoelectric detector 5 which converts it into an electrical signal. The signal is supplied to an evaluating unit (not shown) for further processing. The carrier element 9 together with the parts contained therein is then lowered either by motorized means or by hand until the lower end of the capillary 2 comes into contact with the suspension 21 , containing the beads to be counted, in the container 20. A drop of the suspension 21 is then sucked up, for example by means of the syringe or pump device (not shown) which is connected to the tube 15 at the upper end of the capillary 2, and enters the capillary 2, in which it moves upwards. When the drop, which either consists entirely of solvent or contains at least one bead, enters the measuring light beam, this produces changes in the measuring light M received by the photoelectric detector. That occurrence is shown in Fig. 3. That graph shows the intensity of the measuring light received by the photoelectric detector as a function of the wavelength of the measuring - 15 - light. The wavelength of the measuring light is plotted in nanometers on the axis labelled λ and the absorption of the measuring light is plotted in units of absorption on the axis labelled AS. The zero point of absorption is set as the absorption measured by the photoelectric detector 5 when there is only solvent and no beads inside the measuring light beam in the capillary 2. That scale is, of course, entirely arbitrary. Ethanol is used as solvent in the graph according to Fig. 3. The curve labelled L shows the absorption of the measuring light measured in the case where air is present in the measuring light beam and the curve labelled B shows the absorption of the measuring light in the case where there is one bead in the measuring light beam. It can be seen clearly that, by reference to the absorption of the measuring light, it is possible to distinguish, beyond doubt, whether a bead or air or solvent is present in the measuring light beam. Moreover, the graph in Fig. 3 shows that, by reference to the changes in the intensity of the measuring light received by the photoelectric detector 5, all the possible interfaces and transitions, namely air/solvent, solvent/bead, bead/solvent and solvent/air can be detected beyond doubt. The graph in Fig. 3 also shows that the differences in the absorption of the measuring light between curve B and curve L are dependent on the wavelength. In the application of the device according to the invention, the wavelengths used for the measuring light will, of course, be those with which the differences are most strongly pronounced. Depending on the material of the beads and of the solvent, those wavelengths may be in the infrared range, in the visible range or in the ultraviolet range. The fact that a very wide wave range is shown in Fig. 3 is purely illustrative. In practical application, it is sufficient to detect the measuring light in a distinctly narrower wave range, or alternatively to use monochromatic measuring light. The choice of suitable wavelength depends on the beads and on the solvent used. In principle, the evaluation can be carried out monochromatically, polychromatically or spectrally (see Fig. 3). When the suspension drop moves upwards in the capillary 2, the photoelectric detector connected to the evaluating unit (not shown) registers first the air/solvent transition, and thus the "start" of the drop, by reference to the change in the measuring light it receives. If a bead is present in the drop, the solvent/bead transition is registered next. The bead/solvent transition is then registered and it is thus known that a bead is present in the suspension drop. If further beads are present in the drop, next a solvent/bead transition and then again a bead/solvent transition will be registered, and so on. If, however, the "end" of the drop passes through the measuring light beam, then a solvent/air transition is registered. In that manner, at the end of the operation of suction into the capillary, it is known unambiguously and beyond doubt how many drops are in the capillary 2 and it is also known how many beads each drop contains. Because, according to the invention, the dimensions of the capillary 2 are such that there is room inside it for two beads only one behind the other and not side by side, and additionally that the width of the measuring light beam is such that only one bead can be completely in the measuring light beam at any one time, each individual bead is registered and, as a result, the beads can thus be counted without error. It is, therefore, not the volume of the suspension that is measured or registered but rather the individual bead directly. The difference in the intensity of the measuring light between the case in which there is a bead in the measuring light beam and the case in which there is only solvent in the measuring light beam is so great that it is also possible to detect when two beads are sticking to one another, that is to say are not fully separated from one another by solvent. A particular advantage of the device according to the invention is, moreover, that it is not limited to the use of a specific solvent, but is suitable for all solvents. Moreover, the device does not require the supply of additional solvent and also does not require an additional reservoir for the beads. This reduces the risk of losing individual beads quite considerably. The present invention relates also to a sorting apparatus for distributing small particles, especially carriers (beads), which comprises a device according to the invention for counting small particles. Sorting apparatuses perse are known from the prior art. For example, in the context of combinatory technology, sorting apparatuses for distributing beads are often used, for which the English term "autosampler" has been adopted. Compared with known apparatuses of the generic type, the sorting apparatus according to the invention is improved in that it comprises a device according to the invention for - 17 - counting small particles, as a result of which it is possible to control and determine the number of particles distributed. Fig. 4 shows an embodiment of the sorting apparatus according to the invention in highly schematic form showing the essential parts. The sorting apparatus as a whole has been given the reference numeral 100 and comprises a counting device of the invention according to the first embodiment described above (see Fig. 1). A counting device of the invention according to the second embodiment described above is, of course, also suitable for integration into a sorting apparatus according to the invention. The embodiment of the sorting apparatus according to the invention shown in Fig. 4 also comprises a base plate 101 and a wall element 102 which is fixed to the side of the base plate 101. Movably attached to the wall surface 102 is an arm 103 which extends over the base plate 101 substantially parallel thereto. The arm 103 is movable along the wall surface 102, as shown symbolically in Fig. 4 by the double arrow X. On the arm 103 there is provided a holding and lifting device 104 which extends from the arm 103 towards the base plate 101. The holding and lifting device 104 is movable along the longitudinal direction of the arm 103, as shown symbolically by the double arrow Y. The direction of movement X of the arm 103 and the direction of movement Y of the holding and lifting device 104 are thus perpendicular to one another. Moreover, the holding and lifting device 104 can be extended and retracted, for example in telescopic manner, in the direction of the base plate 101. That direction of movement is indicated symbolically in Fig. 4 by the double arrow Z. Drive means (not shown), for example motorized drive means, are provided for moving the arm 103 and the holding and lifting device 104. For the sake of clarity and because such drive means for moving the arm 103 in direction X and the holding and lifting device 104 in directions Y and Z are sufficiently well known perse, the drive means have not been shown in Fig. 4. Mounted on the end of the holding and lifting device 104 facing the base plate 101 is the carrier element 9 of the counting device, which carrier element contains the capillary 2, the sleeve 8 (Fig. 1 ), the transparent mounting 7 (Fig. 1 ) and also the ends of the delivering and removing optical waveguides 4 and 6. The delivering optical waveguide 4 is connected to the light source 3 and the removing optical waveguide 6 is connected to the photoelectric detector 5. The end of the capillary 2 remote from the base plate 101 is connected via a tube 106 to a pump device 105 which can produce a subatmospheric pressure or a superatmospheric pressure in the capillary 2. The pump device 105 may, for example, comprise a syringe, a micropump or a different pump. There is also provided a control and evaluating unit 107 which, on the one hand, controls the drive devices (not shown) for moving the arm 103 and the holding and lifting device 104, as indicated symbolically by a signal line 108, and which, on the other hand, receives signals from the photoelectric detector 5 via a further signal line 109, which signals are processed further and evaluated by the control and evaluating unit 107 to count the beads or the drops. The unit 107 also controls the pump device 105 by way of a connecting line 110. The operation of that embodiment of the sorting apparatus according to the invention is explained below with reference to an example. A suspension of beads and a solvent is present in an initial container 120. The beads are to be distributed into various target vessels 122, for example the various positions of a microtitre plate 121 or a deep-well plate, in such a manner that a maximum of one bead is then present in each of the target vessels 122. The initial container 120 and the microtitre plate 121 with the target vessels 122 are placed on the base plate 101. First, the arm 103 and the holding and lifting device 104 are so moved that the capillary 2 is above the initial container 120. The carrier element 9 is then lowered by means of the holding and lifting device so that the capillary 2 comes into contact with the suspension in the initial container 120 and sucks up a drop from the suspension by means of the pump device 105. As the drop rises up the capillary 2, the number of beads contained in the drop is counted, as already described in detail above. If the drop does not contain a bead or contains more than one bead, it is ejected back into the initial container 120 by means of a pressure pulse from the pump device 105 and a new drop is then sucked up. If the drop contains precisely one bead, the carrier element 9 with the capillary 2 is raised by the holding and lifting device 104. The holding and lifting device 104 is then moved over an as yet unoccupied target vessel 122 and the carrier element with the capillary 2 is lowered again. Finally, the drop with the bead contained therein is ejected, for example by means of compressed air, by the pump device 105 from the capillary 2 into the target vessel 122. The holding and lifting device 104 then again raises the carrier element 9 with the capillary 2 and is moved back over the initial container 120, from which a new drop can be removed. In that manner it is possible to distribute the beads contained in the initial container 120 individually into the various target vessels 122. Since the counting device according to the invention makes it possible not only to count the beads contained in one drop but also to determine the number of drops sucked into the capillary 2, a plurality of drops can be sucked into the capillary 2 from the initial container 120 in one suction operation. If, for example, three drops are already present in the capillary 2, each one of which contains precisely one bead, and if, for example, two beads are detected in the fourth drop that is sucked in, then it is possible, by a metered supply of pressure, to eject only the fourth drop back into the initial container 120 and to keep the other three drops in the capillary 2. In that manner it is possible to suck up a plurality of drops into the capillary 2, each one of which contains precisely one bead. The capillary 2 is then moved over an as yet empty target vessel 122. By a metered supply of pressure, precisely one of the drops in the capillary 2 is then ejected into the target vessel 122. The capillary is then moved over a different, as yet empty target vessel and the next drop is ejected. That operation is repeated until there are no more drops in the capillary 2. The capillary 2 can then be moved back to the initial container 120 again to take up new drops. The possibility of sucking up a plurality of drops into the capillary is especially advantageous because the sorting process can, as a result, be carried out markedly more rapidly. It will be understood that the sorting apparatus according to the invention is not limited to applications in which the beads are to be distributed individually into target vessels. It is, of course, also possible to distribute the beads in larger portions. It may, for example, be desirable to distribute the beads in such a manner that not more than a predetermined number of beads is present in each of the target vessels. That predetermined number may be up to several dozens. The counting device then registers whether the number of beads sucked into the capillary is greater than the predetermined number. If that is the case, the drops with the beads are ejected back into the initial container 120. If, on the other hand, the number of beads sucked up is smaller than or equal to the predetermined number, then the beads are ejected into an empty target vessel 122. The sorting apparatus according to the invention thus allows small particles, for example beads, to be distributed into target vessels 122 in such a manner that the number of particles introduced into the target vessel 122 can be controlled and determined. In particular, it is possible to distribute a portion of such particles, in a controlled manner, into various target vessels 122 so that a maximum of one particle is then present in each of the target vessels 122. Using the sorting apparatus, the particles can be removed directly from the initial container 120, so that no further reservoirs are necessary. As a result, the risk of losing one of the particles during the distribution operation is reduced quite considerably.

Claims: What is claimed is. 1. A device for counting small particles having a capillary (2) for receiving the particles to be counted, a light source (3) for emitting measuring light (M), light-delivering means which deliver the measuring light (M) emitted by the light source (3) to the capillary (2), and also a photelectπc detector (5) for receiving the measuring light (M) and also light-removing means which deliver the measuring light (M) coming from the capillary (2) to the photoelectric detector (5), in which device the internal diameter (ID) of the capillary (2) is such that the individual particles can enter the capillary (2) only one behind the other and can move along inside it only one behind the other, and the light-delivering means irradiate the capillary (2) with the measuring light (M) transversely to the longitudinal axis (A) thereof, the measuring light (M) being in the form of a light beam (M1 , M2), the width of which is such that only one of the particles can be completely in the light beam (M1 , M2) at any one time. 2. A device according to claim 1 , in which the light-delivering means comprise a delivering optical waveguide (4) and the light-removing means comprise a removing optical waveguide (6), which optical waveguides each comprise one or more optical fibres (42, 62). 3. A device according to claim 1 , in which the light-delivering means and/or the light- removing means each comprise a plurality of delivering or removing optical waveguides (4a, 4b, 4c and 6a, 6b, 6c, respectively). 4. A device according to either claim 2 or claim 3, in which each delivering optical waveguide (4; 4a, 4b, 4c) is arranged parallel to the longitudinal axis (A) of the capillary (2), and in which there is provided in the region of the light-exit surface (43) of each delivering optical waveguide (4; 4a, 4b, 4c) a first reflecting surface (10) which is so arranged that it deflects the emergtng measuring light (M) towards the capillary (2). 5. A device according to either claim 2 or claim 3, in which each removing optical waveguide (6; 6a, 6b, 6c) is arranged parallel to the longitudinal axis (A) of the capillary (2), and in which there is provided in the region of the light-entry surface (63) of each removing optical waveguide (6; 6a, 6b, 6c) a second reflecting surface (11 ) which is so arranged that it deflects the measuring light (M) emerging from the capillary (2) into its associated removing optical waveguide (6; 6a, 6b, 6c). 6. A device according to either claim 2 or claim 3, in which both the light-exit surface of each delivering optical waveguide (4, 4a, 4b, 4c) and the light-entry surface of each removing optical waveguide (6; 6a, 6b, 6c) lie in one plane, which is substantially perpendicular to the longitudinal axis (A) of the capillary (2), one light-entry surface (63) being located opposite each light-exit surface (43) with respect to the longitudinal axis (A) of the capillary (2). 7. A device according to claim 3, in which a separate light source is associated with each delivering optical waveguide (4; 4a, 4b, 4c). 8. A device according to claim 3, in which a separate photoelectric detector is associated with each removing optical waveguide (6; 6a, 6b, 6c). 9. A device according to either claim 2 or claim 3, in which there is provided a transparent mounting (7) which surrounds the capillary (2) and to which there are connected, in a mechanically stable manner, the end of each delivering optical waveguide (4; 4a, 4b, 4c) that comprises the light-exit surface (43) and the end of each removing optical waveguide (6; 6a, 6b, 6c) that comprises the light-entry surface (63). 10. A device according to either claim 2 or claim 3, in which each optical waveguide (4,6; 4a,4b,4c,6a,6b,6c) has a diameter of less than 300 μm. 11. A device according to claim 1 , in which the internal diameter (ID) of the capillary (2) is less than 300 μm. 12. A sorting apparatus for distributing small particles which comprises a device (1) for counting small particles according to claim 1

Inventors: DANIGEL HARALD, HASSLER MARTIN, DANIGEL, HARALD, HASSLER, MARTIN
Applicants: CIBA GEIGY AG, DANIGEL HARALD, HASSLER MARTIN, DANIGEL, HARALD, HASSLER, MARTIN, NOVARTIS AG
Application year: 1997
Main classification: G01N35
IPC codes: B01J19, C40B60, G01N15, G01N21, G01N35