JP5290690B2 - Fine particle screening device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening device of fine particles, capable of efficiently searching targeted fine particles from many fine particles, and selectively and efficiently recovering them. <P>SOLUTION: A measuring section 14 has fine particles M of a tip 90 for measurement irradiated with a light L and makes fluorescence generated from the fine particles M. A moving section 16 is mounted with the tip 90 for measurement and a recovery plate 80, moves in a first direction (X-direction) with respect to the measuring section 14 and the second direction (Y-direction) orthogonal to the first direction, and can be positioned. A recovering section 13 has a suction/delivery capillary 140, that moves in the vertical direction (Z direction) orthogonal to the first direction and the second direction, and can be positioned. The suction/delivery capillary 140 selectively suctions all the fine particles M, including the fine particles M1 that emit fluorescence, and discharges them to a predetermined position of the recovery plate 80; and in this manner, the fine particles M are recovered. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a screening apparatus for fine particles, and in particular, applies light to fine particles (for example, cells) to search for fine particles targeted by using fluorescence emitted from the fine particles as a label. The present invention relates to a fine particle screening apparatus and a fine particle screening method for selectively sucking and collecting.

Search and collection devices that search and collect cells that are target objects include the following. A sample plate on which cells are placed is placed on the sample stage, and the illumination and the objective lens are arranged at positions facing each other across the sample plate. The nozzle for sucking the cells is arranged obliquely downward with respect to the cells so as to be sucked. The sample stage can be moved by an XY stage. In the search and recovery apparatus, one sample plate is placed on the sample stage and illuminated, and the fluorescence of the cells is observed, and the fluorescent cells are collected using a nozzle. In this search and recovery device, cells applied on the slide glass are searched with a microscope, a recovery target is selected from the shape, color, size, etc., and the release liquid is dropped on that portion and then the release liquid is recovered together ( For example, see Patent Document 1). As a technique for classifying a particle sample based on fluorescence information, a cell sorter is widely used (see, for example, Patent Document 1).
JP-A-2005-207986

  In the search and recovery device disclosed in Patent Document 1, the cells applied on the slide glass are searched with a microscope, a recovery target is selected from the shape, color, size, etc. It is to be collected.

However, in this method, when a plurality of cells to be collected are present in the vicinity, it is difficult to selectively collect individual cells. In order to prevent this, a method of lowering the concentration of the sample itself can be considered, but a problem of lowering the throughput occurs. In addition, there is a problem that cells that can be targeted are limited because it is difficult to selectively collect a sample that cannot withstand dry-up and needs to be handled as a suspension. Furthermore, in a screening system using fine particles such as cells, acquisition of information such as fluorescence and chemiluminescence is widely desired.
It is desired that the member on which the sample is placed is accurately positioned with respect to the optical system including the illumination and the objective lens, thereby improving the positioning workability and preventing the defocusing and the deviation of the recognition position.

For example, when measuring with a camera or the like, it is necessary to widen the area that can be measured at a time for high throughput. In order to accurately position this region within the field of view and perform measurement with the observation focus being adjusted over the entire surface, it is necessary to satisfy both the accuracy of the member itself and the positioning accuracy during setting.
Fine particles such as noise from foreign objects are reliably removed by measuring fine particles such as cells that are all measurement targets and checking the overall luminance distribution status, and then selecting the fine particles to be collected. Recovery is desired.

Therefore, in order to solve the above-mentioned problems, the present invention can efficiently search for a target fine particle from a large number of fine particles and selectively recover the fine particle, and a fine particle screening apparatus. An object is to provide a screening method.
The present invention improves the positioning workability by positioning accurately, prevents defocusing and misalignment of the recognition position, and efficiently searches for and selects target fine particles from a large number of fine particles. Another object of the present invention is to provide a fine particle screening apparatus and a fine particle screening method that can be efficiently recovered.

  In the present invention, fine particles such as cells, which are all measurement objects, are measured and the entire distribution situation is confirmed, and then fine particles to be collected are selected and targeted from a large number of fine particles. It is an object of the present invention to provide a fine particle screening apparatus and a fine particle screening method capable of efficiently searching and selectively collecting fine particles.

  In order to solve the above-described problems, the fine particle screening apparatus of the present invention searches for fine particles using light information emitted from the fine particles as a label, and selects fine particles for selectively acquiring the searched fine particles. A screening device, which is formed of a base serving as a base of the device and a light-transmitting material, and in which a well containing the fine particles is formed by injecting a suspension containing the fine particles. And a fine particle obtained by irradiating the measurement chip and the fine particle accommodated in the measurement chip with light guided from at least one light source, and / or an autofocus function. Alternatively, the optical information from each fine particle accommodated in the well by measuring the optical information with a measuring unit that acquires optical information about the well An analysis unit to be extracted, mounted on the base, mounted on the measurement chip, and a collection plate for containing the fine particles selectively acquired from the measurement chip, the measurement chip and the The recovery plate is disposed on the base and movable unit is movable in a first direction and at least a second direction orthogonal to the first direction with respect to the measurement unit, and has a suction / discharge capillary, A recovery unit for sucking fine particles in the well provided in the measurement chip by a discharge capillary and discharging and discharging the fine particles to a predetermined position of the recovery plate; and the position information of the well and the The optical information of the fine particles is collated, and the measurement unit performs measurement in a focused state and monitors the positional relationship between the suction / discharge capillary and the measurement chip. Wherein the determining by.

  In the fine particle screening apparatus of the present invention, preferably, the measurement unit includes a light source and a light detection unit for detecting the optical information, and the light source and the light detection unit include the measurement chip. The suction / discharge capillary is disposed on one side with respect to the plane including the surface, and the suction / discharge capillary is disposed on the other side with respect to the plane including the measurement chip.

  In the fine particle screening apparatus of the present invention, preferably, the moving unit is mounted on the first table, and the second table is positioned on the first table so as to move in the first direction relative to the first table. A table and a mounting table mounted on the second table and capable of positioning by moving in the second direction with respect to the second table, and the measuring chip and the recovery plate are for mounting It is arranged side by side on the mounting surface of the table.

  In the fine particle screening apparatus according to the present invention, preferably, the measurement unit includes at least a light source, an optical filter for selecting only a desired excitation wavelength among light emitted from the light source, and the measurement chip. At least one filter unit comprising an optical filter for selecting only a desired wavelength region of optical information from the light, a dichroic mirror for switching an optical path according to a wavelength difference between the excitation light and the optical information, and the excitation wavelength An objective lens for guiding the light to the measurement chip and collecting optical information of the measurement chip; a focus unit having an autofocus function by moving the objective lens in the optical axis direction; And a light detection unit for detecting the light information.

  In the fine particle screening apparatus of the present invention, preferably, the measurement unit has a half mirror, and the upper surface of the measurement chip is irradiated with a part of light from the light source by switching between the half mirror and the filter unit. At the same time, the shape and position information of the well formed on the upper surface of the measurement chip and the upper surface are measured by guiding a part of the reflected light from the upper surface of the measurement chip to a light detection unit. .

  In the fine particle screening apparatus according to the present invention, preferably, the measurement unit emits light from two or more directions symmetrical to an axis perpendicular to the measurement chip upper surface separately from the light source. It has a light source to irradiate, and measures the shape and position information of the upper surface of the measurement chip and wells formed on this surface by transmitted light from the measurement chip.

  In the fine particle screening apparatus of the present invention, preferably, the measurement unit has an autofocus function using the contrast value of the measurement data as an index, and the measurement chip is placed on the top surface of the measurement chip by autofocus using transmitted light or reflected light during measurement. The position of the tip of the suction / discharge capillary in the measurement field of view and the distance from the measurement chip surface are measured by performing autofocusing of the tip of the suction / discharge capillary using reflected light. And

  In the fine particle screening apparatus of the present invention, preferably, the well formed on the measurement chip has a concave shape, and the recovery plate has a plurality of wells for storing the recovered fine particles. It is characterized by.

  The fine particle screening apparatus of the present invention is preferably characterized in that a plurality of recesses shallower than the depth of the well are formed on the upper surface of the measurement chip.

  The fine particle screening apparatus of the present invention is preferably characterized in that the measurement chip is fixed to the moving part using a fixture.

  In the fine particle screening apparatus of the present invention, it is preferable that the surface of the measuring chip that contacts at least the surface of the measuring chip where the well is formed (hereinafter referred to as a reference surface) is more than the measuring chip. It is made of a highly rigid material, the surface of the measuring chip where the well is formed is brought into contact with the reference surface of the fixture, and elasticity is provided from the surface opposite to the side where the well is formed. It has a sealing function so as to correct the warping of the measuring chip itself by being pressed by the member having it, position the fixing tip relative to the fixture, and further prevent the suspension dripping from the upper surface of the measuring chip. It is characterized by.

  In the fine particle screening apparatus of the present invention, preferably, the optical information is fluorescence, and the well position information of the measuring chip is measured by shape information measurement using reflected light or transmitted light, and optical information measurement is performed using the excitation light. Measurement and analysis are performed using both fluorescence information from the fine particles, and after performing fluorescence measurement by irradiating the excitation light, transmitted light measurement in the same visual field is performed.

  In the fine particle screening apparatus according to the present invention, preferably, the optical information is fluorescence, and the excitation light is further irradiated to a region including the plurality of wells on the measurement chip, so that a plurality of fields can be obtained in one field of view. When measuring the fine particles existing in the region including the well, the fine particles using the luminance distribution detected by the light detection unit when the substantially homogenous autofluorescent member is irradiated with excitation light. When correcting the fluorescence measurement value from the above, pixel noise is removed by averaging processing of a plurality of pixels of the luminance distribution or by a frequency filter.

  The fine particle screening apparatus of the present invention is preferably characterized in that the measurement chip is used as the autofluorescent homogenous member and the luminance distribution is acquired in a state where only the liquid used for suspending the fine particles is injected. To do.

  The fine particle screening apparatus of the present invention is preferably characterized in that the measurement chip is used as the autofluorescent homogenous member to obtain a luminance distribution of a portion where the well is not formed.

  The fine particle screening apparatus of the present invention preferably uses the measurement chip as the autofluorescent homogeneous member, extracts the luminance distribution of the portion other than the well from the actual measurement result, and complements this data. In this way, the luminance distribution of the entire measurement visual field is acquired in real time.

  The fine particle screening apparatus of the present invention preferably uses the measurement chip as the autofluorescent homogenous member, and from the measurement information including the fine particles, the fine particle for the luminance distribution of the entire measurement visual field by data processing. It is characterized by using a result with reduced influence.

  The fine particle screening apparatus of the present invention is preferably recovered by moving the suction / discharge capillary in a direction perpendicular to the measurement chip and moving the moving part in which the measurement chip is arranged. The suction / discharge capillary is brought close to a well in which the target fine particles are stored, the fine particles stored in the well are sucked by a syringe method, and discharged into a predetermined well of the recovery plate It is characterized by doing.

  The fine particle screening apparatus of the present invention is preferably characterized in that the inner diameter of the capillary tip is not more than twice the diameter of the well, and the approach distance to the upper surface of the measuring chip is not more than the well pitch. To do.

  The fine particle screening apparatus of the present invention preferably focuses on a reference surface for adjusting the height of the capillary whose height from the measurement chip surface is known, and holds the objective lens at the focused position. The capillary tip is moved by the recovery unit to a position where the capillary tip is in focus, thereby determining the distance from the upper surface of the measurement chip, and the capillary tip is moved by relative movement from the position coordinate of the recovery unit at this time. Is brought close to a desired distance from the measurement chip surface.

  In the fine particle screening apparatus according to the present invention, preferably, the fixture is provided with the reference surface for height adjustment of the capillary whose distance from the reference surface with which the well surface side of the measuring chip abuts is known. It is characterized by being.

  The fine particle screening apparatus of the present invention is preferably characterized in that the capillary tip position in a measurement visual field is recorded, and the well side to be collected is aligned with the capillary tip position by the moving unit.

  The fine particle screening apparatus of the present invention is preferably characterized in that the tip of the capillary is previously filled with a liquid.

  The fine particle screening apparatus according to the present invention is preferably configured to control the suction amount by the suction / discharge capillary in a stepwise or continuous manner during the suction with a constant suction speed or with reference to the suction speed at the beginning of the suction. It is characterized by reducing the speed.

  The fine particle screening apparatus of the present invention is preferably characterized in that the fine particles are living cells.

  The fine particle screening apparatus of the present invention is preferably characterized in that a speed at which the suction / discharge capillary approaches or leaves the measuring chip is controlled.

  In the fine particle screening apparatus of the present invention, preferably, the suction / discharge capillary is lowered to a place that is not the well of the measurement chip and then moved horizontally with respect to the measurement chip to be the target well. It is characterized by being located immediately above.

  In the fine particle screening apparatus of the present invention, preferably, the suction / discharge capillary images the periphery of the well before and after the operation of sucking the fine particles from the well of the measurement chip, and the analysis unit performs the operation before and after the operation. The suction / discharge capillary determines whether or not the fine particles have been sucked from the well by comparing images around the well.

  The fine particle screening apparatus of the present invention is preferably characterized in that the clogging of the fine particles at the tip of the suction / discharge capillary is estimated from an image of the tip of the suction / discharge capillary.

  The fine particle screening apparatus of the present invention is preferably characterized in that an image is obtained from the bottom surface side of the collection plate and whether or not the fine particles are collected in the well of the collection plate.

  The fine particle screening apparatus of the present invention is preferably characterized in that the fine particles sucked from the plurality of wells of the measuring chip are stored in one well of the recovery plate.

  The fine particle screening apparatus of the present invention is preferably characterized in that a preliminary suction operation is continued when the tip of the suction / discharge capillary approaches the suction position with respect to the measurement chip. .

  The fine particle screening method of the present invention is a fine particle screening method for searching for fine particles using light information emitted from the fine particles as a label, and for selectively acquiring the searched fine particles. It has a base, a measuring chip formed of a material that transmits light, and a well in which the fine particles are stored by injecting a suspension of the fine particles, and an autofocus function. And measuring to obtain optical information on the fine particles and / or wells obtained by irradiating the fine particles accommodated in the measurement chip and the measurement chips with light guided from at least one light source. And an analysis unit for extracting optical information from each of the fine particles stored in the well by collating and analyzing the optical information, A measuring plate and a recovery plate for accommodating the fine particles selectively acquired from the measuring chip are mounted on the measuring chip, and the measuring chip and the recovery plate are mounted on the measuring chip. A moving part movable in a first direction and at least a second direction perpendicular to the first direction with respect to the part, and a suction / discharge capillary installed on the base, between the measuring chip and the recovery plate A collection unit for sucking fine particles in the well provided in the measurement chip by the suction / discharge capillary, and discharging and collecting the fine particles in a predetermined position of the collection plate; And the positional relationship between the suction / discharge capillary and the measuring chip is determined by monitoring.

  In the fine particle screening apparatus of the present invention, preferably, the measurement unit has a movable reflector, and the reflector moves between the measurement chip and the collection unit to emit light from the light source. The light detection unit detects light information that is reflected and the reflected light is transmitted through the measurement chip, thereby measuring the shape and position information of the fine particles, the upper surface of the measurement chip, and the well. And

The fine particle screening apparatus of the present invention is preferably characterized in that the reflecting plate is a mirror surface.
The fine particle screening apparatus of the present invention is preferably characterized in that the reflector is a dichroic mirror.
The fine particle screening apparatus of the present invention is preferably a fine particle screening apparatus according to claim 35, wherein a diffusion plate is disposed between the reflector and the measurement chip.

  In the fine particle screening method of the present invention, preferably, the measurement unit includes a light source and a light detection unit for detecting the optical information, and the light source and the light detection unit include the measurement chip. The suction / discharge capillary is disposed on one side with respect to the plane including the surface, and the suction / discharge capillary is disposed on the other side with respect to the plane including the measurement chip.

  The fine particle screening apparatus according to the present invention is provided on a side surface of the measurement chip so as to be positioned in a direction parallel to the upper and lower surfaces of the measurement chip by contacting the abutting portion of the fixture. It has a projection, and a display part which is formed on the measuring chip and clearly indicates a direction when the measuring chip is set with respect to the fixture. The fine particle screening apparatus of the present invention is preferably characterized in that the protrusion is formed so as to protrude from a side surface of the measuring chip.

  In the fine particle screening apparatus of the present invention, preferably, the measurement chip has an area in which the well is formed having a width of 10 mm square or more, and a part or all of the area around the well is fixed. The flatness of an arbitrary 2 mm square in the area where the well is formed is 10 μm or less by applying a force of an appropriate size from the lower surface side where the well is not formed. And

  The fine particle screening apparatus of the present invention preferably has a thickness variation Δt in an arbitrary area of 2 mm square included in the measurement area where the well of the measurement chip is formed, at 10 / (1-1 / n) [μm] or less (n is the refractive index of the material ratio of the measuring chip).

The fine particle screening apparatus of the present invention preferably has a variation in the inclination angle φ formed by the upper surface and the lower surface in an arbitrary 2 mm square region included in the measurement area in which the well of the measurement chip is formed. , (5 × 10 ⁻⁶ ) / {t (1-1 / n)} [rad] or less (t is the average thickness [mm] of the measurement chip, and n is the refractive index of the material of the measurement chip. ).

  The fine particle screening method of the present invention is a fine particle screening method for searching for fine particles using light information emitted from the fine particles as a label, and selectively acquiring the searched fine particles, for measurement. The chip is formed of a material that transmits light, and a plurality of wells having a size capable of storing at least one of the fine particles are provided. The measurement chip is fixed to a fixture, and the measurement unit However, when the optical signal of the fine particle is obtained by irradiating the fine particle accommodated in the measurement chip and the measurement chip with the light, the protrusion of the measurement chip is connected to the fixing tool. When positioning by abutting against the abutment portion, the positioning device is set with respect to the fixing device based on a display unit that clearly indicates a setting direction with respect to the fixing device. That.

  In the fine particle screening apparatus of the present invention, the well of the collection target candidate is selected by designating one or more luminance regions consisting of a lower limit and an upper limit of the analysis luminance from the analysis result of the optical information. Features. The fine particle screening apparatus of the present invention is preferably characterized in that a luminance distribution of the fine particles in the well to be measured is displayed, and a luminance region is designated from the displayed luminance distribution.

  The fine particle screening apparatus of the present invention preferably measures the fine particles in the well a plurality of times, and collects the recovery based on the analysis result of each measurement and the calculation result calculated from the analysis result of the plurality of measurements. The well of the candidate object is selected.

  The fine particle screening apparatus of the present invention is preferably characterized in that the calculation result calculated from the measurement results of the plurality of times is a difference between measurement results at the same location or a ratio of measurement results at the same location. To do.

  The fine particle screening apparatus of the present invention is preferably a logical sum, a logical product, or a logical sum, a logical product, or the like, from each of the measurement results and the recovery target candidates selected from the calculation results calculated from the measurement results of the plurality of times. The well of the collection target candidate can be determined using a logical expression formed by combination.

  The fine particle screening apparatus of the present invention preferably displays each result of the plurality of times measurement and the calculation result calculated from the plurality of times measurement results in a graph, and a luminance region is measured by the measurer on each graph. The selection is performed.

  The fine particle screening apparatus of the present invention is preferably characterized in that a time interval is provided between the plurality of measurements in order to carry out screening based on temporal changes in fluorescence luminance emitted from the fine particles as the sample. .

  The fine particle screening apparatus according to the present invention is preferably configured such that the reagent is supplied between the multiple measurements in order to perform screening based on reactivity with a specific reagent of fluorescence intensity emitted by the fine particles as the sample. Features.

  The screening apparatus for fine particles of the present invention preferably performs screening with a plurality of types of fluorescence information emitted by the fine particles as the sample, so that it is transmitted through the optical path of irradiation light or fluorescence between the multiple measurements. It has a function of automatically switching at least one of the filters for reflection.

  The fine particle screening apparatus of the present invention preferably designates the upper limit number of the fine particles of the well that is the collection target candidate, and only the upper limit number of the wells of the collection target candidate by specifying the luminance region Is recovered.

The fine particle screening method of the present invention is a fine particle screening method for searching for fine particles using light information emitted from the fine particles as a label, and for selectively acquiring the searched fine particles. It has a base, a measuring chip formed of a material that transmits light, and a well in which the fine particles are stored by injecting a suspension of the fine particles, and an autofocus function. And measuring to obtain optical information on the fine particles and / or wells obtained by irradiating the fine particles accommodated in the measurement chip and the measurement chips with light guided from at least one light source. And an analysis unit for extracting optical information from each of the fine particles stored in the well by collating and analyzing the optical information, A measuring plate and a recovery plate for accommodating the fine particles selectively acquired from the measuring chip are mounted on the measuring chip, and the measuring chip and the recovery plate are mounted on the measuring chip. A moving part movable in a first direction and at least a second direction perpendicular to the first direction with respect to the part, and a suction / discharge capillary installed on the base, between the measuring chip and the recovery plate A collection unit for sucking fine particles in the well provided in the measurement chip by the suction / discharge capillary, and discharging and collecting the fine particles in a predetermined position of the collection plate; And selecting the well as the collection target candidate by designating one or more luminance regions composed of a lower limit and an upper limit of the analysis luminance from the analysis result of the optical information. .

  According to the screening apparatus and screening method for fine particles of the present invention, target fine particles can be efficiently searched from a large number of fine particles and selectively recovered efficiently.

According to the present invention, the measurement unit and the recovery unit can be arranged at the same time to obtain high-intensity optical information such as fluorescence, scattered light, and transmitted light of fine particles, and target from a large number of fine particles. It is possible to efficiently search for fine particles to be selectively recovered.
According to the present invention, by positioning accurately, the positioning workability is improved, the defocusing and the recognition position are prevented from being shifted, and the target fine particles are efficiently searched from a large number of fine particles. It can be selectively and efficiently collected.

  According to the present invention, after measuring fine particles such as cells that are all measurement objects and confirming the overall distribution status, the fine particles to be collected are selected, and a target is selected from a large number of fine particles. It is possible to efficiently search for fine particles to be selectively recovered.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings.
FIG. 1 is a front view showing a preferred embodiment of the fine particle screening apparatus of the present invention. FIG. 2 is a perspective view showing a preferred embodiment of the fine particle screening apparatus of FIG.

  The fine particle screening apparatus 1 shown in FIG. 1 and FIG. 2 uses a fluorescent light emitted from a fine particle as a label by applying light to a plurality of fine particles (for example, living cells) in the measurement chip 90 as a target. This is a device for searching for fine particles and selectively aspirating all fine particles in a well containing fine particles satisfying the collection conditions as a target object and collecting them on a collection plate 80. .

  The fine particle screening apparatus 1 shown in FIG. 1 includes a base 11, a support part 12, a recovery part 13, a measurement part 14, an image analysis part 15, and a moving part 16, as shown in FIG. In addition, each part is covered with a cover 19. The cover 19 prevents light and foreign matter from entering from the outside. The base 11 is a main body frame for holding each element of the screening apparatus 1.

  As shown in FIG. 1, the vertical direction in FIG. 1 is the X direction (first direction), and the left-right direction is the Y direction (second direction). The Z direction is a direction perpendicular to the X direction and the Y direction.

  The base 11 holds the collecting unit 13, the measuring unit 14, and the moving unit 16, and the base 11 has plate members 20, 21, and 22. These plate members 20 and 21 are fixed in parallel by a plurality of vertical members 23, and the plate members 20 and 22 are fixed in parallel by a plurality of members 24. The member 24 is made of a material that adjusts the height and blocks vibration.

  On the uppermost plate member 21, the support portion 12 and the support base 30 are fixed. The support part 12 is vertically arranged on the plate member 21 along the Z direction. The support base 30 has a leg portion 30R and a support plate 30P. The plate members 20, 21, 22 and the support plate 30P are arranged at intervals from each other along the Z direction.

Next, a structural example of the moving unit 16 will be described with reference to FIGS. 1 and 3.
FIG. 3 shows the moving unit 16, the mounting table 4 mounted on the moving unit 16, the recovery plate 80, and the measuring chip 90.

  3 and 1 is fixed to a support 30 of the base 11 shown in FIG. The moving unit 16 is equipped with a mounting table 40 that is a moving object, and the mounting table 40 can be moved and positioned along the X direction and the Y direction, respectively.

  As shown in FIG. 3, the moving unit 16 includes a first table 51 and a second table 52. The first table 51 is fixed to the support base 30 in FIG. 1, and the second table 52 is mounted so as to be positioned by moving along the X direction. The second table 52 is mounted so that the mounting table 4 can be positioned by moving along the Y direction.

  As shown in FIG. 3, guide rails 53 and 53 and a first motor 55 are provided on the upper surface of the first table 51. On the lower surface of the second table 52, members 54 and 54 having a U-shaped section and a nut 57 are provided. Guide rails 53 and 53 are engaged with members 54 and 54, respectively. The feed screw 56 of the first motor 55 is engaged with the nut 57. Thereby, the control part 100 commands and operates the first motor 55 to rotate the feed screw 56, whereby the second table 52 can be moved and positioned along the X direction.

  As shown in FIG. 3, guide rails 63 and 63 and a second motor 65 are provided on the upper surface of the second table 52. On the lower surface of the mounting table 40, U-shaped members 64, 64 and nuts 67 are provided. The guide rails 63 and 63 are engaged with the members 64 and 64, respectively. The feed screw 66 of the second motor 65 is engaged with the nut 67. Thus, the mounting table 40 can be moved and positioned along the Y direction by the control unit 100 instructing to operate the second motor 65 and rotate the feed screw 66.

  As shown in FIG. 3, the first table 51 has a first opening 59, the second table 52 has a second opening 69, and the mounting table 40 has a third opening 79. have. The first opening 59, the second opening 69, and the third opening 79 are sized so that they always overlap even when the second table 52 moves in the X direction and the mounting table 40 moves in the Y direction. It is an opening for guiding light (irradiation light) L from the objective lens 110 side of the measurement unit 14 to fine particles of the measurement chip 90 on the mounting table 40.

  As a result, even if the second table 52 moves in the X direction and the mounting table 40 moves in the Y direction, the light L from the objective lens 110 side causes the first opening 59, the second opening 69, and the third. The fine particles of the measuring chip 90 on the mounting table 40 are irradiated through the opening 79, and fluorescence can be generated from the fine particles.

  The mounting table 40 of the moving unit 16 shown in FIG. 3 can be moved in the X and Y directions using a motor and a feed screw, but is not limited thereto, and a linear motor or the like may be used.

  FIG. 4 shows the mounting table 40, the recovery plate 80, and the measuring chip 90. As shown in FIG. 4, the mounting table 40 is, for example, a rectangular plate-like member. On the mounting surface 70 of the mounting table 40, a collection plate 80 and a measuring chip 90 are detachable in the Y direction. Can be mounted side by side.

  The collection plate 80 is a plate-like member, and a large number of wells 81 are arranged on the collection plate 80 in a matrix form at equal intervals along the X direction and the Y direction. These wells 81 can collect and store fine particles, such as biological cells, which are sequentially discharged and discharged separately from the suction / discharge capillary 140. It is a storage unit. The well 81 is, for example, a substantially U-shaped recess or a cup-shaped recess when viewed in cross section.

  The measurement chip 90 shown in FIG. 4 is fixed by a fixing tool 120, and the fixing tool 120 is positioned and fixed at a predetermined position of the mounting table 40.

  FIG. 5 is a cross-sectional view showing an example of the shape of the measuring chip 90 and the fixture 120 of the measuring chip 90.

  The fixture 120 is for fixing and holding the measuring chip 90 at the position of the reference surface CL having a certain height with respect to the mounting surface 70 of the mounting table 40. In the example of FIG. 5, the fixing member 120 includes a first case part 121, a second case part 122, and an elastic member 123. The elastic member 123 is, for example, a rectangular ring, and the elastic member 123 is fixed to the flat surface 126 inside the second case portion 122.

  The measurement chip 90 is disposed between the first case part 121 and the second case part 122. When the measurement chip 90 is pushed up in the Z1 direction by the elastic member 123, the upper surface 90S of the measurement chip 90 is The first case portion 121 is abutted against the flat inner surface 124.

  As a result, the upper surface 90S of the measurement chip 90 is always pressed against the reference surface 124 with high positional accuracy, and the upper surface 90S of the measurement chip 90 can be positioned on the reference surface CL. Even if there are variations in thickness and warpage, the effects of thickness variation and warpage can be reduced.

  Therefore, the distance in the Z direction between the upper surface 90S of the measuring chip 90 and the objective lens 110 and the collection plate 80 of the measuring unit 14 can be accurately managed. In other words, the distance between the position of the fine particles M in the well 200 of the measurement chip 90 shown in FIG. 7 and the objective lens 110 and the recovery plate 80 of the measurement unit 14 can be accurately managed.

  The first case part 121 can be opened and closed with respect to the second case part 122 using, for example, a hinge mechanism part, whereby the measurement chip 90 in the fixture 120 is taken out and a new measurement chip 90 is obtained. Can be exchanged.

Next, the measurement chip 90 will be described with reference to FIGS. 3, 5, and 7.
An example of the shape of the measurement chip 90 is illustrated in FIG. 7A, and the measurement chip 90 is made of a light-transmitting material such as glass or plastic.

  The measurement chip 90 shown in FIGS. 3 and 5 is made of a material that transmits light, and is, for example, a rectangular plate-like member. A plurality of wells 200 are arranged in the X direction and the Y direction to store a plurality of fine particles, respectively. They are arranged in a matrix at regular intervals along the direction. As shown in FIG. 5, each well 200 has such a size that at least one of the fine particles M can be stored by dispensing or batch-injecting a suspension 500 of fine particles.

  It is desirable that a plurality of recesses shallower than the depth of the well 200 be formed on the upper surface 90S of the measurement chip 90. The shallow concave portions are used as a reference for the measurement unit 14 to autofocus the position of the measurement chip 90 in the Z direction. That is, a recess as an autofocus marking is formed in a portion other than the well on the upper surface of the measurement chip 90, but the depth of the recess is preferably as steep and shallow as possible in contrast analysis by image processing. For example, 3 μm or less is preferable. The concave portion may be formed at a fixed position on the upper surface of the measurement chip 90 or may be formed randomly, but the formation position of the concave portion should be determined in consideration of the adsorptivity of the measurement target. is there.

  As shown in FIGS. 1 and 5, the measuring chip 90 is fixed to the moving unit 16 using a fixing tool 120. In this fixture 120, at least a surface (hereinafter referred to as a reference surface) 124 that is in contact with the surface on which the well 200 of the measurement chip is formed is formed of a material having higher rigidity than the measurement chip 90. The measurement chip 90 abuts the upper surface 90S on which the well 200 is formed on the reference surface CL of the fixture 120 and presses it with the elastic member 123 having elasticity from the surface opposite to the side on which the well 200 is formed. Further, the seal member 222 seals between the reference surface CL of the fixture 120 and the upper surface 90S of the measuring chip 90. With this fixing structure, the warping of the measuring chip 90 itself is corrected, the positioning with respect to the fixing tool 90 is performed, and furthermore, the sealing 500 is surely sealed so as not to drop the suspension 500 dripping from the upper surface 90S of the measuring chip 90 Can do.

  As illustrated in FIG. 7A, each well 200 is a fine particle collection and storage unit for storing fine particles M such as biological cells. For example, the cross-sectional shape is a substantially U-shaped recess or cup. It is a recess of the mold. Each well 200 stores a plurality of fine particles M, and a film of the suspension 500 is formed on the upper surface 90S of the measuring chip 90. The diameter D of each well 200 is, for example, 10 μm, and 500 wells are arranged in the X direction and the Y direction, respectively.

Next, an example of the structure of the collection unit 13 will be described with reference to FIGS. 1 and 6.
The recovery unit 13 shown in FIG. 1 includes a suction / discharge capillary 140 that is disposed on the base 11 and can be positioned by moving in the Z direction orthogonal to the X direction and the Y direction. Thus, the total number of fine particles in the well 200 in which the fine particles M1 satisfying the luminance condition illustrated in FIG. 5 are stored is sucked and discharged to a predetermined position of the collection plate 80 and collected.

  The collection unit 13 has a suction / discharge capillary 140 that can be positioned by moving in the Z direction orthogonal to the X direction and the Y direction. The fine particles satisfying the luminance condition by the suction / discharge capillary 140. The total number M of fine particles in the well in which M1 is stored is sucked and discharged to a predetermined position of the recovery plate 80 for recovery.

  As shown in FIG. 6, the collection unit 13 includes an operation unit 130 and a base 131, and the base 131 is fixed to the support unit 12. The operation unit 130 includes a suction pump 133 and a suction / discharge capillary 140. The suction / discharge capillary 140 is a hollow member tapered in the Z2 direction (downward), and the tip 142 of the suction / discharge capillary 140 is a well of the measurement chip 90 as shown in FIGS. 200, and the rear end 143 of the suction / discharge capillary 140 is connected to the suction pump 133.

  As shown in FIG. 6, the feed screw 252 of the motor 251 is engaged with the nut 253 of the operation unit 130, and the control unit 100 operates the motor 251 to rotate the feed screw 252. Can be positioned by moving up and down along the Z direction (Z1 direction and Z2 direction).

  Accordingly, the collection unit 13 in FIG. 6 can be called a Z stage, and the moving unit 16 in FIG. 3 can also be called an XY stage.

Next, the measurement unit 14 and the analysis unit 15 will be described with reference to FIG.
1 irradiates light L to a region including a plurality of wells 200 of the measurement chip 90, thereby generating fluorescence from the fine particles M in the region, and emitting the fluorescence. Receive light. The fluorescence from the received fine particles M is image-analyzed by the image analysis unit 15. The image analysis unit 15 calculates the fluorescence intensity of the fine particles M1 that emit fluorescence of at least the maximum luminance among the plurality of fine particles M in each well 200.

  As a result, the control unit 100 recognizes the position of the well 200 in which the fine particles M1 that emit fluorescence with the maximum luminance satisfying the collection condition are stored in the plane constituted by the X direction and the Y direction. The control unit 100 gives control drive signals to the first motor 55 and the second motor 65 shown in FIG. 3 so that the well 200 of the measuring chip 90 on the moving unit 16 is directly below the suction / discharge capillary 140. Can be located. That is, the suction / discharge capillary 140 can suck fine particles in the well in units of wells. The suction / discharge capillary 140 sucks all the fine particles in the well containing the fine particles satisfying the collection condition. The suction / discharge capillary 140 can suck all fine particles from a selected well of the plurality of wells, that is, a well containing fine particles satisfying the collection condition.

  The measurement unit 14 shown in FIG. 1 irradiates the measurement chip 90 and the fine particles M accommodated in the measurement chip 90 with light guided from at least one light source, thereby allowing the shape of transmitted light or reflected light to be The position information and the luminance information such as fluorescence and chemiluminescence are acquired with a resolution finer than the average size of the individual fine particles, and the shape of the measurement chip itself and the position and size of the well 200 arranged on the measurement chip 90 are obtained. Get information such as.

  The image analysis unit 15 shown in FIG. 1 analyzes the measured shape information and optical information, so that at least one fine particle M1 satisfying the luminance condition that can be set by the measurer exists in each well 200. Get data to confirm. The fine particle screening apparatus 1 specifies the light information from the fine particles by matching the position recognition information of the well 200 with the transmitted light or reflected light and the light information of the fluorescence / chemiluminescence, and further, the measurement unit 14 has an autofocus function, performs measurement in a focused state, and determines the positional relationship between the tip 142 of the suction / discharge capillary 140 and the upper surface of the measurement chip 90 by performing autofocus on both. Can do.

  As shown in FIG. 1, the measurement unit 14 includes an objective lens 110, and the objective lens 110 guides light to the measurement chip 90. The objective lens 110 is disposed below the measurement chip 90 and the moving unit 16, and the suction / discharge capillary 140 is disposed above the measurement chip 90 and the moving unit 16. As a result, the measuring chip 90 and its moving part 16 can be disposed between the objective lens 110 and the suction / discharge capillary 140.

  In the measurement unit 14 of FIG. 1, for example, a laser light source or a mercury lamp can be used as the excitation light source 260. The shutter unit 261 is disposed between the excitation light source 260 and the fluorescence filter unit 262. When the shutter unit 261 does not irradiate the light L to the fine particles M of the measurement chip 90, the excitation light source 260 is generated. The light L to be blocked can be blocked before the fluorescent filter unit 262.

The measuring unit 14 shown in FIG. 1 will be further described.
As shown in FIG. 1, the measurement unit 14 includes at least an excitation light source 260 as a light source, an optical filter (excitation filter) 720 for selecting only a desired excitation wavelength band from light emitted from the light source, An optical filter (fluorescence filter) 721 for selecting only a desired wavelength band of optical information from the measurement chip 90 and a dichroic mirror 751 for switching the optical path according to the difference in wavelength band between excitation light and optical information. At least one fluorescent filter unit 262, an objective lens 110 for guiding the light emitted from the excitation light source to the measurement chip 90 and collecting optical information obtained from the measurement chip 90, and the objective lens 110 as light A focus unit 265 having an autofocus function that is movable in the axial direction, and optical information from the measurement target are detected. A light receiving section 266 as a light detecting portion for, and a. As shown in FIG. 1, the fluorescent filter unit 262 and the light receiving unit 266 are fixed to the fluorescent incident unit 750.

  Furthermore, the measurement unit 14 includes a half mirror (not shown), and by switching between the half mirror and the fluorescent filter unit 262, the observation target is irradiated with a part of the light from the light source 260 and at the same time. By guiding a part of the reflected light from the light receiving unit 266 that is a light detecting unit, the shape and position information of the upper surface 90S of the measuring chip 90 and the well 200 formed on the upper surface 90S can be measured. The half mirror is an optical component that generally has a function of splitting incident light by 50% intensity, but in this embodiment, the light intensity of the spectrum does not necessarily need to be 50%. However, since the measurement accuracy improves as the light intensity incident on the light receiving unit 266 increases, it is preferable to use a branching mirror having a high light intensity split into the light receiving unit 266.

  In the fluorescence filter unit 262, only the wavelength component of the excitation light is transmitted through the optical filter 720 (excitation filter), reflected (transmitted) by the dichroic mirror 751, and incident on the objective lens 110. 90 is irradiated. The light that returns from the measurement chip 90 as reflected light is reflected (transmitted) by the dichroic mirror 751 again, returns to the light source 260 side, and does not go to the light receiving unit 266 side.

On the other hand, since the wavelength of fluorescence is longer than that of the excitation light, the light receiving unit that transmits (reflects) the dichroic mirror 751 and detects only the wavelength band desired to be detected by the optical filter 721 (fluorescent filter) is a detection unit (CCD camera or the like). 266. That is, when the fluorescent filter unit 262 is used, the reflected light is basically not propagated to the detection unit and is difficult to detect. When measuring the shape and position of the measurement chip surface, the well, and the tip of the collection unit by reflected light, only the half mirror is used instead of the fluorescent filter unit 262. When the half mirror is used in this way, the half mirror is placed at the location of the dichroic mirror 751, and the other two optical filters 720 and 721 are basically not necessary, but the filter at a position corresponding to the excitation filter in the fluorescence filter unit. It is more preferable to use a filter that selectively transmits a wavelength that does not easily fade the fluorescence of the measurement target.

Further, an ND filter may be used to adjust the light amount, or an ultraviolet light cut filter may be used so as not to damage fine particles.
Thus, the measurement of position information by reflected light using a half mirror has an advantage that a light source common to fluorescence measurement can be used. This is not limited to a screening apparatus having a measurement function and a recovery function, and is a common advantage even in an apparatus that only measures.

  The plurality of objective lenses 110 and 264 of the measurement unit 14 shown in FIG. 1 can be rotated at a revolver type, for example, so that an objective lens having a necessary magnification can be positioned at a position below the measurement chip 90. The focus unit 265 moves and positions, for example, the objective lens 110 disposed in the lower position of the measuring chip 90 along the Z direction by operating the motor 265M according to a command from the control unit 100, for example. The focus adjustment of the objective lens 110 with respect to the fine particles M of the measurement chip 90 can be performed.

FIG. 28 is a detailed explanatory diagram of the measurement unit 14.
In the fine particle screening apparatus 1 </ b> B shown in FIG. 28, the light reflector 1100 is disposed between the collection unit 13 and the measurement chip 90.

Here, the fine particle screening apparatus 1B includes a measurement chip 90, a moving unit 16 for moving the measurement chip 90, an objective lens 110, an operation unit 130 of the recovery unit 13, an excitation light source 260, It has a fluorescent filter unit 262, a light receiving unit 266 such as a CCD camera, a reflecting plate 1100, a reflecting plate moving device 1110, and a control unit 100.
The excitation light source 260 and the light receiving unit (an example of a light detection unit) 266 are disposed on the same side with respect to the measurement chip 90 and the reflection plate 1100, that is, below the measurement chip 90.
The reflector 1100 is disposed between the suction / discharge capillary 140 of the operation unit 130 of the collection unit 13 and the measurement chip 90. The reflection plate 1100 may be anything as long as it can reflect and obtain transmitted light information. For example, a mirror surface or a dichroic mirror can be used.

  Further, the reflecting plate 1100 is connected to the reflecting plate moving device 1110, and the reflecting plate moving device 1110 can move the reflecting plate 1100 in the Y direction by a command from the control unit 110. The movement in the Y direction is not limited to the linear direction as shown in FIG. Thereby, the reflector 1100 can be disposed between the suction / discharge capillary 140 of the operation unit 130 of the recovery unit 13 and the measurement chip 90, and conversely, the suction / discharge capillary 140 of the operation unit 130 and the measurement chip 90. Can be withdrawn from between.

In a state where the reflector 1100 is moved and positioned between the suction / discharge capillary 140 of the operation unit 130 and the measurement chip 90, the light of the excitation light source 260 passes through the excitation filter 270 and passes through the dichroic mirror ( (Or a half mirror) 751 is reflected, passes through the objective lens 110, the opening of the moving unit 16, and the measuring chip 90, and reaches the reflecting plate 1100.
Then, the light reaching the reflecting plate 1100 is reflected by the reflecting plate 1100 and passes through the measuring chip 90 in the direction opposite to the previous transmission. The transmitted light information can be detected by the light receiving unit 266 after passing through the measurement chip 90, the dichroic mirror (or half mirror) 751, and the absorption filter 721. The image analysis unit 15 measures the shape and position information of the fine particles M in the measurement chip 90 well 200, the upper surface 90S of the measurement chip 90, and the well 200 as shown in FIG. 29 based on the transmitted light information. It has become.

   As a result, the light transmitted through the measuring chip 90 can be acquired by the light receiving unit 266 using the reflector 1100. The light source 250 shown in FIG. 31 emits light from two or more directions symmetric with respect to an axis perpendicular to the upper surface 90S of the measurement chip 90 without using a reflector, and the transmitted light from the measurement chip 90 is transmitted. As a result, a high-contrast image, for example, about twice as high as that obtained by measuring the shape and position information of the upper surface 90S of the measuring chip 90 and the well 200 formed on the upper surface 90S can be obtained. In this way, by using one excitation light source 260, the measurement chip 90 can acquire fluorescence and transmitted light. In addition, since the light from the excitation light source 260 passes through the measurement chip 90 once, ultraviolet light is cut from the transmitted light information of the measurement chip 90, so that the fluorescence is not easily faded and the fine particles are not damaged.

  FIG. 30A shows an example in which nothing is arranged between the reflector 1100 and the measurement chip 90, and FIG. 30B shows an example between the reflector 1100 and the measurement chip 90. Further, an example in which a diffusion plate 1200 is arranged is shown.

  As described above, in the example shown in FIG. 30A, the reflected light from the reflecting plate 1100 is biased toward the central portion of the measuring chip 90, whereas in the example shown in FIG. By being arranged, the reflected light from the reflecting plate 1100 can be transmitted to the measuring chip 90 as uniform light with respect to the measuring chip 90, so that transmitted light information without bias can be obtained. .

  The measurement unit 14 has an autofocus function using the contrast value of the measurement data as an index, maintains the focus on the upper surface 90S of the measurement chip 90 by the autofocus by transmitted light or reflected light during measurement, and reflects the reflected light. By performing auto-focusing of the tip / end portion 142 of the suction / discharge capillary 140, the position of the tip portion 142 of the suction / discharge capillary 140 in the measurement visual field and the distance from the measurement chip surface can be measured.

  In the fine particle screening apparatus 1, the light information is fluorescence, and the position information of the well 200 of the measurement chip 90 by shape information measurement by reflected light or transmitted light and the fluorescence from the fine particles by optical information measurement by excitation light. Measurement and analysis are performed using both information, and after measuring fluorescence by irradiating excitation light, transmitted light measurement in the same visual field is performed. That is, the fine particle screening apparatus 1 analyzes only the portion of the position of the well 200 specified by the shape information in the fluorescence measurement data. At that time, if the shape information is measured first, the fluorescence of the fine particles to be measured fades, and accurate information cannot be obtained. Therefore, the fluorescence luminance is measured by reflected light or transmitted light. It is effective to measure earlier.

  Further, by irradiating the region including the plurality of wells 200 on the measurement chip 90 with the excitation light, fine particles existing in the region including the plurality of wells 200 are measured in one field of view. At this time, when correcting the fluorescence measurement value from the fine particles using the luminance distribution detected by the light receiving unit 266 that is the light detection unit when the excitation light is irradiated to the substantially autofluorescent homogeneous member, Pixel noise is removed by averaging processing of a plurality of pixels in the luminance distribution or by a frequency filter.

  As a method for acquiring correction data, for example, using a measuring chip 90 as a homogenous member of autofluorescence, a method of acquiring an excitation light intensity distribution in a state where only a liquid used for suspending fine particles is injected, Extracting the luminance distribution of the part other than the well 200 from the measurement result, and complementing this data to obtain the luminance distribution of the entire measurement visual field, and further the luminance of the part of the chip surface where the well is not formed There is a method to acquire information. Furthermore, from the measurement information including the fine particles, the result of reducing the influence of the fine particles on the luminance distribution of the entire measurement visual field by data processing can be used.

  As the autofluorescent homogeneous member, a luminance distribution of a portion where no well is formed is obtained using a measurement chip.

  The target of correction is not only the intensity distribution (non-uniformity) of the excitation light irradiated in the measurement field of view, but also the non-uniformity of detection efficiency due to the transmission efficiency of each optical system such as a lens or filter. . These non-uniformities are highly dependent on the radial distribution of the excitation light itself and the radial distribution of optical components such as lenses that guide the excitation light and fluorescence. It is preferable to remove high frequency components. Even if the original image is obtained from measurement information containing fine particles, the effect of fine particle brightness can be substantially eliminated during the process, so depending on the required accuracy, information for correction can be obtained in advance. Rather than doing so, it is possible to correct simultaneously with the measurement. As a processing method, it is conceivable to use data processing using averaging, frequency analysis, cluster analysis, or the like. These multiple methods should be selected according to the required correction accuracy.

  In FIG. 1, when the shutter of the shutter unit 261 is opened, the light of the excitation light source 260 is transmitted only through the excitation wavelength band by the fluorescence filter 720 of the fluorescence filter unit 262, reflected by the dichroic mirror 751, and transmitted through the objective lens 110. The measurement chip 90 is irradiated. As a result, when the fine particles M in the measurement chip 90 generate fluorescence, for example, the fluorescence passes through the dichroic mirror 751, passes through only the arbitrary wavelength band by the fluorescent filter 721, and is received by the light receiving unit 266. The signal-electrical signal is converted, and the fluorescence is subjected to image analysis by the image analysis unit 15.

  Next, using the fine particle screening apparatus 1 shown in FIG. 1, the total number of fine particles including the fine particles emitting fluorescence in the well of the measuring chip 90 are selected from the fine particles in the other wells. An example of a screening method for sucking and collecting on the collecting plate 80 will be described with reference to FIGS.

  FIG. 7A is a cross-sectional view showing a part of the measurement chip 90, and fine particles are not yet contained in the well 200 of the measurement chip 90. FIG. 7B shows a state in which a plurality of fine particles M are arranged in each well 200 of the measurement chip 90.

  FIG. 8A shows a case where a plurality of fine particles M in the well 200 are irradiated with light (irradiation light, excitation light) L, and the plurality of fine particles M emit fluorescence. An example in which M1 emits fluorescence is shown. FIG. 8B shows a state in which the suction / discharge capillary 140 approaches the respective wells 200 in order and sucks and collects all the fine particles M including the fine particles M1 emitting fluorescence with the maximum luminance. Show.

  FIG. 9 shows an example in which all the fine particles including the fine particles M1 emitting fluorescence with the maximum luminance are sucked from the well 200 of the measurement plate 90 and collected in the well 81 of the collection plate 80.

  As shown in FIG. 1, on the mounting surface 70 of the mounting table 40, a recovery table 80 and a fixture 120 for the measuring chip 90 are respectively arranged at predetermined positions. The fixture 120 is disposed above the objective lens 11. The suction / discharge capillary 140 is positioned on the measurement chip 90.

  The light from the excitation light source 260 is propagated to the objective lens 11 as described above, and is applied to the measurement chip 90 as light L. As a result, the plurality of fine particles M in the measurement chip 90 shown in FIG. 8A generate fluorescence. As described above, the fluorescence of the plurality of fine particles M passes through the dichroic mirror 751. Then, only the wavelength band desired to be detected is transmitted by the optical filter (fluorescence filter) 721, received by the light receiving unit 266, converted into an optical signal-electric signal, and the fluorescence is image-analyzed by the image analysis unit 15.

  The image analysis unit 15 analyzes the fluorescence intensity of at least the maximum brightness fine particles M1 among the plurality of fine particles M in each well 200. Based on the analysis result, fine particles satisfying the luminance condition are collected from the well 200 in which the fine particles are stored.

  Based on the result of the image analysis unit 15, the control unit 100 operates the first motor 55 in response to the command of the control unit 100 shown in FIG. Thus, the second table 52 is moved and positioned along the X direction, and the second motor 65 is operated to move and position the mounting table 40 along the Y direction. Then, when the control unit 100 shown in FIG. 6 instructs and operates the motor 251, the suction / discharge capillary 140 is lowered in the Z2 direction.

  As a result, the suction / discharge capillary 140 can be positioned in any one well by bringing the tip 142 thereof close to each well 200 of the measurement plate 90 as shown in FIG. 8B.

  Thereafter, as shown in FIG. 9, when the control unit 100 commands and operates the suction pump 133, the suction / discharge capillary 140 causes the total number of fine particles including the fine particles M <b> 1 emitting fluorescence with the maximum luminance. A total number of fine particles M including the fine particles M1 are selectively sucked from the wells 200 having M separately from the fine particles of the other wells 200.

  Furthermore, when the control unit 100 shown in FIG. 6 instructs and operates the motor 251, the suction / discharge capillary 140 is raised in the Z1 direction. When the control unit 100 shown in FIG. 3 instructs and operates the first motor 55, the second table 52 is moved and positioned along the X direction, and the second motor 65 is operated, thereby mounting table. By moving and positioning 40 along the Y direction, the suction / discharge capillary 140 is relatively moved and positioned on an arbitrary well 81 of the recovery plate 80. Then, when the controller 100 shown in FIG. 6 instructs and operates the motor 251, when the suction / discharge capillary 140 is lowered in the Z <b> 2 direction, the distal end portion 142 of the suction / discharge capillary 140 is predetermined on the recovery plate 80. It approaches the well 81 at the position.

  The well 81 is preliminarily filled with a liquid LD such as a culture solution, and the tip 142 of the suction / discharge capillary 140 enters the liquid LD.

  By operating the suction pump 133 in reverse, the suction / discharge capillary 140 can discharge all the fine particles M including the fine particles M1 into the liquid in the well 81 at a predetermined position of the collection plate 80. As a result, the total number of fine particles M including the fine particles M 1 emitting fluorescence with the maximum luminance can be collected in the well 81 of the collection plate 80.

  The above-described suction and collection operations for all the fine particles including the fine particles M1 emitting fluorescence with the maximum luminance are performed in order for each well 200 of the measurement chip 90 in which the fine particles satisfying the collection conditions are stored. Do.

  In this way, the total number of fine particles including the fine particles M 1 emitting fluorescence in each well 200 is moved by the moving unit 16 in the X and Y directions and sucked by the collecting unit 13. By the operation of moving the discharge capillary 140 up and down in the Z direction, it is possible to recover from the well 81 at a predetermined position of the recovery plate 80 reliably and efficiently. As a result, target fine particles can be efficiently searched from a large number of fine particles and selectively recovered efficiently.

  In the fine particle screening apparatus 1 of the present invention, the suction / discharge capillary 140 moves in the vertical direction (Z direction) with respect to the measurement chip 90 and approaches each well 200 of the measurement chip 90. As a result, the tip 142 of the suction / discharge capillary 140 approaches each well 200 with certainty, and the total number of fine particles including the target fine particles M1 from the well 200 to the fine particles in the other wells. It can be collected by aspiration separately.

  FIG. 10 shows an example of a reference surface for height adjustment of the tip 142 of the suction / discharge capillary 140. This height matching reference surface 890 is set on the fixture 121, and the height from the surface of the measurement chip 90 is known. The reference surface 890 for height adjustment coincides with the inner surface 124 of the fixture 121. The objective lens 110 shown in FIG. 1 focuses on the reference surface 890 for height adjustment, and with the objective lens 110 held at that position, the collection unit 13 reaches a position where the tip 142 of the suction / discharge capillary 140 is in focus. Move. Thus, the distance from the upper surface 90S of the measurement chip 90 is determined, and the tip 142 of the suction / discharge capillary 140 is moved from the upper surface 90S of the measurement chip 90 by relative movement from the position coordinates of the recovery unit 13 at this time. The desired distance V is lowered to the position 891 along the Z2 direction to approach the upper surface 90S. As a result, the tip 142 of the suction / discharge capillary 140 can be reliably aligned along the Z direction using the height matching reference surface 890.

  FIG. 11 shows the recovery success rate when the fine particles are recovered by the syringe method using the suction / discharge capillary 140. The definition of the recovery success rate refers to the probability that all the fine particles in the well to be recovered are recovered by the suction / discharge capillary 140, and no single fine particle is recovered from the adjacent well.

The number of trials is 10 under each condition, and suction is by stroke control. The amount of suction was set such that the recovery rate was maximized under each condition. The suction speed was constant. As the suction target, immobilized yeast cells in PBS (Phosphate buffered saline) were used. The well diameter is about 10 μm and the well pitch is about 30 μm. The approach distance (interval) of the suction / discharge capillary 140 to the measurement chip is set to four conditions of 10, 20, 30, and 40 μm, and the inner diameter of the tip 142 of the suction / discharge capillary 140 is 10, 20, 30, and 40 μm. These four conditions were used.

  Referring to FIG. 11, the recovery success rate is higher as the approach distance (μm) to the measurement chip is smaller, and the recovery success rate is higher as the inner diameter (μm) of the tip 142 of the suction / discharge capillary 140 is smaller.

  That is, the inner diameter of the tip 142 of the suction / discharge capillary 140 is preferably not more than twice the diameter of the well, and the approach distance to the measuring chip is preferably not more than the well pitch. By doing so, the suction / discharge capillary 140 can more reliably collect fine particles from the target well by the syringe method.

  The suction / discharge capillary 140 is recorded by recording the tip 142 of the suction / discharge capillary 140 in the measurement field of view and aligning the well side to be collected of the measurement chip 90 with the tip of the capillary by the moving unit 16. Align the center of the tip 142 with the center of the well. Thereby, the total number of fine particles in the well can be recovered more reliably.

  When the amount of suction is controlled when the fine particles are sucked by the suction / discharge capillary 140, the suction speed is constant or the speed is decreased stepwise or continuously during the suction with reference to the suction speed at the beginning of the suction. . That is, depending on the type of fine particles, the fine particles may be adsorbed on the wall surface of the well, and a suction force that exceeds this adsorption force is required. No suction is required. On the contrary, after that, it is necessary to reduce the suction force in order to prevent erroneous suction from the adjacent well. Thus, when sucking, the fine particles can be reliably sucked from the well by controlling the suction force.

  As shown in FIG. 9, the tip portion 142 of the suction / discharge capillary 140 can be prefilled with a suspension 500 that is an example of a liquid. Thus, if the liquid is not put in the tip part 142 in advance, the liquid flows from the tip part 142 of the suction / discharge capillary 140 by capillary action unless the airtightness of the recovery part 13 is perfect. The speed of the inflow of the liquid depends on the degree of airtightness of the recovery unit 13, but the final inflow amount depends only on the physical properties of the suction / discharge capillary 140 and the liquid and the diameter of the suction / discharge capillary 140 and flows in Enters the position of the weight and balance of the liquid. For this reason, unnecessary fine particles also flow in with the liquid. The liquid to be put in is preferably used for actually suspending the measurement fine particles, but is not limited thereto.

  The filling amount is preferably a balance position by capillary action, which is determined by the physical properties of the solution (viscosity / density), the affinity between the suction / discharge capillary 140 and the solution, and the inner diameter of the capillary, but there is an error in the filling amount. Even in this case, the inflow speed can be reduced, and the accuracy of the suction / discharge operation can be increased.

  FIG. 12 shows an example of a phenomenon in which the fine particles M1 are blown out from the target well 200. This is a phenomenon that occurs because the liquid in the capillary flows downward from the capillary tip due to inertial force. In FIG. 12, adjacent wells 200 are arranged for each particle storage pitch PT. The inner diameter ST of the distal end portion 142 of the suction / discharge capillary 140 is 1.5 times or more the average diameter of the fine particles and is equal to or less than the particle storage pitch PT. The distance SL between the tip portion 142 of the suction / discharge capillary 140 and the tip 90 is set to be equal to or less than the particle storage pitch PT.

  As shown in FIG. 12B, when the suction / discharge capillary 140 approaches the target well 200, the fine particles M1 may be blown out of the target well 200. The phenomenon that such fine particles M1 are blown out from the target well 200 is that the amount of the liquid 330 in the suction / discharge capillary 140 is large when the suction / discharge capillary 140 approaches and stops rapidly. This is likely to occur when there are many, and when the tapered portion of the tip 142 of the suction / discharge capillary 140 is long.

Therefore, by performing the operation of collecting the fine particles M1 as shown in FIG. 13, the fine particles M1 are prevented from being blown out from the target well 200.
As shown in FIG. 13A, before the tip 142 of the suction / discharge capillary 140 lands on the suspension 500, the suction / discharge capillary 140 performs idle suction, and the suction / discharge capillary 140 moves in the Z2 direction. To the target well 200 at high speed. As shown in FIG. 13B, after the tip 142 of the suction / discharge capillary 140 has landed on the suspension 500, the tip 142 of the suction / discharge capillary 140 is brought into a high-speed approach stage by slowing down the speed. Then, the approach speed is lowered in multiple stages to approach the target well 200 at a low speed. In this manner, the tip 142 of the suction / discharge capillary 140 is approached at a speed of at least two steps of approaching from a high speed approach to a low speed approach. The speed of high-speed approach is not particularly limited.

  Thereafter, as shown in FIG. 13C, the tip 142 of the suction / discharge capillary 140 approaches at a low speed of, for example, 1 mm / s or less, and the suction / discharge capillary 140 performs preliminary suction. Thereby, the fine particles M1 are not blown out from the target well 200.

It should be noted that the average approach speed from when the tip 142 of the suction / discharge capillary 140 approaches 0.2 mm or less from the upper surface (target position) of the chip 90 until it stops is 0.5 mm / s or less. More preferred. The magnitude of the downward deceleration and the upward acceleration when the distal end portion 142 of the suction / discharge capillary 140 approaches 0.1 mm or less from the upper surface (target position) of the chip 90 is 20 μm / s ² or less. It is desirable. The preliminary suction operation is continued at least when the tip portion 142 of the suction / discharge capillary 140 is decelerated before it finally stops.

  As described above, the suction / discharge capillary 140 can control the speed at which it approaches or leaves between the tip portion 142 and the tip 90, but this speed is variable or continuous in steps. Variable control is possible.

  As shown in FIG. 13A, before the tip 142 of the suction / discharge capillary 140 lands on the suspension 500, the suction / discharge capillary 140 performs air suction in the air. It is preferable that (total suction amount in liquid <total discharge amount). This is because the liquid level in the capillary rises as the recovery operation is repeated, thereby preventing the phenomenon that the fine particles are easily ejected from the target well, and that the degree becomes severe. This is to prevent it.

  The suction / discharge capillary 140 in the up-and-down operation of each stop position, speed, acceleration / deceleration, suction / discharge capillary 140 suction / discharge amount, speed, and timing of the capillary up-and-down operation are aspirated. It is preferable that the discharge parameter has a function that can be changed by the measurer during the operation of collecting the fine particles. It is further preferable that automatic adjustment can be performed.

  Thereafter, as shown in FIG. 13D, the tip 142 of the suction / discharge capillary 140 sucks the fine particles M1 from the target well 200. As shown in FIG. 13 (E), the tip 142 of the suction / discharge capillary 140 is positioned in the well 81 of the recovery plate 80, and the fine particles M1 are put into the well 81 by repeating the specified number of discharges and suction operations. Spit out. By repeating the discharge and suction operations for the designated number of times as described above, it is possible to prevent the fine particles M1 from remaining in the tip portion 142. By repeating such a series of operations, the target particles can be reliably and repeatedly collected while preventing the fine particles M1 from being blown out of the target well 200.

  As another method for preventing the fine particles M1 from being blown out from the target well 200, as shown in FIG. 14A, the tip 142 of the suction / discharge capillary 140 is adjacent to the target along the Z2 direction. The tip 142 of the suction / discharge capillary 140 is moved horizontally along the Y1 direction after approaching the protruding portion 209 between the wells 200 and 200, so that the target well 200 is targeted. Position. As described above, when the tip 142 of the suction / discharge capillary 140 is lowered to a place other than the well and then moved horizontally and positioned right above the target well, the fine particles M1 are blown out of the target well 200. Can be prevented.

  Furthermore, another method for preventing the fine particles M1 from being blown out from the target well 200 is to set the shape of the distal end portion 142 of the suction / discharge capillary 140. FIGS. 15A and 15B show examples of the shape of the distal end portion 142 of the suction / discharge capillary 140. In the shape of the distal end portion 142 of FIG. 15A, the inner diameter ST is 0 from the distal end 142S. The axial length DP until reaching 7 mm is 4 mm, and in the shape of the tip 142 in FIG. 15B, the axial length DP is 5.0 mm. For example, if the length DP from the tip 142S is 5 mm or less, and particularly preferably 4 mm to 5.0 mm, fine particles are ejected even if the tip 142 of the suction / discharge capillary 140 approaches the target well at high speed. It is less likely to be done. This is because the fluid resistance against the inertial force acting on the liquid in the capillary increases. FIG. 16 shows an example of the relationship between the shape of the tip 142 of the suction / discharge capillary 140 and the extent to which fine particles are ejected from the target well. The actually curved curve CM1 is larger than the curved curve CM2 having a smaller tilt. The amount of fine particles ejected can be reduced.

  In addition, the perpendicularity of the tip surface of the suction / discharge capillary 140 with respect to the axial direction is desirably 20 μm or less. The outer diameter of the suction / discharge capillary 140 is preferably 10 μm or more and 60 μm or less with respect to the inner diameter.

  This is because the tip end surface of the capillary is easy to observe (vertical shape with a cross-sectional area secured), and AF (autofocus) accuracy is improved, so that positioning accuracy is improved. This is to improve the recovery success rate. The reason why the outer diameter of the suction / discharge capillary 140 is set to 60 μm or less is that a part of the capillary tip may be located in the adjacent well.

  Furthermore, in order to improve the recovery rate of the fine particles M1, as shown in FIG. 17, the suction / discharge capillary 140 is made to correspond to each of the plurality of target wells 200, respectively. The tip portion 142 of the capillary 140 can suck the fine particles in the target well 200 and store them in one well of the recovery plate. By doing so, for example, it is conceivable that cells having the same brightness, for example, can be conveniently handled later if they are collected in the same well of the recovery plate.

  Next, with reference to FIG. 18, an operation example for confirming whether or not the distal end portion 142 of the suction / discharge capillary 140 has sucked (picked up) the fine particles will be described.

  FIG. 18A shows an example in which the fine particles M remain in the target well 200, and FIG. 18B shows an example in which the fine particles M do not remain in the target well 200. In FIG. 18A, the light receiving unit 266 receives the state in which the fine particles M remain in the target well 200 via the objective lens 110. In FIG. 18B, the light receiving unit 266 receives a state in which the fine particles M are not left in the target well 200 via the objective lens 110. In this way, the analysis unit can photograph the surroundings of the well before and after the pickup operation of the fine particles, and the comparison can determine whether the fine particles are successfully picked up, and the fine particles M are sucked and discharged into the target well 200. It can be optically confirmed whether or not the tip portion 142 of the capillary 140 is reliably sucked.

  If it is determined that the fine particle pickup has failed, the recovery rate can be improved by repeating the pickup from the same well a predetermined number of times. The analysis unit can estimate the clogging of the distal end portion 142 of the suction / discharge capillary 140 from the history of success or failure of the pickup.

Similarly, the light receiving unit 266 detects the clogged state in the distal end portion 142 of the suction / discharge capillary 140 by receiving the state in the distal end portion 142 of the suction / discharge capillary 140 through the objective lens 110. be able to.
As shown in FIGS. 19A and 1, the plurality of light sources 250 irradiate light from two or more directions symmetric with respect to an axis perpendicular to the upper surface 90 </ b> S of the measurement chip 90 to measure the measurement chip 90. The transmission light source is configured to transmit light to the light receiving unit 266 through the objective lens 110.

  FIG. 19B shows the case where the objective lens 110 is in focus with respect to the well 200 of the chip 90 and the case where it is not in focus. When the objective lens 110 is in focus with respect to the well 200, a single ring-shaped image 200J is received, but when the objective lens 110 is not in focus with respect to the well 200, the object lens 110 is separated. A double ring-shaped image 200K is received. When the set angle θ with respect to the vertical line of the light source 250 is known in advance, the analysis unit calculates the distance from the separation distance VN of the separated double ring-shaped image 200K to the focus of the objective lens 110 by image processing. Can be calculated. This method can significantly reduce the time required for autofocus.

  FIG. 20 shows an arrangement example of the wells 200 in the measurement chip 90. The well 200 in the measuring chip 90 is divided into regions 919 corresponding to the observation field of the optical system of the light receiving unit 266 and the objective lens 110 in FIG. 1, and each region 919 is arranged in a matrix with an interval MB. It is arranged. In each region 919, a plurality of wells 200 are arranged in a matrix. The interval MB between the adjacent regions 919 and 919 is larger than the arrangement pitch of the wells in the same region 919. Accordingly, the position of each region 919 can be recognized by at least one position CV of the wells at the four corners of each region 919, and the region 919 can be moved to the center of the visual field of the optical system.

The state of the remaining fine particles in the well can be monitored and confirmed in real time by an image of the optical system, and the clogging in the tip 142 of the suction / discharge capillary 140 can be monitored and confirmed in real time by the image of the optical system. Parameters such as the speed of the tip 142 of the suction / discharge capillary 140, the stop position, the suction speed, and the degree of deceleration can be updated.
The analysis unit can capture the tip 142 of the suction / discharge capillary 140 and estimate the clogging of the tip 142 of the suction / discharge capillary 140 from the image. It is also possible to confirm the storage of the collected fine particles by a photographed image from the bottom surface side of the collection plate 80.

  In the embodiment of the present invention, the suction / discharge capillary is approached to the measuring chip at a speed of at least two stages of high speed → low speed, so that the fine particles are prevented from coming out from the well. The recovery rate of particles can be improved. In addition, the suction / discharge capillary is lowered to a location other than the well of the measurement chip, and then moved horizontally with respect to the measurement chip to be positioned directly above the target well, thereby allowing fine particles to enter from within the well. Outflow is further prevented, and the recovery rate of fine particles can be further improved.

  The suction / discharge capillary draws fine particles from the well of the measurement chip and images the surroundings of the well before and after the operation. The analysis unit compares the images of the surroundings of the well before and after the operation, so that the suction / discharge capillary is removed from the well. Determine whether fine particles have been aspirated. Thereby, the collection rate of fine particles can be improved.

The clogging of fine particles at the tip of the suction / discharge capillary is estimated from the image of the tip of the suction / discharge capillary. Thereby, the collection operation can be performed smoothly.
Obtain an image from the bottom side of the collection plate and check whether fine particles are collected in the well of the collection plate. Thereby, it can be confirmed by an image whether fine particles are recovered in the well of the recovery plate.

  The tip of the suction / discharge capillary approaches the well of the measurement chip at a low speed and the suction / discharge capillary is preliminarily sucked. Thereby, it can prevent that a fine particle comes out from the inside of a well, and can improve the collection | recovery rate of a fine particle.

FIG. 21 shows a preferred shape example of the measuring chip 90.
The measurement chip 90 shown in FIG. 21 is a rectangular flat plate as described above and has the well region 200R, and is, for example, a resin molded product. For example, two positioning protrusions 720 protrude from one long side portion 710 of the measuring chip 90, and one positioning protrusion 721 protrudes from one short side portion 711, for example. Is formed. The protrusions 720 and 721 have, for example, a rectangular shape, the protrusion 720 has an abutting surface 720B, and the protrusion 721 has an abutting surface 721B. That is, the protrusions 720 and 721 are formed so as to protrude from the two side surfaces of the measuring chip 90, respectively.
The abutting surface 720B of the protrusion 720 abuts against the positioning portion 120F inside the fixture 120, and the abutting surface 721B of the projection 721 abuts against the positioning portion 120G inside the fixture 120.

  As a result, the measuring chip 90 can be reliably positioned by abutting along the abutting direction GH against the positioning portions 120F and 120G inside the fixture 120, so that the positioning can be performed with good reproducibility. Improves. On the other hand, when no projection is used, the entire corresponding side surface of the measuring chip 90 is brought into contact for positioning by abutment with the fixture, but accuracy is ensured over the entire side surface. It is very difficult.

  Further, the display unit 750 of the measurement chip 90 is formed on the upper surface 90S side of the measurement chip 90. The display unit 50 has an arrow shape, for example, and faces the abutting direction GH. The display unit 50 may be provided on the upper surface 90S, the lower surface, or the outer peripheral portion of the measurement chip 90, and is a mark such as a pattern or a notch that clearly indicates the direction (front and back and direction) when the measurement chip 90 is set. .

Next, FIG. 22 shows that the warpage of the measurement chip 90 is eliminated when the measurement chip 90 is fixed to the fixture 120.
FIG. 22A is an embodiment of the present invention, and FIG. 22B is a comparative example.
22A shows the objective lens 110, the light receiving portion 266, and the fixture 120. FIG. The upper and lower surfaces of the measuring chip 90 are examples in which a low-order aspect is formed as a shape as typified by, for example, warping due to a temperature change of a material after release in molding.

  The upper surface 90S of the measuring chip 90 is applied to the inner surface 124 of the fixture 120, and the warping of the measuring chip 90 is corrected by applying a force FC from the lower surface 90D side of the measuring chip 90. That is, the region including the measurement area on the upper surface 90S side where the well is formed is pressed against the inner surface 124 which is a flat rigid plane of the fixture 120 by applying a force from the lower surface 90D which is the opposite surface. Correct 90 warpage. The parallelism of an arbitrary 2 mm square region of the measurement area with respect to the inner surface 124 that is a flat rigid plane is 10 μm or less (excluding minute undulations and protrusions such as the inclination of the well itself). Thereby, the positional accuracy of the well with respect to the optical system is improved, and focusing can be ensured in the entire region within the observation field.

On the other hand, in the comparative example shown in FIG. 22B, there are small irregularities on the upper surface 90SN and the lower surface 90DN of the measuring chip 90N, and even if the upper surface 90SN of the measuring chip 90N is the fixing tool 120. Even if a force FC is applied from the lower surface 90DN side of the measuring chip 90N against the inner surface 124, the warping of the measuring chip 90 cannot be corrected, and within one observation field (for example, 2 mm square). Focus cannot be ensured throughout.
Next, FIG. 23 shows the shift of the observation focus caused by the thickness variation of the measurement chip 90.

  23A to 23C, the distance between the upper surface of the measurement chip (the surface on which the well is formed) and the optical system are all the same, but the focal position varies depending on the thickness of the chip. Show. That is, in FIG. 23B, the focal point MP of the optical system 976 is accurately positioned on the upper surface 90S, whereas in FIG. 23A, the focal point MP is on the upper side of the upper surface 90S, and FIG. ) Are located on the lower side, and both are out of focus from the measurement target position. If the amount of deviation exceeds the depth of field, normal measurement is impossible. In the case of a general measurement optical system for securing an observation field of view of about 2 mm square, it is necessary to suppress the focus shift within a range of 10 μm, and an allowable thickness fluctuation amount is calculated as follows.

23, t is the thickness [mm] of the measuring chip 90, Δt is the thickness variation [mm] of the measuring chip 90, and L ₀ and L are the focal length [mm of the lens 110B of the objective lens 110. , And ΔL is a focus shift [μm].
Here, the focal length L has the relationship of Formula 1.
L = L ₀ + t × (1-1 / n) (1)
From Equation 1,
ΔL = Δt × (1-1 / n)... Equation 2
Is obtained.
In order to make the focus shift within ± 5 μm (the focus shift width within one field of view is within 10 μm),
Δt ≦ 10 / (1-1 / n)... Formula 3
It becomes.

  When the measuring chip 90 is made of polystyrene (refractive index n = 1.59), Δt is within ± 13.5 μm and the thickness variation Δt is in order to make the focal shift ΔL within ± 5 μm. It is within 27 μm.

  As a result, even if the thickness of the measuring chip 90 varies within this range, it is possible to suppress the focal shift within an area of any 2 mm square within ± 5 μm. Can be prevented.

  FIG. 24 shows an error in the observation position due to the parallel displacement between the upper surface 90S and the lower surface 90D of the measuring chip 90. FIG. In this fine particle screening device, the position of the well and the suction / discharge capillary is grasped and corrected based on the observation image, and the position recognition accuracy by the image requires ± 5 μm. Therefore, the distortion of the observation image is below this level. It is necessary to keep it down.

24A shows an example in which the upper surface 90S and the lower surface 90D are parallel, and FIG. 24B shows an example in which the upper surface 90S and the lower surface 90D are not parallel. The observation target MJ is a well or the tip of a suction / discharge capillary. On the upper surface 90S on which at least the well of the measurement chip 90 is formed, the upper surface 90S and the lower surface 90D are used so that the position recognition accuracy within an arbitrary 2 mm square region included in the measurement area of the optical system is within ± 5 μm. The inclination angle (displacement from parallel) φ is less than (5 × 10 ⁻⁶ ) / {t (1-1 / n)} [rad] (t is the average thickness of the measuring chip [ mm] and n indicate that the refractive index of the material of the measurement chip is required.

Δ indicates the deviation of the observation position of the observation object MJ, t indicates the average thickness of the measurement chip 90, n indicates the refractive index of the material of the measurement chip 90, and φ indicates the inclination angle of the lower surface 90D.
The deviation Δ of the observation position of the observation object MJ can be expressed by Equation 4.
Δ = t · tan [φ−sin ⁻¹ {(sin φ) / n}] ≈t · φ · (1-1 / n) Equation 4
In order to make Δ ≦ 5 μm,
φ ≦ (5 × 10 ⁻⁶ ) / {t (1-1 / n)} [rad] Expression 5
It becomes.

  Thereby, the position recognition accuracy can be within ± 5 μm at least in the entire observation field (for example, 2 mm square), and the observation target in the entire observation field (for example, 2 mm square) can be reduced. The position recognition accuracy is improved.

  FIG. 25 shows an example of the relationship between the inclination angle φ for satisfying the position recognition accuracy within ± 5 μm and the deviation Δ of the observation position, the chip thickness t is 1 mm, and the refractive index n corresponds to polystyrene. This is the case of 1.59. Since the deviation Δ (μm) of the observation position is within 5 μm, the variation in the tilt angle is 0.8 degrees or less.

In the embodiment of the present invention, the measurement chip 90 has an area in which a well is formed having a width of 10 mm square or more, and an appropriate part or all of the surrounding area not used for measurement is the fixture 120. Is provided. An appropriate size from the lower surface side where the well of the measuring chip 90 is not formed in a direction to be pressed against a reference surface (corresponding to the inner surface 124) that can be regarded as a substantially rigid body included in one plane of the fixture 120. By applying the force FC, the flatness of an arbitrary 2 mm square region included in the entire region where the well is formed with respect to the reference surface (corresponding to the inner surface 124) can be 10 μm or less. For this reason, positioning with high accuracy can improve positioning workability and prevent defocusing and deviation of the recognition position.
FIG. 26 is a diagram illustrating an example of a graph when the optical signal of fine particles is measured once. In the example shown in FIG. 26, the vertical axis represents the frequency at which the fluorescence brightness of the fine particles is detected, and the horizontal axis represents the measured value (brightness).

  The image analysis unit 15 in FIG. 1 displays the measurement curve MK1 in the measurement result of one graph 860 shown in FIG. 26, and arbitrarily designates the luminance region TR1 and the luminance region TR2 for this measurement curve MK1. can do. The luminance region TR1 is a region composed of a lower limit 871 and an upper limit 872, and the luminance region TR2 is a region composed of a lower limit 873 and an upper limit 874. The luminance region TR1 and the luminance region TR2 are a fine particle collection target FH1 and a collection target FH2, respectively.

As described above, the image analysis unit 15 in FIG. 1 provides the graph 860 from the result of the analysis of the optical information of the fine particles, on which the measurer has one or more luminance regions including the lower limit and the upper limit of the analysis luminance. By setting, it is possible to designate a well to be collected. That is, the graph 860 of FIG. 26 displays the luminance distribution of the measurement target in a graph, and the luminance region of the fine particles to be collected can be easily specified using the graph 860.
FIG. 27 is a diagram illustrating an example of a graph when the optical signal of fine particles is measured a plurality of times. In the example shown in FIG. 27, the vertical axis represents the frequency at which the fluorescence brightness of the fine particles is detected, and the horizontal axis represents the measured value (brightness).

In the example shown in FIG. 27, the image analysis unit 15 in FIG. 1 provides three graphs 881, 882, 883. A graph 881 shows the measurement result (1), a graph 882 shows the measurement result (2), and a graph 883 shows the calculation result (3) of the measurement results (1) and (2).
In the example of FIG. 27, the same sample (fine particles) is measured a plurality of times (in this example, twice), and is calculated from the analysis results (1) and (2) of each measurement and the analysis results of the plurality of measurements. Based on the calculation result (3), the collection target can be selected.

  The measurement result (1) shown in FIG. 27A is the same as the measurement result shown in FIG. In the measurement result (2) shown in FIG. 27B, the luminance region TR3 is set for the measurement curve MK2. The luminance region TR3 is a region having a lower limit 888 and an upper limit 889. The calculation result (3) shown in FIG. 27C shows the calculation result (difference) between the measurement result (1) shown in FIG. 27A and the measurement result (2) shown in FIG. A luminance region TR4 is set for the measurement curve MK3. The luminance region TR4 is a region having a lower limit 898 and an upper limit 899. That is, the result calculated from a plurality of measurement results is a difference between measurement results at the same location (same well). The measurement result (1) shown in FIG. 27A, the measurement result (2) shown in FIG. 27B, and the calculation result (3) shown in FIG. 27C are one or more logical sums or logical products. Can be arbitrarily combined to select and determine the collection target of fine particles in the well. The measurement result (1) shown in FIG. 27 (A) and the measurement result (2) shown in FIG. 27 (B) have different measurement conditions for the same sample, for example, elapsed time or before dropping a specific reagent. It is an example of measurement when conditions such as whether or after are different. For example, the luminance region set in each of the measurement result (1) shown in FIG. 13A, the measurement result (2) shown in FIG. 27B, and the calculation result (3) shown in FIG. Fine particles in wells satisfying all conditions can be collected.

  In the example described above, the calculation result (3) shown in FIG. 27C is the calculation result (difference) between the measurement result (1) shown in FIG. 27A and the measurement result (2) shown in FIG. Is shown. However, as another embodiment, the calculation result calculated from a plurality of measurement results may be a ratio of measurement results at the same location.

  For example, when the measurement result (1) shown in FIG. 27A is obtained and then the measurement result (2) shown in FIG. 27B is obtained as the measurement result after a predetermined time has elapsed, the fluorescence emitted by the fine particles is obtained. In order to carry out screening based on changes in luminance over time, a predetermined time interval can be provided between a plurality of measurements.

For example, when obtaining the measurement result (1) shown in FIG. 27 (A) and obtaining the measurement result (2) shown in FIG. 27 (B), screening based on the reactivity with the specific reagent of the fluorescence luminance emitted by the fine particles is performed. Therefore, a predetermined reagent can be supplied manually or automatically between these measurements.
For example, when the measurement result (1) shown in FIG. 27 (A) is obtained and the measurement result (2) shown in FIG. 27 (B) is obtained, the optical path of irradiation light or fluorescence is used during these multiple measurements. By automatically switching at least one of the filters for transmitting or reflecting the light, screening by measuring a plurality of types of fluorescence can be performed.

  The recovery unit 13 shown in FIG. 1 has a function of specifying the upper limit number of fine particles in the recovery target candidate well and actually recovering only the upper limit number of the recovery target candidate wells by specifying the luminance region. Have. This is a particularly effective function for efficiently performing large-scale screening in which, for example, the number of wells is several hundred thousand or more.

  As represented by a flow cytometer (cell sorter), when the collection condition luminance is given at the beginning, it is necessary to confirm the luminance in advance with the same sample and determine the condition. In this case, it takes time, and if the quality is different between the confirmation sample and the actual measurement sample, such as fading or foreign matter, the desired sample may not be collected.

  However, in the embodiment of the present invention, since the distribution of the measurement result of the population itself including the sample to be collected can be directly selected, the quality of the sample itself (for example, the color fading status, the degradation status, the foreign matter, etc.) It is possible to select the collection target according to the contamination status.

  In the embodiment of the present invention, the luminance distribution is more important than the luminance value itself in the graph showing the measurement result, and the fluorescence luminance of the fine particles in all the wells is measured, and the entire luminance such as the peak luminance of the frequency is measured. By confirming the distribution status and selecting a collection target well from the distribution itself, it is possible to specify a collection condition in which a noise portion or the like is reliably removed.

  As described above, the image analysis unit 15 in FIG. 1 measures the luminance distribution a plurality of times to accurately control, for example, the measurement time difference, the measurement timing after the reagent is dropped, or the history is accurately recorded. can do. In the case of measuring a plurality of times, the measurement is not limited to three times as shown in FIG. 13, but may be two times or four times or more.

Next, another embodiment of the present invention will be described with reference to FIG.
The fine particle screening apparatus 1C shown in FIG. 31 is different from the fine particle screening apparatus 1B shown in FIG. 28 in that a plurality of light sources 250 are provided above the measurement chip 90 in addition to the light source 260. The light source 250 irradiates light from two or more directions symmetrical with respect to an axis perpendicular to the upper surface 90S of the measurement chip 90. The shape and position information of the upper surface 90S of the measurement chip 90 and the well 200 formed on the upper surface 90S can be measured by the transmitted light from the measurement chip 90. In this measurement, a fluorescent filter is not required, but by selecting the light source wavelength and the amount of light so that a sufficient amount of light remains even if it passes through the dichroic mirror and the fluorescent filter, this can be done with the fluorescent filter unit placed in the optical path. There is an advantage that observation is possible without removing or switching.
Since the light source 250 can be arranged avoiding the collection unit, the collection can be arranged while the transmission light source is arranged.

By the way, this invention is not limited to the said embodiment, A various modified example is employable.
For example, in the embodiment of the present invention, biological cells are exemplified as target fine particles, but the present invention is not limited to this, and other types of fine particles may be used.

  Further, as shown in FIG. 3, one collection plate 80 is arranged on the mounting plate 40, but a plurality of collection plates may be arranged side by side.

As the drive system for linear movement of the moving unit 16, a rotary electric motor, a feed screw, and a guide rail are used, but a linear motor or the like can be used instead of the rotary electric motor.

  The cross-sectional shape of the well 81 of the recovery plate 80 and the cross-sectional shape of the well 200 of the measuring chip 90 are not limited to the illustrated example, and other shapes such as a hemispherical shape can also be adopted.

  The fine particle screening apparatus of the present invention is a field that requires testing, analysis and analysis of biopolymers of genes, immune systems, proteins, amino acids, and sugars, such as engineering, food, agriculture, fishery processing, etc. It can be applied to various fields such as pharmaceutical field, hygiene, health, immunity, plague, genetic field such as heredity, and scientific field such as chemistry or biology.

It is a front view which shows preferable embodiment of the screening apparatus of the fine particle of this invention. It is a perspective view which shows the screening apparatus of the fine particle of FIG. It is a perspective view which shows the mounting table, collection | recovery plate, and measurement chip | tip mounted on the moving part and the moving part. It is a figure which shows the mounting table, the collection | recovery plate, and the chip | tip for a measurement. It is a figure which shows the example of a shape of the measuring chip and the holding member of this measuring chip. It is a perspective view which shows the structural example of a collection | recovery part. FIG. 7A is a cross-sectional view showing a part of the measurement chip, in which fine particles are not yet contained in the well of the measurement chip. FIG. 7B is a diagram showing a state where fine particles M are inserted into the wells of the measurement chip. FIG. 8A is a diagram showing an example in which the light L is irradiated to the fine particles M in the well and a part of the fine particles M emits fluorescence. FIG. 8B is a diagram illustrating a state where the suction / discharge capillary approaches the selected well and sucks and collects all the fine particles. FIG. 9 is a diagram showing an example in which all the fine particles including the fluorescent fine particles M1 are sucked from the wells of the measurement plate and collected in the wells of the collection plate. It is a figure which shows the example of the reference surface for capillary height adjustment. The collection success rate when collecting fine particles with a capillary is shown. It is a figure which shows the example of the phenomenon in which a fine particle is blown out from an object well. It is a figure which shows collection | recovery operation | movement of a fine particle. It is a figure which shows another method which prevents blowing out a fine particle from an object well. It is a figure which shows another method which prevents blowing out a fine particle from an object well. It is a figure which shows the relationship between the shape of the front-end | tip part of a suction / discharge capillary, and a fine particle being ejected from an object well. It is a figure which improves the collection | recovery rate of a fine particle. It is a figure which shows the operation | movement which confirms the success or failure of whether the front-end | tip part of the suction / discharge capillary has attracted | sucked the fine particle. It is a figure which shows the merit in the case of using the several light source. It is a figure which shows the example of an arrangement | sequence of the well in a measurement chip | tip. It is a figure which shows the preferable example of a shape of the chip | tip for a measurement. It is a figure which shows that the curvature of a measurement chip is eliminated when the measurement chip is fixed to a fixture. It is a figure shown about the focus shift by the fluctuation | variation of the thickness of the chip | tip for a measurement. It is a figure about the error of the observation position by the parallel shift of the upper surface and lower surface of a measurement chip. It is a figure which shows the example of relationship between inclination-angle (phi) and the observation position shift (DELTA) for satisfy | filling the position recognition accuracy within +/- 5micrometer. It is a figure which shows the example of a graph at the time of measuring a sample once. It is a figure which shows the example of a graph when the same sample is measured in multiple times. It is detailed explanatory drawing of a measurement part. It is a figure which shows the example of the chip | tip for a measurement which permeate | transmits transmitted light in embodiment of FIG. It is a figure which shows the example by which the diffusion plate is arrange | positioned between the reflecting plate and the chip | tip for a measurement. It is a figure which shows another embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fine particle screening apparatus 11 Base 12 Support part 13 Collection | recovery part 14 Measurement part 15 Image analysis part 16 Moving part 19 Cover 30 Support stand 40 Mounting table 51 1st table 52 2nd table 55 1st motor 56 Feed screw 57 Nut 65 Second motor 66 Feed screw 67 Nut 70 Mounting surface 80 Collection plate 81 Well of collection plate 90 Measuring chip 100 Control unit 110 Objective lens 120 Measuring chip fixture 130 Operation unit 133 Suction pump 140 Suction / discharge capillary 142 Suction / discharge capillary 142 Discharge capillary tip 200 Measuring chip well 251 Motor 252 Feed screw 253 Nut 260 Excitation light source 261 Shutter unit 262 Fluorescence filter unit 264 Objective lens 500 Suspension LD Liquid L Light (Excitation (Emitted light, irradiated light)
X 1st direction Y 2nd direction Z 3rd direction (vertical direction)
CL Reference surface on top of measurement chip

Claims (8)

A fine particle screening device for performing a search for fine particles using light information emitted from the fine particles as a label, and selectively acquiring the searched fine particles,
A base that is the basis of the device,
A measuring chip formed of a material that transmits light and injecting a suspension containing the fine particles to form a well in which the fine particles are stored; and
Light detecting means for detecting a light source, the light information related to the fine particles and / or well obtained by irradiating the measuring chip and light emitted from the light source to the fine particles contained in the well And a measurement unit having an autofocus function ,
By analyzing the optical information measured by the measurement unit, an analysis unit that extracts light information from each of the fine particles stored in the well;
The measurement chip and the collection plate for accommodating the fine particles selectively acquired from the measurement chip are mounted on the base, and the measurement chip and the collection plate are measured with the measurement chip. A moving part movable in a first direction and at least a second direction perpendicular to the first direction with respect to the part;
It has a suction / discharge capillary installed on the base, and fine particles in the well provided in the measuring chip are sucked by the suction / discharge capillary and discharged to a predetermined position of the recovery plate for recovery. A collection unit for
With
The light source and the light detection unit are arranged on one side of a space defined by a plane including the measurement chip upper surface, and the suction / discharge capillary is the other side of the space defined by a plane including the measurement chip upper surface. Placed in
Furthermore, the measurement unit has a movable reflector, and the reflector is moved between the measurement chip and the recovery unit and is emitted from the light source in a state of being focused on the measurement chip upper surface. The light detection unit detects light that has passed through the measurement chip, reflected by the reflection plate, and again transmitted through the measurement chip, thereby providing positional information on the fine particles and the upper surface of the measurement chip. , Measuring the shape and position information of the well, and focusing on the tip of the suction / discharge capillary for sucking the fine particles, the positional relationship between the suction / discharge capillary and the measurement chip Is to measure,
A screening apparatus for fine particles, characterized by determining whether a positional relationship between the suction / discharge capillary and the measurement chip is appropriate . The light source of the measurement unit and the light detection unit for detecting the optical information are disposed below a space defined by a plane including the measurement chip upper surface, and the suction / discharge capillary is used for the measurement 2. The fine particle screening apparatus according to claim 1, wherein the fine particle screening apparatus is disposed above a space defined by a plane including a chip upper surface and further above the reflector . The apparatus for screening fine particles according to claim 1 or 2, wherein the moving direction of the reflecting plate is at least one of a linear direction and a rotating direction. The said reflecting plate is a mirror surface, The screening apparatus of the fine particle as described in any one of Claims 1-3 characterized by the above-mentioned. The said reflecting plate is a dichroic mirror, The screening apparatus of the fine particle as described in any one of Claims 1-3 characterized by the above-mentioned. The screening apparatus for fine particles according to any one of claims 1 to 5, wherein a diffusion plate is disposed between the reflection plate and the measurement chip. The measurement unit, of the light emitted from the front Symbol light source, an optical filter for selecting only light of a desired wavelength band, only light information of a desired wavelength band of the optical information from the measurement tip And at least one filter unit comprising a dichroic mirror for switching an optical path according to a difference in wavelength band between the light in the desired wavelength band and the optical information,
An objective lens for condensing optical information from the measurement chip while guiding the light in the desired wavelength band to the measurement chip;
Screening apparatus of the fine particles according to any one of claims 1 to claim 6, characterized in that it comprises a focusing unit having an autofocus function by which the movable said objective lens in the optical axis direction. The measurement unit includes a half mirror, and switches the half mirror and the filter unit to irradiate a part of light from the light source onto the measurement chip upper surface, and at the same time, reflects light from the measurement chip upper surface. The fine particle screening apparatus according to claim 7 , wherein the shape and position information of the well formed on the top surface of the measurement chip and the top surface are measured by guiding a part of the surface to a light detection unit.

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