JP2006112993A - Color bead discrimination device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To shorten vibration detection time to perform analysis at a high speed, using a readily discriminable reference bead. <P>SOLUTION: This color bead discriminating device produces a plurality of kinds of color beads, while changing the mixing ratio of semiconductor nanoparticles for emitting different light to be applied to a surface, specifies mixing percentage of the semiconductor nanoparticles applied to the color beads from a fluorescent image of the color beads, and discriminates the kind of the color beads. In the fluorescent image, obtained by forming the reference bead using the beads with a light transmissivity different from that of the beads and by radiating excitation light to a sample mixed with the color beads, by detecting vibration from the fluorescent image of the reference bead, the vibration detection time is shortened, to perform analysis at a high speed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a color bead discrimination device for gene expression analysis and the like.

  Conventionally, in gene analysis, a test object is stained with a fluorescent dye, and a flow cytometer or the like is used. However, the fluorescence intensity of the fluorescent dye has light dependency and is attenuated even when exposed to sunlight. Therefore, there is a problem that the fluorescence intensity is attenuated when laser light is irradiated for measurement.

  In order to solve this problem, a technique for analyzing genes using semiconductor nanoparticles whose fluorescence intensity does not attenuate even when irradiated with light is known. Fluorescence generated from semiconductor nanoparticles is photographed by multiple CCD cameras via optical filters, so that multiple inspection objects can be measured at one time. Since the intensity is not attenuated, highly reliable measurement can be performed (for example, Patent Document 1).

  In addition, semiconductor nanoparticles emit fluorescence of different colors depending on the size of the particles when irradiated with excitation light, so changing the compounding ratio of semiconductor nanoparticles of different sizes and applying them to the surface of the beads makes them various colors. Multiple types of beads that emit light can be created. A gene can be analyzed by binding a different gene to each bead and detecting the gene and determining the kind of the bead (see, for example, Patent Document 2).

Using the above technology, semiconductor nanoparticles applied to each bead by binding different genes to multiple types of beads created by changing the mixing ratio of semiconductor nanoparticles with different sizes and applying them to the surface of the beads. By photographing the fluorescence emitted by the particles with a plurality of CCD cameras, an apparatus for analyzing more types of genes at high speed has become possible.
JP 2003-28797 A (page 4-6, FIG. 2) JP 2002-311027 (page 3-4, FIG. 3)

  However, since Patent Document 1 uses a plurality of expensive CCD cameras, the cost of the apparatus becomes expensive. Therefore, an apparatus configuration with one CCD camera can be considered. In this configuration, a plurality of optical filters created so as to pass only light of a specific wavelength are used, and images of the inspection target beads are sequentially captured by one CCD camera while switching each. Among the plurality of optical filters, if there is a vibration from the outside in photographing with a specific optical filter, only a photographed image with the optical filter is affected by the vibration. In order to detect this vibration, at least one bead that emits light within the passband of the optical filter is required.

  For example, if vibration is detected by selecting beads that emit light only at a specific wavelength, vibration cannot be detected with an optical filter having a pass band other than that wavelength. Therefore, in order to accurately detect vibrations, it is necessary to examine images of all inspection target beads photographed for each optical filter. In general, there are about several hundred beads to be inspected. Therefore, if vibration of the bead to be inspected under all photographing conditions (types of optical filters) is detected, an enormous amount of time is required for the processing. The present invention solves the above-described problems, and an object thereof is to shorten the processing time of vibration detection when photographing a test target bead.

  In order to solve the above-described conventional problems, the colored bead discriminating apparatus of the present invention includes a plurality of bead particles in which semiconductor nanoparticles emitting fluorescence having different wavelengths according to the particle size are attached to the surface of a substantially spherical bead particle. Irradiate transmitted light to a sample bead, a reference bead prepared by changing the light transmittance of the bead particles of the color bead, and a target object in which the sample to be measured is mixed with the color bead and the reference bead. Transmitted light irradiating means for obtaining a transmitted image; excitation light irradiating means for irradiating the object to be measured with excitation light to obtain a fluorescence image; a plurality of filter means for passing light in a specific wavelength region; Imaging means for capturing a transmission image and a fluorescence image of a measurement object, storage means for storing the captured transmission image and fluorescence image, and transmission image detection means for detecting the center point and gradation of the bead particles in the transmission image; , A fluorescent image detecting means for detecting a gradation of each color bead from the fluorescent image, a bead distinguishing means for distinguishing the colored bead from the reference bead, and a colored bead for identifying the type of the colored bead It is characterized by comprising discrimination means and vibration detection means for detecting the vibration of the object to be measured using the reference beads.

  According to the color bead discriminating apparatus of the present invention, the time required for vibration detection is shortened by creating reference beads using beads having different light transmittances and detecting vibration from the fluorescence image of the reference beads.

  Embodiments of the color bead discrimination device of the present invention will be described below in detail with reference to the drawings.

  1 is a block diagram of a colored bead discriminating apparatus according to a first embodiment of the present invention, FIG. 2 is a block diagram of a writing means, FIG. 3 is a block diagram of vibration detecting means, and FIG. 4 is a diagram of beads according to an embodiment of the present invention. The schematic diagram of the color bead which apply | coated the semiconductor nanoparticle and the reference | standard bead is shown. Table 1 shows the types of colored beads that can be produced when the blending ratio of the three types of semiconductor nanoparticles emitting red, green, and blue is divided into three stages for each color.

  In FIG. 4, 201 and 301 are semiconductor nanoparticles emitting blue, 202 and 302 are semiconductor nanoparticles emitting green, and 203 and 303 are semiconductor nanoparticles emitting red. In FIG. 4a, a colored bead 200 is shown. The colored beads are made of beads having a particle size of 10 μm, which can capture mRNA and are transparent (transmittance of light having a wavelength of 660 nm is 70% or more). The semiconductor beads that emit red, green, and blue light are applied to the transparent beads at a predetermined ratio, whereby the colored beads 200 are formed. In FIG. 4b, a reference bead 300 is shown. This reference bead 300 is made on the basis of black beads (with a transmittance of 0% for light having a wavelength of 660 nm of 0%) and a particle diameter of 10 μm. By applying blue, green, and red semiconductor nanoparticles to the black beads in a mixing ratio of (1: 1: 1), the reference beads 300 can be formed.

  Next, the blending ratio of the semiconductor nanoparticles will be described. There are three colors of semiconductor nanoparticles, and there are three levels of mixing for each color, so there are a total of 27 combinations. However, when the luminance ratio of blue: green: red is (0: 0: 1) and (0: 0: 2), both are coated only with red semiconductor nanoparticles. It becomes. Similarly, when the luminance ratio of blue: green: red is (1: 1: 1) and (2: 2: 2), both blue, green and red semiconductor nanoparticles are used (1: 1: 1). Both are the same beads. Therefore, 20 types of beads can be created by removing the combination that results in the same beads. Table 1 shows the types of beads.

  Here, the colored beads 200 are prepared by blending the semiconductor nanoparticles at the blending ratios shown in Table 1 and applying them to the bead surface. In addition, since the colored beads 200 and the reference beads 300 have different light transmittances, the beads can be easily distinguished from the difference in light transmittance.

  The operation when the color bead reading device detects vibration based on the fluorescence of the reference bead 300 will be described with reference to FIGS. 1, 2, 3, 4, 5, 6, and 7. In FIG. 1, a well plate 101 is composed of a plurality of wells 102 for injecting an observation target (not shown; mRNA or protein combined with semiconductor nanoparticles or beads). It can be moved in the two-dimensional plane XY direction. The CPU 104 controls the entire apparatus. First, the CPU 104 controls the well plate driving means 103 to move the well plate 101 so that the well 102 into which the observation target is injected is positioned directly above the objective lens 105, and the optical filter 108 is mounted. The filter wheel 114 is rotated by controlling the filter wheel driving means 115 so that the non-existing portion is positioned directly above the objective lens 105.

  Next, the CPU 104 turns on the LED 106 and irradiates the well 102 with LED light. The transmission image of the observation target in the well 102 by the LED light is enlarged by the objective lens 105, passes through the dichroic mirror 107 and the filter wheel 114 where the optical filter 108 is not mounted, and is further collected by the imaging lens 109. The CCD camera 110 is reached. The objective lens 105 is a lens for enlarging an image to be observed in the well 102, and the dichroic mirror 107 is a mirror that reflects light having a wavelength less than a predetermined wavelength and passes light having a wavelength greater than or equal to a predetermined wavelength.

  The optical filter 108 is a band-pass filter that passes only in a predetermined wavelength range. In this embodiment, it is assumed that bandpass filters for blue, green, and red are attached and the optical filter 108 is not used during transmission image shooting. The imaging lens 109 is a lens for collecting light. The CCD camera 110 is an image detection unit that converts the light collected by the imaging lens 109 into two-dimensional image data and outputs it. The CPU 104 gives a command to the CCD camera control unit 111 and takes a transmission image of the observation object by the CCD camera 110.

  An automatic focus adjustment program (not shown) of the CPU 104 calculates the contrast value of the image from the transmission image and finds a position where the contrast value is maximized, thereby focusing the objective lens 105. The CPU 104 automatically adjusts the focus of the objective lens 105 that maximizes the contrast value while moving the objective lens 105 in the direction (Z-axis direction) perpendicular to the two-dimensional plane XY by the Z-axis driving unit 112. Find the position. The CPU 104 stores the transmission image of the observation object captured with the objective lens 105 in focus as the BFI (Bright Field Image) in the storage unit 130 through the data transfer unit 126.

  The transmission image detection unit 150 reads the BFI from the storage unit 130 through the reading unit 140, performs binarization processing on all pixels, and stores the binarized data in the storage unit 130. In order to prevent erroneous detection due to dust, the transmission image detecting means 150 extracts the contour of the object from the binarized image, obtains the area and aspect ratio of the object, and does not fall within a certain size range. Items are excluded from inspection. Further, in order to prevent the measurement result of the observation object from being influenced by the surrounding observation objects, the objects that are not separated by a certain distance or more are excluded, and the positions of the color beads 200 and the reference beads 300 that are finally the inspection objects are excluded. The information is transferred to the bead discriminating means 170.

  The bead discriminating unit 170 obtains the data of each bead from the binarized BFI image stored in the storage unit 130 based on the positional information of the color beads 200 and the reference beads 300 transferred from the transmission image detecting unit 150. read out. The BFI image after binarization is shown in FIG. The colored beads 200 serve as a lens and collect light, so that the central part is bright and the peripheral part is dark. Further, since the reference bead 300 does not transmit light, the whole becomes dark. The bead discriminating unit 170 determines that the color bead 200 is bright when the pixel data at the center of the bead is bright, and determines the reference bead 300 when the pixel data at the center of the bead is dark. The position information x is created and stored in the bead type storage unit 180.

  Next, the CPU 104 controls the filter wheel driving unit 115 to rotate the filter wheel 114 so that the portion where the optical filter 108 is mounted is located directly above the objective lens 105, and turns off the LED 106. The excitation light is irradiated from the excitation light source 113. The excitation light is short-wavelength light such as blue laser light, and when it hits the dichroic mirror 107, the excitation light reflects in the direction of the objective lens 105 due to the property that the dichroic mirror 107 reflects light having a predetermined wavelength or less. To do.

  The objective lens 105 focuses the light from the dichroic mirror 107 onto the observation target in the well 102. The semiconductor nanoparticles attached to the observation target in the well 102 are excited by the light irradiated from the objective lens 105 and emit light having a predetermined wavelength or more that can pass through the dichroic mirror 107. The light emitted from the semiconductor nanoparticles attached to the observation target passes through the objective lens 105, the dichroic mirror 107, and the optical filter 108, and is collected by the imaging lens 109, similarly to the transmitted light of the observation target. The CCD camera 110 is reached.

  At this time, since the optical filter 108 has a property of passing only a specific wavelength range, only the specific wavelength range of light emitted from the semiconductor nanoparticles attached to the observation target reaches the CCD camera 110. In this state, the CPU 104 gives a command to the CCD camera control unit 111, and among the light emitted from the semiconductor nanoparticles attached to the observation target by the CCD camera 110, the light of only a specific wavelength range that has passed through the optical filter 108. Two-dimensional image data is stored in the storage means 130 through the CCD camera control means 111 and the writing means 120.

  There are a plurality of optical filters 108 having different pass wavelength ranges, and they are attached to the filter wheel 114. The filter wheel 114 can be rotated by the filter wheel driving means 115 to switch the optical filter 108. While switching the optical filter 108, the CPU 104 captures images of various wavelength ranges of light emitted from the semiconductor nanoparticles attached to the observation target by the above method, and repeats the same processing.

  For the data input to the writing unit 120, the average value of all pixels is calculated by the average luminance calculation unit 122 and the average luminance is stored in the average luminance holding unit 123. Based on the value set by the CPU 104 in the writing means CPU interface 121, the low brightness judgment reference holding means 124 calculates the brightness as a judgment reference for selecting the background from the acquired image and outputs it to the vibration detection means 190. To do.

  The CPU 104 sets the output of the average luminance holding unit 123 as it is in the low luminance judgment reference holding unit 124 for the writing unit CPU interface 121 and sets the output of the average luminance holding unit 123 in the writing unit CPU interface 121. One mode is selected from a mode in which the value amplified / attenuated at the ratio is held in the low luminance judgment criterion holding unit 124 and a mode in which the luminance set in the writing unit CPU interface 121 is held in the low luminance judgment criterion holding unit 124. Can be set.

  Similarly, the high-brightness determination criterion holding unit 125 calculates a luminance serving as a determination criterion for selecting the color beads 200 and the reference beads 300 from the acquired image from the values set by the CPU 104 in the writing unit CPU interface 121. And output to the vibration detecting means 190.

  The CPU 104 sets the output of the average luminance holding unit 123 as it is in the high luminance judgment reference holding unit 125 for the writing unit CPU interface 121 and sets the output of the average luminance holding unit 123 in the writing unit CPU interface 121. It is possible to select and set a mode in which the value amplified / attenuated at the above ratio is held in the high luminance judgment reference holding unit 125 and a mode in which the luminance set in the writing unit CPU interface 121 is held in the high luminance judgment reference holding unit 125. it can.

  FIG. 6 is an image diagram showing the positions of the color beads 200 and the reference beads 300 to be inspected obtained from the BFI. The hatched area is the position of the colored bead 200 to be measured, and the place painted black is the position of the reference bead 300.

  7A to 7C are configuration diagrams of the vibration detection inner circle and the vibration detection outer circle according to the present invention. In FIG. 7 a, 300 is a reference bead, 310 has the same center point as the reference bead 300, a vibration detection inner circle whose radius is two pixels shorter than the reference bead 300, and 320 has the same center point as the reference bead 300. This is a vibration detection outer circle whose radius is two pixels longer than that of the reference bead 300.

  The vibration detection circle calculation means 193 determines each reference bead from the radius of the vibration detection inner circle 310 set in the vibration detection CPU interface 191 by the CPU 104 and the position information of the center point of the reference bead 300 from the transmission image detection means 150. A point on the vibration detection inner circle 310 with respect to 300 (hereinafter referred to as a vibration detection point) is calculated, and luminance information of the vibration detection point in the two-dimensional fluorescence image is read from the storage unit 130 via the reading unit 140. The vibration generation detection unit 192 compares the read luminance information with the luminance information output from the low luminance determination criterion holding unit 124. If the luminance output from the low luminance determination criterion holding unit 124 is higher, the vibration generation detection unit 192 vibrates. Is detected, the CPU 104 is notified.

  Similarly, the vibration detection circle calculation unit 193 determines each of the radius of the vibration detection outer circle 320 set in the vibration detection CPU interface 191 from the CPU 104 and the position information of the center point of the reference bead 300 from the transmission image detection unit 150. The vibration detection point on the vibration detection outer circle 320 with respect to the reference bead 300 is calculated, and the luminance information of the vibration detection point in the two-dimensional fluorescence image is read from the storage unit 130 via the reading unit 140. The vibration generation detection unit 192 compares the read luminance information with the luminance information output from the high luminance determination criterion holding unit 125, and if the luminance output from the high luminance determination criterion holding unit 125 is lower, the vibration generation detection unit 192 vibrates. Is detected, the CPU 104 is notified.

  FIG. 7 b shows the relationship between the reference bead 300 and the vibration detection inner circle 310 and the vibration detection outer circle 320 in the two-dimensional fluorescence image when vibration occurs, and FIG. 7 c shows the reference bead 300 in the two-dimensional fluorescence image when there is no vibration. And the relationship between the vibration detection inner circle 310 and the vibration detection outer circle 320 are shown. For example, four vibration detection points (represented by asterisks in FIG. 7) on the vibration detection inner circle 310 and four vibration detection points (in FIG. 7) on the vibration detection outer circle 320 are detected. (Represented by a triangle).

  In FIG. 7 b, the vibration detection point 315 having a lower luminance than the luminance information output from the low luminance determination criterion holding unit 124 exists on the vibration detection inner circle 310 and the luminance output from the high luminance determination criterion holding unit 125. Since the vibration detection point 325 having a luminance higher than that of the information exists on the vibration detection outer circle 320, it can be determined that the apparatus is vibrating. In FIG. 7c, there is no vibration detection point on the vibration detection inner circle 310 whose luminance is lower than the luminance information output from the low luminance determination criterion holding unit 124, and the luminance output from the high luminance determination criterion holding unit 125. Since there is no vibration detection point on the vibration detection outer circle 320 having a luminance higher than that of the information, it can be determined that the apparatus is not vibrating.

  FIG. 8 shows the flow of processing when vibration is detected. Consider a case where vibration is detected when a fluorescent image is captured using the green optical filter 108. When vibration is detected by the vibration detection unit 190, vibration information is transferred to the CPU 104. Since the vibration is detected, the CPU 104 captures the fluorescent image again with the green optical filter 108 on the optical axis without rotating the filter wheel 114. If vibration does not occur when the image is taken again using the green optical filter 108, the filter wheel driving means 115 is controlled to rotate the filter wheel 114 so that the blue optical filter 108 is on the optical axis. . A fluorescent image is captured using the blue optical filter 108. If no vibration is detected, the current well 102 has been photographed and the next well 102 is photographed. By re-acquiring the image in this way, the time required for re-acquiring the image can be shortened even when there is vibration.

  The fluorescence image detection unit 160 reads the position information x of the colored beads 200 from the bead type storage unit 180. The fluorescence image detection means 160 reads a fluorescence image photographed using the red optical filter 108 from the storage means 130, and has a radius 10 centered on the center point of the colored beads 200 obtained from the position information x previously read. The total luminance (hereinafter referred to as red luminance value) of each pixel included in the pixel circle is obtained for each color bead 200.

  Next, a fluorescent image photographed using the green optical filter 108 is read out from the storage unit 130, and each pixel included in a circle having a radius of 10 pixels centered on the center point of the colored bead 200 obtained from the position information x. Is calculated for each colored bead 200 (hereinafter referred to as a green luminance value).

  Similarly, a fluorescent image photographed using the blue optical filter 108 is read out from the storage unit 130, and each pixel included in a circle corresponding to a radius of 10 pixels centered on the center point of the colored bead 200 obtained from the position information x. Is calculated for each colored bead 200 (hereinafter referred to as a blue luminance value).

  When the red luminance value is not close to 0, the fluorescent image detecting means 160 divides the green luminance value of each colored bead 200 by the red luminance value.

When the division result is 0 to 0.1, green luminance value: red luminance value = 0: 1
When the division result is 0.49 to 0.51, the green luminance value: the red luminance value = 1: 2.
When the division result is 0.98 to 1.02, green luminance value: red luminance value = 1: 1
When the division result is 1.96 to 2.04, green luminance value: red luminance value = 2: 1
And

  When the division result does not fall within the above range, it is excluded from the inspection target as an abnormal bead. Similarly, the ratio between the blue luminance value and the red luminance value can be obtained by dividing the blue luminance value by the red luminance value. From the ratio of these (green luminance value: red luminance value) and (blue luminance value: red luminance value), (red luminance value: green luminance value: blue luminance value) is obtained. Next, when the red luminance value is near 0 and the green luminance value is not near 0, the blue luminance value of each color bead 200 is divided by the green luminance value.

When the division result is 0 to 0.1, blue luminance value: green luminance value = 0: 1
When the division result is 0.49 to 0.51, blue luminance value: green luminance value = 1: 2.
When the division result is 0.98 to 1.02, blue luminance value: green luminance value = 1: 1
When the division result is 1.96 to 2.04, blue luminance value: green luminance value = 2: 1
It becomes.

  When the red luminance value is not close to 0 for each colored bead 200, the colored bead discriminating unit 195 is represented by the ratio (red luminance value: green luminance value: blue luminance value) calculated by the fluorescent image detecting unit 160. The types 6 to 19 of coated beads shown in FIG. For each color bead 200, the color bead discriminating means 195 is calculated by the fluorescence image detecting means 160 when the red luminance value is near 0 and the green luminance value is not near 0 (green luminance value: blue luminance). The types 2 to 5 of coated beads shown in Table 1 are determined from the ratio of (value). If the red luminance value, the green luminance value, and the blue luminance value are around 0 for each colored bead 200, the colored bead discriminating means 195 determines that the type of coated beads shown in Table 1 is 0, If the blue luminance value is not near 0, it is determined that the type of coated beads is 1.

  In this example, the reference beads are obtained by applying blue, green, and red semiconductor nanoparticles at a ratio of (1: 1: 1), but the semiconductor nanoparticles that emit light of each color are applied to the beads. In this case, since the reference beads can be photographed using the optical filter 108 that transmits light of blue, green, and red wavelengths, the ratio of application of each semiconductor nanoparticle is not a problem.

  In this example, three types of semiconductor nanoparticles and three types of optical filters 108 are shown. However, the number of semiconductor nanoparticles and optical filters 108 may be less than three. Also good.

  The second embodiment of the present invention differs from the first embodiment of the present invention only in the operation at the time of occurrence of vibration. Only the operation when vibration is generated will be described with reference to FIG.

  Considering the case where vibration is detected while taking a fluorescent image using the green optical filter 108, when vibration is detected by the vibration detection unit 190, vibration information is transferred to the CPU 104. . Since the vibration is detected, the CPU 104 controls the filter wheel driving unit 115 to rotate the filter wheel 114 so that the portion where the blue optical filter 108 is not mounted is on the optical axis. Then, in the order of the BFI image, the red fluorescent image, the green fluorescent image, and the blue fluorescent image, image capturing is started again from the beginning while detecting vibration in the same manner as the first. By performing the image reacquisition in this way, the process for image reacquisition becomes the same regardless of the process in which vibration occurs, so that the control can be simplified.

  The colored bead discriminating apparatus according to the present invention is useful as a gene analyzing apparatus or the like that can shorten the time required for vibration detection of the apparatus and performs gene analysis at a higher speed.

The block diagram of the color bead discrimination device in Example 1 and 2 of this invention Block diagram of writing means in Embodiments 1 and 2 of the present invention The block diagram of the vibration detection means in Example 1 and 2 of this invention Schematic diagram of colored beads and reference beads in Examples 1 and 2 of the present invention The image figure of the transmission image in Example 1 of this invention The image figure which shows the position of the color bead and the reference | standard bead in Example 1 and 2 of this invention Configuration diagram of vibration detection inner circle and vibration detection outer circle in the first and second embodiments of the present invention Processing diagram when vibration occurs in Embodiment 1 of the present invention Processing diagram when vibration occurs in Embodiment 2 of the present invention

Explanation of symbols

101 well plate 102 well 103 well plate driving means 104 CPU
105 Objective lens 106 LED
DESCRIPTION OF SYMBOLS 107 Dichroic mirror 108 Optical filter 109 Imaging lens 110 CCD camera 111 CCD camera control means 112 Z-axis drive means 113 Excitation light source 114 Filter wheel 115 Filter wheel drive means 120 Writing means 121 Writing means CPU interface 122 Average brightness calculation means 123 Average brightness Holding means 124 Low luminance judgment standard holding means 125 High luminance judgment standard holding means 126 Data transfer means 130 Storage means 140 Reading means 150 Transmission image detection means 160 Fluorescence image detection means 170 Bead distinction means 180 Bead type storage means 190 Vibration detection means 191 Vibration detection CPU interface 192 Vibration generation detection means 193 Vibration detection circle calculation means 195 Color bead discrimination means 200 Color beads 201, 3 1 Semiconductor nanoparticles that emit blue light 202, 302 Semiconductor nanoparticles that emit green light 203, 303 Semiconductor nanoparticles that emit red light 300 Reference beads 310 Inner circle of vibration detection 315 Within vibration detection whose luminance is lower than the average luminance of the entire pixel Point on circle 320 Outer circle of vibration detection 325 Point on outer circle of vibration detection having luminance higher than average luminance of entire pixel

Claims (12)

Colored beads composed of a plurality of bead particles in which semiconductor nanoparticles emitting fluorescence of different wavelengths depending on the particle size are attached to the surface of a substantially spherical bead particle;
Reference beads prepared by changing the light transmittance of the bead particles of the color beads,
A transmitted light irradiating means for obtaining a transmitted image by irradiating a measured object mixed with the sample to be measured with the colored beads and the reference beads;
Excitation light irradiation means for irradiating the object to be measured with excitation light to obtain a fluorescence image;
A plurality of filter means for passing light in a specific wavelength range;
Imaging means for capturing a transmission image and a fluorescence image of the object to be measured;
Storage means for storing the photographed transmission image and fluorescence image;
Transmission image detection means for detecting the center point and gradation of the bead particles in the transmission image;
Fluorescence image detection means for detecting a gradation for each fluorescence wavelength for each color bead from the fluorescence image;
Bead distinguishing means for distinguishing between the colored beads and the reference beads;
Color bead discriminating means for discriminating the type of the color beads;
Vibration detecting means for detecting vibration of the object to be measured using the reference beads;
A colored bead discriminating apparatus comprising: The bead discriminating unit detects the gradation for each of the color beads and the reference beads detected by the transmission image detecting unit, and compares the color beads with the reference beads by comparing with a predetermined gradation value. The colored bead discrimination device according to claim 1. The colored bead discriminating means compares the gradation ratio of each fluorescent bead for each color wavelength detected by the fluorescent image detecting means with a predetermined gradation ratio of the bead for each fluorescent wavelength. The color bead discrimination device according to claim 1, wherein the color beads are distinguished. The vibration detection means includes a vibration detection inner circle having the same center as the center point calculated by the transmission image detection means and a radius shorter than that of the reference bead, and a center having the same center as the center point and a radius greater than that of the reference bead. A vibration detection circle calculating means for obtaining a long vibration detection outer circle,
When the luminance of at least one point on the vibration detection inner circle of the fluorescence image of the reference bead is lower than a predetermined low luminance determination criterion, or the luminance of at least one point on the vibration detection outer circle is a predetermined high 2. The colored bead discriminating apparatus according to claim 1, further comprising a vibration generation detecting unit that determines that vibration has occurred when the luminance is higher than a criterion for determination of luminance. 5. The colored bead discriminating apparatus according to claim 4, wherein the low luminance judgment standard is an average luminance value of each of the fluorescent images photographed for each of the filter means. 5. The colored bead discriminating apparatus according to claim 4, wherein the low luminance judgment standard can be set from outside. 5. The color bead discriminating apparatus according to claim 4, wherein the high luminance judgment criterion is an average luminance value of each of the fluorescent images photographed for each of the filter means. 5. The colored bead discriminating apparatus according to claim 4, wherein the high luminance judgment standard can be set from outside. 5. The colored bead discriminating apparatus according to claim 4, wherein a value of a radius of the vibration detection inner circle can be set from outside. The color bead discriminating apparatus according to claim 4, wherein a value of a radius of the vibration detection outer circle can be set from outside. 2. The colored bead discriminating apparatus according to claim 1, wherein when vibration is detected from the fluorescence image of the reference bead by the vibration detection unit, only the fluorescence image in which vibration is detected is re-acquired. 2. The transmission image including the reference beads in which vibration is detected and all the fluorescence images are re-acquired when vibration is detected from the fluorescence image of the reference beads by the vibration detection unit. The described colored bead discrimination device.

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