Auxiliary member and observation method

Abstract

To provide a technique capable of achieving both cell observation by transmitted light and strict fluorescence / luminescence observation when performing fluorescence observation or luminescence measurement of a biological sample such as a cell.SOLUTION: Fluorescence observation or luminescence measurement is performed on a biological sample stored in a storage part 90 by using an auxiliary member 1 attachable to and detachable from a container 9 including the storage part 90 having a light transmissive bottom surface 91 and a light shielding wall surface 92. The auxiliary member 1 has a light-shielding portion 11 that comes into close contact with the bottom surface 91 when attached to the container 9. As a result, when the auxiliary member 1 is not attached, cell observation by transmitted light is possible, and when the auxiliary member 1 is attached, strict fluorescence / luminescence observation can be performed.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an auxiliary member that is attached to a container that holds a biological sample together with a culture medium inside, and an observation method for observing a biological sample using the container and auxiliary member. [Background technology]

[0002] Conventionally, methods for observing and measuring cells include fluorescence observation and luminescence measurement. A conventional multi-well plate having a plurality of storage sections for holding biological samples is described in, for example, Patent Document 1.

[0003] Fluorescence observation, for example, involves immunostaining using antibodies and fluorescent substances, and detecting fluorescence to confirm the presence or absence of target proteins, etc. Luminescence measurement, which utilizes chemiluminescence, has been used for a long time. For example, in cells into which a luciferase gene has been introduced, when luciferin, the basis of luciferase, is added, ATP (adenosine triphosphate) present in the living cells reacts with luciferin via the enzyme luciferase to produce oxyluciferin. Since the oxyluciferin produced by this reaction continues to emit light to stabilize energy, the ATP activity of the cells can be examined by measuring the amount of luminescence.

[0004] When performing observations or measurements under such fluorescence / luminescence conditions, if a transparent container is used, the fluorescent or luminescent light will diffuse in multiple directions through the container, making accurate measurements difficult. Therefore, when observing or measuring fluorescent or luminescent light, it is preferable to contain the sample in an opaque container. For example, well plates for fluorescence observation are made of black materials for both the bottom and walls of the container to suppress autofluorescence. Furthermore, for luminescence measurements, for example, the bottom and walls of the container are made of white materials to facilitate light collection when measuring the luminescence intensity from above the well plate. However, such opaque containers do not allow for normal bright-field observation using transmitted light. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-543048 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, in recent years, well plates with opaque walls and transparent bottoms have been sold. This allows bright-field observation using transmitted light, while the opaque walls reduce the diffusion of fluorescent and luminescent light. However, this is not sufficient when the intensity of the fluorescence or luminescence is low. For example, accurate measurement becomes difficult when precise measurement is required under limited conditions for biological samples such as patient specimens, where the luminescence intensity is low.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a technology that enables both cell observation using transmitted light and strict fluorescence and luminescence observation when performing fluorescence observation or luminescence measurement on biological samples such as cells. [Means for solving the problem]

[0008] In order to solve the above problem, the first invention of the present application is an auxiliary member that can be attached and detached to a container having a storage section with a light-transmitting bottom surface and light-shielding wall surfaces, and has a light-shielding section that adheres to the bottom surface when attached to the container.

[0009] A second invention of the present application is the auxiliary member of the first invention, wherein the container has a plurality of the storage sections, and the light-shielding section is in close contact with the bottom surfaces of all of the storage sections.

[0010] The third invention of the present application is an auxiliary member of the first invention, which has a notch arranged on the edge on the outside of the light-shielding portion, and the notch fits into a downward-protruding rib on the container.

[0011] The fourth invention of the present application is an observation method for observing a biological sample contained in a container having a storage section with a light-transmitting bottom surface and light-shielding wall surfaces, comprising the steps of: a) attaching to the container an auxiliary member having a light-shielding section that is in close contact with the bottom surface; and b) performing fluorescence observation or luminescence measurement of the biological sample contained in the container with the auxiliary member attached.

[0012] A fifth invention of the present application is the observation method of the fourth invention, wherein the step a) includes the steps of: a1) placing the auxiliary member on the upper surface of an attachment jig; a2) lowering the container from above the auxiliary member and the attachment jig, pressing the bottom surface of the container against the auxiliary member, and attaching the auxiliary member to the container; and a3) raising the container, and separating the container with the attached auxiliary member from the attachment jig. [Effects of the Invention]

[0013] According to the first to fifth aspects of the present invention, by using a container with a storage section having a light-transmitting bottom surface and light-shielding wall surfaces and an auxiliary member, it is possible to simultaneously perform cell observation using transmitted light and strict fluorescence and luminescence observation of a biological sample held in the storage section. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 2 is a perspective view of a well plate and an auxiliary member according to the first embodiment. [Figure 2] FIG. 2 is a perspective view of a well plate and an auxiliary member according to the first embodiment. [Figure 3] FIG. 3 is a cross-sectional view showing a state in which an auxiliary member is attached to the well plate according to the first embodiment. [Figure 4] FIG. 3 is a cross-sectional view showing a state in which an auxiliary member is attached to the well plate according to the first embodiment. [Figure 5] 10 is a flowchart showing a flow of sample observation using a well plate and an auxiliary member according to the first embodiment. [Figure 6] 10 is a graph showing experimental results. [Figure 7] FIG. 10 is a cross-sectional view of a well plate and an auxiliary member according to a second embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[0016] 1. First Embodiment <1-1. Configuration of well plate and auxiliary parts> 1 and 2 are perspective views of an auxiliary member 1 according to the first embodiment and a well plate 9 to which the auxiliary member 1 is attached. FIGS. 3 and 4 are cross-sectional views showing how the auxiliary member 1 is attached to the well plate 9.

[0017] The well plate 9 is a container for containing and holding biological samples. The well plate 9 is a generally plate-shaped container having a plurality of wells (recesses) 90. The wells 90 are containers for containing and holding biological samples. As shown in FIG. 1, the plurality of wells 90 are regularly arranged on the top surface of the well plate 9. In this embodiment, the shape of the wells 90 when viewed from above is circular. However, the shape of the wells 90 when viewed from above may be other shapes, such as rectangular. Each well 90 has a light-transmitting bottom surface 91 and a light-blocking wall surface 92.

[0018] The well plate 9 is composed of a first member 80 made of a light-blocking resin, and a sheet-like second member 70 made of a transparent resin that transmits light.

[0019] The first member 80 has a main body 81, an upper plate 82, a side plate 83, and a plurality of ribs 84. The main body 81 is a plate-like portion that is thick in the vertical direction. The main body 81 has a thickness that is the same as the internal height of the well 90. The main body 81 is provided with a plurality of cylindrical through holes 810 that constitute the well 90. The internal space of these through holes 810 is the internal space of the well 90, and the inner circumferential surface of the through holes 810 is the wall surface 92 of the well 90.

[0020] 3, a sheet-like second member 70 is attached to the bottom surface of the main body 81. As a result, the second member 70 serves as the light-transmitting bottom surface 91 of each well 90.

[0021] The upper plate portion 82 is a plate-shaped portion that extends outward from the upper edge of the main body portion 81. The thickness of the upper plate portion 82 is smaller than the thickness of the main body portion 81. The upper surfaces of the main body portion 81 and the upper plate portion 82 form the upper surface of the well plate 9 that extends horizontally.

[0022] The side plate 83 is a cylindrical portion extending downward from the outer edge of the upper plate 82. The ribs 84 are plate-shaped portions that are approximately perpendicular to the upper plate 82 and the side plate 83. That is, the ribs 84 protrude downward from the lower surface of the upper plate 82 and protrude inward from the inner surface of the side plate 83. In this embodiment, a total of ten ribs 84 are provided on the well plate 9, three on each longitudinal side and two on each lateral side.

[0023] As shown in Figures 3 and 4, a biological sample S to be observed is held in each well 90 together with a culture solution. This allows the biological sample S to be cultured in each well 90. The biological sample S may be a single cell, or may be a cell mass (spheroid or organoid) formed by multiple cells. The biological sample S may also be a tissue or a portion of a tissue from a living organism.

[0024] The auxiliary member 1 is a plate-shaped member. The auxiliary member 1 is made of a resin having light-blocking properties. The color of the auxiliary member 1 is, for example, white or black. The color of the auxiliary member 1 is preferably the same as the color of the side wall surface 92 of the well plate 9. The auxiliary member 1 is attached to the well plate 9 so that its upper surface contacts the lower surface of the main body 81 of the well plate 9.

[0025] The auxiliary member 1 has a light-shielding portion 11 and an edge portion 12. The light-shielding portion 11 is a portion that comes into close contact with the bottom of the well 90 when attached to the well plate 9. In other words, the light-shielding portion 11 is a portion that overlaps with the main body portion 81 when the auxiliary member 1 is attached to the well plate 9. The edge portion 12 is a portion that is outside the light-shielding portion 11. The edge portion 12 has a plurality of notches 10. In other words, the notches 10 are arranged outside the light-shielding portion 11. When the auxiliary member 1 is attached to the well plate 9, the notches 10 engage with the ribs 84. As a result, even when the well plate 9 to which the auxiliary member 1 is attached is lifted, the auxiliary member 1 is held on the underside of the well plate 9 and will not fall off.

[0026] <1-2. Observation of samples using auxiliary materials> Next, the flow of a sample observation process using the well plate 9 and the auxiliary member 1 will be described with reference to Figures 3 to 5. Figure 5 is a flowchart showing the flow of a sample observation process using the well plate 9 and the auxiliary member 1.

[0027] 5, when performing fluorescence observation or luminescence measurement of a sample, first, the auxiliary member 1 is attached to the well plate 9 (step S10: auxiliary member attachment step). Then, with the auxiliary member 1 attached to the well plate 9, fluorescence observation or luminescence measurement of the biological sample contained in each well 90 of the well plate 9 is performed (step S20: observation step).

[0028] In step S10, when attaching the auxiliary member 1 to the well plate 9, it is necessary to attach the auxiliary member 1 without misalignment to the underside of the well plate 9 without tilting the well plate 9. For this purpose, it is recommended to use an attachment jig 6 as shown in Figures 3 and 4.

[0029] 3, first, the auxiliary member 1 is placed on the upper surface of the mounting jig 6 (step S11). Then, the well plate 9 is lowered from above the auxiliary member 1 and the mounting jig 6, and the bottom surface of the well plate 9, i.e., the bottom surfaces of the multiple wells 90, is pressed against the auxiliary member 1, and the auxiliary member 1 is attached to the well plate 9 (step S12). Specifically, the rib 84 is inserted into the notch 10, and the rib 84 and the notch 10 fit together, and the upper surface of the auxiliary member 1 comes into close contact with the bottom surface of the well 90.

[0030] 4, the well plate 9 is raised to separate the well plate 9 with the auxiliary member 1 attached thereto from the attachment jig 6 (step S13). By attaching the auxiliary member 1 to the well plate 9 using the attachment jig 6 in this way, the attachment of the auxiliary member 1 to the well plate 9 can be performed while maintaining a stable posture of the well plate 9. In other words, it is possible to prevent the posture of the well plate 9 from becoming unstable and the biological sample or culture medium held in the well 90 from spilling out of the well 90.

[0031] In this way, the auxiliary member 1, which can be attached to and attached to the bottom of the well 90, can be detached from the well plate 9, in which the wells 90 have transparent bottoms. This allows for cell observation of the biological sample held in the well 90 using transmitted light when the auxiliary member 1 is not attached, and allows for strict fluorescence and luminescence observation of the biological sample held in the well 90 when the auxiliary member 1 is attached.

[0032] <1-3. Effect of auxiliary materials: luminescence measurement experiment> Next, the effect of such auxiliary member 1 was verified by carrying out a luminescence measurement experiment on cells. Figure 6 is a graph showing the results of the luminescence measurement experiment described below.

[0033] The sample observed in this experiment was prepared by adding the luminescence measurement reagent CellTiter-Glo (registered trademark) to CHO-K1 cells, a commercially available cell line. The container in which the sample was stored was a well plate with the same shape as the well plate 9 according to the first embodiment. Using this sample, the amount of luminescence was measured for multiple cell seeding numbers under the following conditions 1 and 2. Condition 1: The bottom of the well is colorless and transparent Condition 2: A white sheet is attached to the bottom of the well

[0034] Condition 1 above is a case where a commercially available well plate is used as is. This well plate has white walls and a colorless, transparent bottom. Condition 2 is a case where a white sheet is attached to the bottom of the well of a well plate of the same type as the well plate used in Condition 1. In other words, Condition 2 is a condition similar to the case where the auxiliary member 1 according to the first embodiment is attached to the bottom of a well with a transparent bottom.

[0035] As a result of this experiment, as shown in Figure 6, with the same number of cells seeded, the amount of luminescence measured under condition 2 was approximately twice that of condition 1. In other words, by adhering an opaque material to the bottom of the well, it is possible to perform precise fluorescence and luminescence observation of the biological sample held in the storage section. It is expected that a similar effect will be obtained when auxiliary member 1 is used.

[0036] 2. Second Embodiment 6 is a cross-sectional view of a well plate 9A and an auxiliary member 1A according to the second embodiment. The bottom surface of each well 90 in the well plate 9 according to the first embodiment is flat. In contrast, the transparent bottom surface 91A of each well 90A in the well plate 9A according to the second embodiment is curved.

[0037] The auxiliary member 1A according to the second embodiment is a plate-like member that is thicker than the height of the curved bottom surface of the well 90A. The upper surface of the auxiliary member 1A is provided with a plurality of recesses 13A that are shaped to fit the curved bottom surface 91A of the well 90A. As a result, when the auxiliary member 1A is attached to a well plate 9A, the curved surfaces that make up the recesses 13A come into close contact with the bottom surface 91A of each well 90A. In other words, the curved surfaces that make up the recesses 13A of the auxiliary member 1A serve as light-shielding portions that come into close contact with the transparent bottom surface 91A of the well 90A.

[0038] In this way, the bottom surface of well 90A does not have to be flat. The shape of the light-shielding part of auxiliary member 1A can be appropriately changed to match the shape of the bottom surface of well 90A.

[0039] <3. Modifications> Although one embodiment of the present invention has been described above, the present invention is not limited to the above embodiment.

[0040] In the first embodiment, each well 90 of the well plate 9 has a circular shape when viewed from above. However, each well 90 may have another shape when viewed from above, such as a rectangle with rounded corners.

[0041] In the above embodiment, the well plate 9 has been described as having a plurality of wells 90. However, the sample container of the present invention may be a Petri dish or a flask having one storage portion.

[0042] Furthermore, the elements appearing in the above-described embodiments and modifications may be combined as appropriate within the scope of not causing any contradiction. [Explanation of symbols]

[0043] 1,1A Auxiliary parts 6 Mounting jig 9,9A well plate 10 Cutout 11 Light blocking section 12 Edge 13A Recess 81 Main body 82 Upper plate 83 Side plate part 84 Ribs 90,90A well 91,91A bottom 92 Wall

Claims

1. An auxiliary member detachable from a container having a storage section with a light-transmitting bottom surface and a light-shielding wall surface, a light-shielding portion that is in close contact with the bottom surface when attached to the container; An auxiliary member having:

2. 2. The auxiliary member according to claim 1, the container has a plurality of the storage portions, The light-shielding portion is an auxiliary member that is in close contact with the bottom surface of all of the storage portions.

3. 2. The auxiliary member according to claim 1, a notch disposed on the outer edge of the light blocking portion; and The notch is an auxiliary member that fits onto a downwardly protruding rib on the container.

4. 1. A method for observing a biological sample contained in a container having a container part with a light-transmitting bottom surface and a light-shielding wall surface, comprising: a) attaching an auxiliary member having a light-shielding portion that is in close contact with the bottom surface of the container; b) performing fluorescence observation or luminescence measurement of the biological sample contained in the container to which the auxiliary member is attached; An observation method comprising:

5. The observation method according to claim 4, The step a) a1) placing the auxiliary member on an upper surface of a mounting jig; a2) lowering the container from above the auxiliary member and the mounting jig, pressing the bottom surface of the container against the auxiliary member, and attaching the auxiliary member to the container; a3) raising the container to separate the container to which the auxiliary member is attached from the mounting jig; An observation method, including:

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