JP2007298380A - Sample holding member - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample holding member which enables the efficient measurement of emission under various conditions such as the measurement of multicolor emission or the like. <P>SOLUTION: The sample holding member 2 is constituted of a microplate 20 having a plurality of wells 21, which respectively hold a sample, arranged thereto in a two-dimensional array state and the filter member 30 provided so as to be fixed at a predetermined position where the emission from the sample held in each of the wells 21 of the microplate 20 transmits. Further, the filter member 30 has a plurality of optical filter parts 31 arranged in a two-dimensional array state so as to correspond to a plurality of the wells 21 of the microplate 20 and a plurality of the optical filter parts 31 respectively have light transmission characteristics with respect to the wavelength corresponding to a wavelength selectivity necessary for measuring emission. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a sample holding member that holds a sample to be subjected to luminescence measurement.

As a method for measuring luminescence generated when a reagent is injected into a sample such as a cell in the field of drug discovery or the like, a luminescence measurement method using a sample holding member such as a microplate is used (for example, Reference 1: Japanese Patent Application Laid-Open No. 2005-318867). 2001-188044, literature 2: JP-A-9-26426). A microplate is a plate-shaped sample holding member provided with a plurality of wells (test holes) each capable of holding a sample, and the evaluation of the sample is performed by measuring light emission from the sample held in each well. In addition, it is possible to efficiently perform necessary light emission analysis (see, for example, Document 5: Japanese Translation of PCT International Publication No. 2005-502896, Document 6: Japanese Patent Application Laid-Open No. 2002-350349).
JP 2001-188044 A Japanese Patent Laid-Open No. 9-26426 Japanese Patent No. 3,452,566 JP 2002-310894 A JP-T-2005-502896 JP 2002-350349 A

  In the luminescence measuring apparatus described in the above-mentioned document 1, a sample is held by a microplate having a bottom surface so as to transmit light emitted from the sample when a reagent is injected, and light detection means installed below the sample is held. Luminescence is detected. On the other hand, in such luminescence measurement, various types of luciferase and the like have recently been used as a reagent, and various colors such as multicolor and multiple types of luminescence phenomena having different wavelength spectra are used for the measurement. Luminescence measurement is being performed under various conditions.

  In connection with this, the structure corresponding to the light-emission measurement on various conditions, such as a multicolor light-emission measurement, is also calculated | required also in the light emission measuring apparatus. With regard to such a configuration, for example, Document 3: Japanese Patent No. 3452666 describes a configuration in which a rotating disk for switching a wavelength selection filter is disposed in front of a camera. Document 4: Japanese Patent Laid-Open No. 2002-310894 describes a configuration in which two cameras having different wavelengths of detectable light are installed. However, these configurations complicate the configuration of the apparatus including the wavelength selection filter and the camera. In addition, for example, when performing multicolor luminescence measurement, there is a problem in terms of measurement efficiency of luminescence, such as being unable to simultaneously measure the multicolor luminescence phenomenon from the sample.

  The present invention has been made to solve the above problems, and an object of the present invention is to provide a sample holding member capable of efficiently performing luminescence measurement under various conditions such as multicolor luminescence measurement. And

  In order to achieve such an object, the sample holding member according to the present invention is a sample holding member for holding a sample, and (1) a plurality of wells each capable of holding a sample to be subjected to luminescence measurement are two-dimensional. A microplate arranged in an array, and (2) a filter member fixed to the microplate at a predetermined position where light emitted from a sample held in a well of the microplate is transmitted, (3) The filter member has a plurality of optical filter portions arranged in a two-dimensional array so as to correspond to the plurality of wells of the microplate, and each of the plurality of optical filter portions has a wavelength required for light emission measurement. It is characterized by having a light transmission characteristic for a wavelength corresponding to selectivity.

  In the sample holding member described above, the microplate having a plurality of wells is placed at a predetermined position on the optical path to the image acquisition means of the two-dimensional light image of the microplate including light emission from the sample at the time of light emission measurement. A sample holding member is configured by providing a filter member fixed to the plate. The optical filter portion of the filter member corresponding to the well of the microplate is configured to exhibit wavelength selectivity (spectral characteristics) corresponding to the conditions of light emission measurement performed on the sample.

  As described above, according to the sample holding member provided with the filter function for light emission, the configuration of the apparatus including, for example, an image acquisition unit and a light guide optical system can be simplified when applied to light emission measurement. In addition, the filter member including the optical filter portion arranged in a two-dimensional array corresponding to the well in which the sample is held is fixed to the microplate, so that the light emission is measured in the light emission measurement under various conditions. It is possible to improve the measurement efficiency, the working efficiency at the time of light emission measurement, and the like.

  Here, in the sample holding member having the above-described configuration, the filter member includes at least a first filter region and a second filter region, each of which has a different light transmission characteristic, with respect to light emission from the sample. Thus, it is preferable that the light emission measurement can be performed for each of the first light component transmitted through the first filter region and the second light component transmitted through the second filter region.

  As described above, the optical filter portion of the filter member corresponding to the well of the microplate has a configuration in which a plurality of filter regions having different wavelength selectivity are provided, and each filter region is transmitted when applied to luminescence measurement. By performing the light emission analysis process on the light component, it is possible to simultaneously measure a plurality of types of light emission phenomena having different wavelength spectra. This makes it possible to efficiently perform multicolor light emission measurement and the like with a simple configuration.

  In this case, in the optical filter portion of the filter member, the areas of the first filter region and the second filter region are preferably set in accordance with the measurement conditions of the first light component and the second light component. . Thus, referring to the wavelength components in the light emission corresponding to the light transmission characteristics in the filter region, each area of the plurality of filter regions in the optical filter unit and the area ratio thereof according to the measurement conditions of each light component By setting, it is possible to perform light emission measurement with sufficient accuracy for each wavelength component selected in each filter region.

  As for the installation configuration of the filter member with respect to the microplate, a configuration in which the filter member is attached on the bottom surface of the microplate serving as an emission surface of light emission from the sample can be used. Alternatively, it is possible to use a configuration in which the filter member is provided integrally with the bottom surface portion of the microplate that serves as an emission surface of light emission from the sample.

  In addition, the microplate may have a configuration in which the side wall that separates adjacent wells is impermeable to light emitted from the sample. According to such a configuration, when performing luminescence measurement simultaneously on the samples held in each of the plurality of wells, the occurrence of crosstalk between adjacent wells can be suppressed.

  According to the sample holding member of the present invention, a filter member is provided at a predetermined position with respect to a microplate having a plurality of wells, and light emission performed on the sample is performed on the optical filter portion of the filter member corresponding to the well of the microplate. By configuring so as to show wavelength selectivity according to the measurement conditions, the configuration of the apparatus when applied to luminescence measurement can be simplified, and the luminescence measurement can be performed in luminescence measurement under various conditions. It is possible to improve the efficiency, the working efficiency at the time of measuring luminescence, and the like.

  Hereinafter, preferred embodiments of a sample holding member according to the present invention will be described in detail with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described.

  FIG. 1 is a configuration diagram schematically showing one embodiment of a luminescence measuring apparatus to which a sample holding member according to the present invention is applied. FIG. 2 is a perspective view showing an example of the configuration of the sample holding member according to the present invention. 3 is a side sectional view showing a sectional structure of the sample holding member shown in FIG. The luminescence measuring apparatus 1 according to the present embodiment uses a sample holding member 2 including a microplate 20 as a sample holding member, and starts from a sample S (see FIG. 3) arranged at a measurement position P while being held by the microplate 20. It is an apparatus for measuring the luminescence phenomenon.

  The sample S is not particularly limited as long as it is a target for luminescence measurement, and examples thereof include biological samples such as cells and antibodies. As an example of the light emission phenomenon from the sample S to be measured, light emission generated when a reagent is injected into the sample S can be given. Hereinafter, the configuration of the luminescence measurement apparatus 1 will be described. The luminescence measurement device 1 shown in FIG. 1 includes a data acquisition device 10 and a data analysis device 50.

  First, the configuration of the data acquisition device 10 used for acquiring the measurement data of the light emission of the sample S and the configuration of the sample holding member 2 that holds the sample S will be described. The data acquisition device 10 includes a dark box 15 in which a sample holding member 2 including a microplate 20 that holds a sample S to be subjected to luminescence measurement is housed, and a dark box 15 that is placed in a measurement position P. And an image acquisition unit 40 used for detecting light emission from the sample S.

  In the present embodiment, the microplate 20 constituting the sample holding member 2 is a plate-like member in which a plurality of wells (test holes) 21 are arranged in a two-dimensional array as shown in FIGS. Each of the plurality of wells 21 is configured to be able to hold a sample S to be subjected to luminescence measurement. In the configuration example shown in FIG. 2, 8 × 12 = 96 wells 21 are arranged in a two-dimensional array as a plurality of wells 21. The bottom surface 22 of the microplate 20 is formed of a material that can transmit light emitted from the sample S. Further, a filter member 30 (described later) is fixed to the microplate 20 on the bottom surface 22 which is the surface of the microplate 20 on the image acquisition unit 40 side and serves as an emission surface of light emission from the sample S at the time of light emission measurement. Has been provided. In the present embodiment, the microplate 20 and the filter member 30 constitute a sample holding member 2 for holding the sample S.

  In the dark box 15, the sample holding member 2 including the microplate 20 is held by the microplate holder 11. In addition, a microplate transport mechanism 12 is installed for the sample holding member 2 and the holder 11 so that a predetermined direction in the dark box 15 is a transport direction (a direction from the right side to the left side in FIG. 1). ing.

  A predetermined number (for example, 25) of the sample holding member 2 before the measurement in which the sample S is held is stocked on one side of the dark box 15 which is the carry-in side with respect to the transfer direction of the microplate 20 in the transfer mechanism 12. A carry-in side microplate stocker 13 is provided for the purpose of storage. Further, on the other side of the dark box 15 on the carry-out side with respect to the conveyance direction of the microplate 20, a carry-out microplate stocker 14 for stocking the sample holding member 2 after measurement is installed.

  In such a configuration, the sample holding member 2 carried into the dark box 15 from the carry-in microplate stocker 13 is held by the microplate holder 11 and carried by the carrying mechanism 12. Then, the sample holding member 2 is temporarily stopped at the measurement position P, and in this state, necessary light emission measurement is performed on the sample S held by the microplate 20. After the measurement is completed, the sample holding member 2 is transported again by the transport mechanism 12 and is transported to the transport-side microplate stocker 14. In FIG. 1, for the transport mechanism 12 and the stockers 13 and 14, a specific configuration for carrying in, transporting, and unloading the sample holding member 2 including the microplate 20 is omitted.

  Luminescence measurement is performed on the sample S in each well 21 of the microplate 20 above the measurement position P with respect to the measurement position P where the microplate 20 and the sample S held by the microplate 20 are arranged at the time of performing the luminescence measurement. A dispensing device 16 for injecting a reagent for use is installed. Examples of the luminescent reagent used for luminescence measurement include luciferase, aequorin, luminol, and the like. Further, FIG. 1 shows a state in which the dispensing device 16 is at the injection position above the microplate 20. However, in addition to the injection position, the dispensing device 16 has a dispensing device replacement position and a reagent suction. It is preferable that the position and the cleaning position are configured to be movable.

  Below the measurement position P, an image acquisition unit 40 used for measurement of luminescence emitted from the sample S accommodated in the well 21 through the bottom surface 22 of the microplate 20 and the filter member 30 is installed. . In the present embodiment, the image acquisition unit 40 includes an imaging device 45 that can acquire a two-dimensional light emission image by light emission from the sample S. The imaging device 45 can be configured using, for example, a highly sensitive two-dimensional photon counting camera. Further, if necessary, the image acquisition unit 40 may be configured by arranging an image intensifier tube, a relay lens, or the like before the two-dimensional camera.

  A light guide optical system 41 is installed between the measurement position P where the sample holding member 2 is arranged at the time of light emission measurement and the imaging device 45 of the image acquisition unit 40. The light guide optical system 41 guides a two-dimensional light image obtained by viewing the microplate 20 holding the sample S in each of the plurality of wells 21 from the bottom surface 22 side through the filter member 30 to the image acquisition unit 40. It is an optical system. The image acquisition unit 40 detects a two-dimensional light image of the microplate 20 including light emission from the sample S guided by the light guide optical system 41 under a predetermined light guide condition, and performs two-dimensional light emission necessary for light emission measurement. Get an image. In FIG. 1, the optical path of the two-dimensional light image in the light guide optical system 41 is schematically shown by a solid line.

  The specific configuration of the light guide optical system 41 is appropriately determined by an optical element capable of realizing a necessary function (for example, a condensing function, an optical image reduction function, etc.) according to the configuration of the sample holding member 2 and the imaging device 45, and the like. What is necessary is just to comprise. In particular, the light guide optical system 41 may include a reduction optical system that guides the image acquisition unit 40 while reducing the two-dimensional light image of the microplate 20 in order to appropriately acquire the light emission image in the image acquisition unit 40. preferable.

  FIG. 4 is a configuration diagram illustrating an example of an optical system including the sample holding member 2, the light guide optical system 41, and the image acquisition unit 40. In the present configuration example, as the light guide optical system 41, a fiber optical block 42 disposed between the sample holding member 2 composed of the microplate 20 and the filter member 30 on the bottom surface 22 side and the image acquisition unit 40 is used. ing. In the above configuration, the filter member 30 is provided at a position on the optical path of the two-dimensional light image from the microplate 20 to the image acquisition unit 40.

  The fiber optical block (fiber optical plate) 42 is an optical element formed so as to be able to transmit an optical image by bundling a large number of optical fibers. Further, the fiber optical block 42 here is formed in a tapered shape having a cross-sectional area that decreases from the sample holding member 2 side to the image acquisition unit 40 side, and functions as a reduction optical system. In FIG. 4, for the sake of explanation, the sample holding member 2 and the fiber optical block 42, and the fiber optical block 42 and the image acquisition unit 40 are illustrated in a spaced arrangement, but these are directly optical It is also possible to have a configuration that is connected to each other.

  Next, the filter member 30 that is fixed to the microplate 20 in the sample holding member 2 according to the present invention that holds the sample S will be described. FIG. 5 is a diagram illustrating an example of the configuration of the microplate 20 and the filter member 30. Here, for convenience of explanation, the microplate 20 having a configuration in which 8 × 12 = 96 wells 21 are arranged in a two-dimensional array is illustrated, and a part of the structure is shown in a simplified manner. The planar shape of each well 21 is rectangular.

  5A shows a side cross-sectional structure of the microplate 20 and the filter member 30, and FIG. 5B shows a planar structure of the filter member 30 as viewed from the image acquisition unit 40 side. The microplate 20 in the sample holding member 2 of the present configuration example is formed of a material capable of transmitting light emitted from the sample S together with a bottom surface portion 23 constituting the bottom surface 22 and a side wall portion 24 separating adjacent wells 21. Has been. The filter member 30 is attached to the microplate 20 on the bottom surface 22 of the microplate 20 serving as an emission surface of light emission from the sample S.

As shown in FIG. 5 (b), the microplate 20 has a two-dimensional structure composed of ₁₂ well rows L ₁ , L ₂ ,..., L _i ,. It has a well structure. For such micro-plate 20, provided on its bottom surface 22, preferably in the sheet-shaped filter member (filter sheet) 30 is formed so as to correspond to the well string L ₁ ~L ₁₂ described above In addition, 12 rows of optical filter portions 31 _{1 to} 31 ₁₂ are provided.

The optical filter portion ₃₁ of the filter member 30 i (i = 1 to 12), the arrangement direction of the well 21 in the corresponding wells column _{L i} is formed in a shape extending along a (vertical direction in the drawing). As a result, the optical filter unit 31 _i is a combination of the eight optical filter units corresponding to the eight wells 21 constituting the well row L _i . In other words, the configuration of the filter member 30 having the 12 rows of optical filter portions 31 _{1 to} 31 ₁₂ includes 96 optical filter portions arranged in a two-dimensional array so as to correspond to the wells 21 arranged in a two-dimensional array. It is equivalent to the provided configuration.

  Further, as shown in FIG. 5, each of the optical filter portions 31 arranged so as to correspond to the plurality of wells 21 as described above has a first filter region 31a (see FIG. 5) with the center line of the well row L as a boundary. And a second filter region 31b (right region in the drawing). These filter regions 31a and 31b are formed so as to have different light transmission characteristics with respect to wavelengths.

  Next, the data analysis apparatus 50 used for analysis of measurement data will be described. The data analysis device 50 is an analysis unit that performs an analysis process on measurement data including a light emission image acquired by the image acquisition unit 40. Further, the data analysis device 50 controls the light emission measurement for the sample S in the light emission measurement device 1 by controlling the operation of each part of the data acquisition device 10. In FIG. 1, a display device 71 that displays measurement results and an input device 72 that is used for inputting data and instructions necessary for light emission measurement are connected to the data analysis device 50. Yes.

  FIG. 6 is a block diagram showing an example of the configuration of the data analysis device 50 used in the luminescence measurement device 1 shown in FIG. The data analysis device 50 according to the present embodiment includes an analysis processing unit 51 and an analysis region setting unit 56. The analysis processing unit 51 is an analysis processing unit that performs a light emission analysis process on the sample S using the light intensity distribution in the light emission image acquired by the image acquisition unit 40 of the data acquisition device 10 as analysis data. In the present configuration example, the analysis processing unit 51 includes a light emission intensity calculation unit 52 and an analysis data selection unit 53.

  The analysis data selection unit 53 refers to the analysis region set for the emission image of the measurement data input from the data acquisition device 10 and selects necessary data based on the analysis region from the image data of the emission image. Use analysis data. In addition, the emission intensity calculation unit 52 uses the analysis data selected by the analysis data selection unit 53 and performs a process of calculating the emission intensity as an emission analysis process for the sample S (analysis processing step). This emission intensity is calculated as, for example, the average light intensity in the light intensity distribution in the analysis region.

  Further, in the analysis processing unit 51 shown in FIG. 6, a measurement data correction unit 54 is provided for measurement data such as a light emission image input from the data acquisition device 10. The measurement data correction unit 54 performs necessary corrections on the measurement data such as dark correction and shading correction on the light emission image acquired by the image acquisition unit 40. Here, the dark correction is correction for subtracting the output of the imaging device 45 in the dark from the measurement data. The shading correction is correction for data variation caused by an optical system including the imaging device 45 and the light guide optical system 41.

  An analysis region applied to the emission image in the analysis processing unit 51 is set in the analysis region setting unit 56. In the present embodiment, this analysis region is set so as to correspond to the configuration of the optical filter portion 31 of the filter member 30 disposed for each well 21 of the microplate 20 (analysis region setting step).

  That is, as shown in FIG. 5B, the optical filter unit 31 corresponding to the well 21 is divided into two filter regions 31a and 31b having different light transmission characteristics with respect to wavelengths. At this time, with respect to light emission from the sample S in the well 21, the first light component transmitted through the first filter region 31a and the second light component transmitted through the second filter region 31b in the optical filter unit 31 are: Wavelength components having different light emission are selected light components. The first light component image (the image of the first filter region 31a) and the second light component image (the image of the second filter region 31b) are two-dimensionally acquired by the image acquisition unit 40. In the luminescent image, images are formed in different areas.

  On the other hand, in the analysis region setting unit 56, the first analysis region for performing the light emission analysis processing on the first light component and the light emission analysis on the second light component on the light emission image obtained by the image acquisition unit 40. A second analysis region for performing processing is set. Thereby, in the analysis process part 51, the light emission analysis process is performed about each of the 1st light component which permeate | transmitted the 1st filter area | region 31a, and the 2nd light component which permeate | transmitted the 2nd filter area | region 31b.

  In the data analysis device 50 shown in FIG. 6, a measurement data storage unit 61 and a correction data storage unit 63 are provided as data storage means. The measurement data storage unit 61 is used to store measurement data input from the data acquisition device 10 or measurement data corrected by the measurement data correction unit 54. The correction data storage unit 63 is used for storing correction data and the like necessary when the measurement data correction unit 54 corrects the measurement data.

  FIG. 7 is a block diagram illustrating an example of a hardware configuration of the data analysis device 50 illustrated in FIG. 6. The data analysis device 50 in this configuration example includes a bus 80 and a calculation unit 81 such as a CPU connected to the bus 80. The bus 80 is connected to an image memory 82, an external device connection interface 83, a program storage unit 84, and a data storage unit 85, thereby configuring the data analysis device 50.

  The image memory 82 is used for inputting image data such as a light emission image acquired by the image acquisition unit 40 of the data acquisition device 10. The interface 83 is used to connect each device such as the data acquisition device 10 constituting the luminescence measurement device 1. The program storage unit 84 stores programs necessary for the operation of the data acquisition device 10, analysis processing of measurement data acquired by the data acquisition device 10, and the like, and a work area, a parameter area, and the like are provided as necessary. . The data storage unit 85 corresponds to, for example, the data storage units 61 and 63 in the configuration shown in FIG.

  Further, a monitor 73 used as a display device 71, a keyboard 74 used as an input device 72, a mouse 75, and the like are connected to the bus 80 of the data acquisition device 50. Further, if necessary, an external storage device such as a hard disk may be further connected.

  In the above configuration, when the sample holding member 2 including the microplate 20 in which the sample S is held in the well 21 is arranged at the measurement position P in the dark box 15, the data analysis device 50 is connected to the dispensing device 16. Instruct the dispensing of the reagent. Receiving the instruction, the dispensing device 16 dispenses the reagent used for the luminescence measurement to each well 21 of the microplate 20 (reagent injection step). At this time, light is generated in the well 21 as a result of the interaction between the sample S and the injected reagent.

  A two-dimensional light image including light emission from the sample S generated in the well 21 is sent to the image acquisition unit 40 via the bottom surface portion 23, the filter member 30, and the light guide optical system 41 that constitute the bottom surface 22 of the microplate 20. And a two-dimensional light emission image is acquired by the imaging device 45 (image acquisition step). Further, the measurement data including the light emission image acquired by the image acquisition unit 40 is sent to the data analysis device 50. Then, the data analysis device 50 performs analysis processing necessary for the evaluation of the sample S or the like on the input measurement data as described above with reference to FIG. 6 (data analysis step).

  The effects of the sample holding member 2, the luminescence measuring apparatus 1 using the sample holding member 2, and the luminescence measuring method will be described.

  In the luminescence measuring apparatus 1 and the sample holding member 2 applied to the luminescence measuring method shown in FIGS. 1 to 7, the luminescence from the sample S during the luminescence measurement with respect to the microplate 20 having the plurality of wells 21. The sample holding member 2 is configured by providing the filter member 30 fixed to the microplate 20 at a predetermined position on the optical path to the image acquisition unit 40 of the two-dimensional optical image of the microplate 20 including the microplate 20. For the optical filter portion 31 of the filter member 30 corresponding to the well 21 of the microplate 20, it is necessary for wavelength selectivity according to the specific conditions of the luminescence measurement performed on the sample S, that is, for the luminescence analysis on the sample S. It is configured to exhibit excellent spectral characteristics.

  Thus, according to the sample holding member 2 to which an optical filter function for light emission from the sample S is applied, for example, when applied to the light emission measurement of the sample S by the light emission measuring device 1, for example, the image acquisition unit 40 or the guide The configuration of the apparatus including the optical optical system 41 and the like can be simplified. In addition, the filter member 30 including the optical filter unit 31 arranged in a two-dimensional array corresponding to the well 21 in which the sample S is held is fixed to the microplate 20, thereby emitting light under various conditions. In the measurement, it is possible to improve the measurement efficiency of light emission, the work efficiency at the time of light emission measurement, and the like.

  Moreover, in the said embodiment, the optical filter part 31 in the filter member 30 is comprised by the 1st filter area | region 31a and the 2nd filter area | region 31b from which a light transmission characteristic differs mutually. Thereby, the sample holding member 2 of the present embodiment, for the light emission from the sample S, each of the first light component transmitted through the first filter region 31a and the second light component transmitted through the second filter region 31b. It becomes the structure which can perform luminescence measurement about.

  As described above, when the optical filter unit 31 of the filter member 30 corresponding to the well 21 of the microplate 20 is provided with a plurality of filter regions 31a and 31b having different wavelength selectivity, when applied to luminescence measurement, By performing the light emission analysis process on the light components transmitted through the respective filter regions, it is possible to simultaneously measure a plurality of types of light emission phenomena having different wavelength spectra. This makes it possible to efficiently perform multicolor light emission measurement and the like with a simple configuration.

  In addition, the luminescence measuring apparatus 1 shown in FIG. 1 to which the sample holding member 2 is applied has an imaging device 45 capable of acquiring a two-dimensional luminescent image as a light detection means for detecting luminescence from the sample S. While using the image acquisition part 40, the filter member 30 for selecting the wavelength component to measure among the light emission from the sample S is arrange | positioned on the optical path of the two-dimensional optical image from the microplate 20 to the image acquisition part 40. It becomes the composition. Thereby, in the luminescence measurement under various conditions, it is possible to improve the measurement efficiency of the luminescence, the working efficiency at the time of luminescence measurement, and the like.

  Furthermore, in the above-described embodiment, the sample holding member 2 has a configuration in which a plurality of filter regions 31 a and 31 b having different wavelength selectivity are provided for the optical filter portion 31 of the filter member 30 corresponding to the well 21 of the microplate 20. Yes. As described above, the light emission image from the microplate 20 is acquired by the image acquisition unit 40 via the filter member 30, and each of the first filter region 31a and the second filter region 31b is obtained using the obtained light emission image. According to the configuration capable of performing light emission measurement on the transmitted first light component and second light component, it is possible to simultaneously measure a plurality of types of light emission phenomena having different wavelength spectra. This makes it possible to efficiently perform multicolor light emission measurement.

  Examples of such a luminescence phenomenon include a luciferase reaction (under neutrality) with an emission wavelength of 565 nm and a luminol reaction (in aqueous solution) with an emission wavelength of 430 nm. Moreover, as an optical filter that can be used in the filter regions 31a and 31b for these light emission phenomena, for example, a sharp cut filter (FUJI SC 56, manufactured by Fuji Film) that cuts a light component having a wavelength of 560 nm or less, and There is a band-pass filter (FUJI BPB 42, manufactured by Fuji Film) that transmits light components having a wavelength of around 420 nm.

  In the data analysis apparatus 50 having the configuration shown in FIG. 6, the analysis region setting unit 56 has a first analysis region for the first light component transmitted through the first filter region 31 a for the two-dimensional emission image, and A light emission analysis process is performed by setting a second analysis region for the second light component transmitted through the second filter region 31b. As described above, by performing a light emission analysis process by setting a plurality of analysis regions corresponding to the configuration of the optical filter unit 31 in the filter member 30 with respect to the light emission image, multicolor light emission measurement can be performed reliably and easily. Can be done by the method.

  However, the light emission analysis processing for the first light component and the second light component is not limited to the method of setting the analysis region as described above, and the light emission analysis processing may be performed by another method. In general, the analysis processing unit 51 of the data analysis device 50 uses the light intensity distribution in the light emission image as analysis data, and the optical filter unit 31 for light emission from the sample S held in the well 21 of the microplate 20. It is sufficient that the light emission analysis process is performed for each of the first light component transmitted through the first filter region 31a and the second light component transmitted through the second filter region 31b.

  Specifically, for example, a sheet-like filter member can be used as the filter member 30 provided to be fixed to the microplate 20 in the sample holding member 2. Further, regarding the installation configuration of the filter member 30 with respect to the microplate 20, in the configuration example shown in FIG. 5, the filter member 30 is fixedly mounted on the bottom surface 22 of the microplate 20 that is the emission surface of light emission from the sample S. It is configured.

  In such a configuration, specifically, the filter member 30 is a member different from the microplate 20, and the filter member 30 selected on the bottom surface 22 of the microplate 20 according to the wavelength selectivity necessary for light emission measurement. The structure which makes the sample holding member 2 by attaching and fixing this sheet | seat can be used. In this case, depending on the type of filter sheet attached as the filter member 30, it is possible to cope with light emission phenomena having various wavelength components in the light emission measurement of the sample S. Further, by appropriately selecting the filter member 30, it is easy to cope with the type of the microplate 20, for example, the number of wells of the microplate such as 96 wells, 384 wells, or 1536 wells. Further, regarding the specific configuration of the sample holding member 2, various configurations other than the configuration example shown in FIG. 5 may be used.

  FIG. 8 is a diagram illustrating another example of the configuration of the sample holding member. In the sample holding member 2b of the present configuration example, the microplate 20b in which the bottom surface portion 23b and the side wall portion 24b are both formed of a material that can transmit light emitted from the sample S is used. Further, the filter member 30b is configured to be integrated with the bottom surface portion 23b on the bottom surface 22 side of the microplate 20b. In this case, the microplate 20b itself becomes a sample holding member whose bottom surface portion 23b functions as an optical filter.

  In such an integrated configuration, the well 21 in the microplate 20b constituting the sample holding member 2b can be reliably aligned with the optical filter portion 31 in the filter member 30b, and the corresponding light emission is performed. There is an advantage that the analysis area can be easily set in the analysis processing. Further, the portion of the bottom surface portion 23b of the microplate 20b that functions as the filter member 30b may be a part of the bottom surface portion 23b (for example, the lower portion of the bottom surface portion 23b) as shown by a dotted line in FIG. Alternatively, the entire bottom surface portion 23b may function as the filter member 30b.

  FIG. 9 is a diagram showing still another example of the configuration of the sample holding member. In the sample holding member 2c of this configuration example, the bottom surface portion 23c is formed of a material that can transmit light emitted from the sample S, and the side wall portion 24c that separates the adjacent wells 21 is not resistant to light emission from the sample S. A microplate 20c formed of a transparent material (for example, a black material) is used. Further, the filter member 30 is configured to be mounted on the bottom surface 22 of the microplate 20c.

  According to such a configuration, when the light emission measurement is performed simultaneously on the sample S held in each of the plurality of wells 21, the light transmission between the adjacent wells 21 is suppressed, whereby the light emission measurement is performed. The occurrence of crosstalk can be suppressed. Such a configuration can also be used in combination with the configuration shown in FIG.

  FIG. 10 is a configuration diagram illustrating another example of the optical system including the sample holding member 2, the light guide optical system 41, and the image acquisition unit 40. In this configuration example, a lens optical system 43 having a function as a reduction optical system is used as the light guide optical system 41. Further, the installation configuration of the filter member 30 is a configuration in which the filter member 30 is mounted on the bottom surface 22 of the microplate 20 as in the configuration of FIG. 4. As described above, for the light guide optical system 41 that guides the two-dimensional light image from the microplate 20 to the image acquisition unit 40, optical systems having various configurations can be used. Such a configuration can also be used in combination with the configuration shown in FIGS.

  The configuration of the optical filter unit 31 in the filter member 30 of the sample holding member 2, the setting of the analysis region in the light emission analysis process, and the like will be described more specifically. The analysis region in the light emission analysis process is set on a two-dimensional light emission image including the image of the filter member 30 acquired by the image acquisition unit 40. In the following, for convenience of explanation, a filter is used. The configuration of the member 30 and the set analysis region are shown in correspondence on the same drawing. In the configuration example of the filter member 30 shown below, the optical filter portions 31 are arranged in a two-dimensional array so as to correspond to the wells 21 of the microplate 20 on a one-to-one basis.

  FIG. 11 is a diagram illustrating an example of the configuration of the filter member 30. In this configuration example, the optical filter unit 31 corresponding to the well 21 is divided with the vertical center line of the square outer shape as a boundary, and the two filters of the first filter region 31a and the second filter region 31b. It is composed of areas. On the other hand, the first analysis region Ra and the second analysis region Rb on the light emission image are set to regions where the light components transmitted through the corresponding filter regions 31a and 31b are selected. Then, by performing emission analysis processing for each of these analysis regions Ra and Rb, for example, as shown in graphs Ga and Gb of examples of temporal changes in emission intensity in FIG. 12, each wavelength component of emission from the sample S is shown. Information on the light emission characteristics for (two colors) is obtained.

  When the optical filter unit 31 is configured by two filter regions in this way, as shown in FIG. 5, the array of wells in the well row including the plurality of optical filter units 31 corresponding to the well row. It is also possible to adopt a configuration of an optical filter portion having a shape extending along the direction. However, even in this case, the analysis region is set individually so as to correspond to each well as shown in FIG. In FIG. 11, the analysis regions Ra and Rb are set to have slightly smaller areas than the filter regions 31a and 31b. However, the entire regions corresponding to the filter regions 31a and 31b are set as analysis regions Ra and Rb. The regions Ra and Rb may be set so as to be directly adjacent to each other.

  FIG. 13 is a diagram illustrating another example of the configuration of the filter member 30. In the present configuration example, the optical filter unit 31 is divided with the vertical center line and the horizontal center line of the square outer shape as boundaries, and the first filter region 31a, the second filter region 31b, and the third filter The region is composed of four filter regions, a region 31c and a fourth filter region 31d. On the other hand, the first analysis region Ra, the second analysis region Rb, the third analysis region Rc, and the fourth analysis region Rd on the luminescent image are transmitted through the corresponding filter regions 31a, 31b, 31c, and 31d, respectively. The selected light component is set in the selected region. Then, by performing emission analysis processing on each of these analysis regions Ra, Rb, Rc, and Rd, for example, as shown in graphs Ga, Gb, Gc, and Gd of examples of emission intensity over time in FIG. Information on the light emission characteristics for each wavelength component (four colors) of light emission from S is obtained.

  FIG. 15 is a diagram showing still another example of the configuration of the filter member 30. The optical filter unit 31 in this configuration example has the same configuration as that shown in FIG. 13, but differs in that the outer shape is not a square but a circle. Such a configuration is applicable, for example, when the planar shape of the well 21 in the microplate 20 is not a square but a circle.

  In the filter member 30 shown in FIGS. 5, 11, 13, and 15, the plurality of filter regions of the optical filter unit 31 have the same area. On the other hand, the optical filter unit 31 in the filter member 30 has a plurality of filter regions (for example, the first filter region 31a and the second filter region 31b) each having a plurality of light components (for example, a plurality of light components) , The first light component and the second light component) are preferably set according to the respective measurement conditions.

  Thus, referring to the wavelength components in the light emission corresponding to the light transmission characteristics in the filter region, each area of the plurality of filter regions in the optical filter unit and the area ratio thereof according to the measurement conditions of each light component By setting, it is possible to perform light emission measurement with sufficient accuracy for each wavelength component selected in each filter region. Here, as measurement conditions to be referred to when setting the area of the filter region, for example, spectral sensitivity characteristics of a camera used as the imaging device 45 in the image acquisition unit 40, light emission expected in the light emission phenomenon in the sample S Examples include intensity balance (for example, intensity difference caused by gene expression level, luminous efficiency, etc. in sample S).

  FIG. 16 is a graph illustrating an example of spectral sensitivity characteristics of the imaging device 45 in the image acquisition unit 40. In this graph, the horizontal axis indicates the wavelength (nm) of light, and the vertical axis indicates the relative sensitivity (%) in the imaging device 45. In such spectral sensitivity characteristics, if the wavelength range (blue) of the light component selected in the first filter region 31a is Sa and the wavelength range (red) of the light component selected in the second filter region 31b is Sb, The relative sensitivity ratio is approximately blue: red = 4: 1.

  In contrast, as shown in FIG. 17, the optical filter unit 31 is configured so that the area of the first filter region 31a: the area of the second filter region 31b = 1: 4. By adopting such a configuration, it is possible to match the measurement sensitivity for each light component of light emission as a whole. The method for setting the area of such a filter region is the same for the setting according to the emission intensity balance in the sample S. Further, in this configuration example, the configuration of the optical filter unit in the case of having two filter regions is shown. However, for example, the configuration in the case of having four filter regions in FIG. 18 is not limited to the number of filter regions. The above-described method for setting the area of the filter region can be applied.

  The sample holding member according to the present invention is not limited to the above-described embodiments and configuration examples, and various modifications are possible. For example, in the above embodiment, the optical filter unit 31 in the filter member 30 is configured to include at least the first filter region 31a and the second filter region 31b having different light transmission characteristics from each other. In addition to such a configuration, various configurations may be used according to specific conditions of the target luminescence measurement. In general, the filter member has a plurality of optical filter portions arranged in a two-dimensional array so as to correspond to a plurality of wells of the microplate, and each of the plurality of optical filter portions has a wavelength required for light emission measurement. What is necessary is just to have the light transmission characteristic about the wavelength corresponding to selectivity. Even with such a configuration, in the luminescence measurement under various conditions, it becomes possible to improve the measurement efficiency of the luminescence, the working efficiency at the time of luminescence measurement, and the like.

  Moreover, about the specific structure of the light emission measuring apparatus to which the sample holding member is applied, the light emission measuring apparatus 1 shown in FIG. 1 shows an example of the structure. Generally, the light guide optical system and the image are displayed. Various configurations may be used according to the configuration of the optical system including the acquisition unit, the configuration of the dispensing device, the conditions of the luminescence measurement, and the like. Further, the data analysis device 50 that performs analysis processing on the measurement data including the acquired luminescence image may be provided separately from the luminescence measurement device 1.

  The present invention can be used as a sample holding member capable of efficiently performing luminescence measurement under various conditions such as multicolor luminescence measurement.

It is a block diagram which shows typically one Embodiment of the light emission measuring device with which a sample holding member is applied. It is a perspective view which shows an example of a structure of a sample holding member. It is side surface sectional drawing which shows the cross-section of the sample holding member shown in FIG. It is a block diagram which shows an example of the optical system containing a sample holding member, a light guide optical system, and an image acquisition part. It is a figure which shows an example of a structure of a microplate and a filter member. It is a block diagram which shows an example of a structure of a data analyzer. FIG. 7 is a block diagram illustrating an example of a hardware configuration of the data analysis apparatus illustrated in FIG. 6. It is a figure which shows the other example of a structure of a sample holding member. It is a figure which shows the other example of a structure of a sample holding member. It is a block diagram which shows the other example of the optical system containing a sample holding member, a light guide optical system, and an image acquisition part. It is a figure which shows an example of a structure of a filter member. It is a graph which shows the example of the light emission measurement using the filter member shown in FIG. It is a figure which shows the other example of a structure of a filter member. It is a graph which shows the example of the light emission measurement using the filter member shown in FIG. It is a figure which shows the other example of a structure of a filter member. It is a graph which shows an example of the spectral sensitivity characteristic of the imaging device in an image acquisition part. It is a figure which shows the other example of a structure of a filter member. It is a figure which shows the other example of a structure of a filter member.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Luminescence measuring device, 10 ... Data acquisition device, 11 ... Microplate holder, 12 ... Conveyance mechanism, 13 ... Carry-in side microplate stocker, 14 ... Carry-out side microplate stocker, 15 ... Dark box, 16 ... Dispensing device, 2 DESCRIPTION OF SYMBOLS ... Sample holding member, 20 ... Microplate, 21 ... Well, 22 ... Bottom, 23 ... Bottom part, 24 ... Side wall part, 30 ... Filter member, 31 ... Optical filter part, 31a ... 1st filter area | region, 31b ... 2nd Filter area 31c Third filter area 31d Fourth filter area 40 Image acquisition unit 41 Light guiding optical system 42 Fiber optical block 43 Lens optical system 45 Imaging device
DESCRIPTION OF SYMBOLS 50 ... Data analysis apparatus, 51 ... Analysis processing part, 52 ... Luminescence intensity calculation part, 53 ... Analysis data selection part, 54 ... Measurement data correction part, 56 ... Analysis area setting part, 61 ... Measurement data storage part, 63 ... Correction Data storage unit 71 ... Display device 72 ... Input device 73 ... Monitor 74 ... Keyboard 75 ... Mouse 80 ... Bus 81 ... Calculation unit 82 ... Image memory 83 ... Interface 84 ... Program storage unit 85: Data storage unit.

Claims (6)

A sample holding member for holding a sample,
A microplate in which a plurality of wells each capable of holding a sample to be subjected to luminescence measurement are arranged in a two-dimensional array;
A filter member fixed to the microplate at a predetermined position through which light emitted from the sample held in the well of the microplate is transmitted;
The filter member has a plurality of optical filter portions arranged in a two-dimensional array so as to correspond to the plurality of wells of the microplate, and each of the plurality of optical filter portions is necessary for light emission measurement. A sample holding member having a light transmission characteristic for a wavelength corresponding to wavelength selectivity.   The filter member includes at least a first filter region and a second filter region in which each of the plurality of optical filter portions has different light transmission characteristics, and the first filter region with respect to light emission from the sample. 2. The sample holding member according to claim 1, wherein luminescence measurement can be performed for each of the first light component transmitted through the first filter and the second light component transmitted through the second filter region.   The optical filter unit is characterized in that the areas of the first filter region and the second filter region are set according to the measurement conditions of the first light component and the second light component, respectively. The sample holding member according to claim 2.   The sample holding member according to any one of claims 1 to 3, wherein the filter member is mounted on a bottom surface of the microplate that is an emission surface of light emitted from the sample.   The said filter member is provided in the bottom face side of the said microplate used as the emission surface of the light emission from the said sample integrally with the bottom face part. Sample holding member.   The sample holding member according to any one of claims 1 to 5, wherein in the microplate, a side wall that separates adjacent wells is impermeable to light emitted from the sample.

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