JP2018183070A - Observation device - Google Patents

Abstract

Kind Code: A1 Even if an objective lens comes into contact with a member corresponding to a cover glass and slightly presses it, the member is not damaged, and can be used not only for observation of culture environments but also for real-time observation of permeability tests. To provide an observation device capable of A plate (10) having a through hole (11) and supporting portions (12) having a diameter larger than the diameter of the through hole at both ends of the through hole; A flexible connection member 20 having a hole 22, a transmission member 31 having an outer edge 31 bonded around the transmission hole of the connection member, and a stepped portion 13 formed by recessing a support portion from the surface of the plate. and , wherein the height H1 of the shoulder is higher than the height of one or both of the connecting member and the transmissive member from the support to provide the viewing device 100 . [Selection drawing] Fig. 2

Description

  The present invention relates to an observation apparatus, and more particularly to an observation apparatus for observing a culture environment and a permeability test.

  Conventionally, techniques related to an observation apparatus for observing a culture environment and an observation apparatus for observing a permeability test are known. For example, Patent Document 1 discloses a culture container, a culture observation apparatus, and a culture observation method suitable for maintaining a culture environment. The culture container includes a first transparent member, a second transparent member, a housing member, and a sealing member. The first transparent member can be maintained at a set temperature. The second transparent member faces the first transparent member. The casing member is joined to the first transparent member and the second transparent member, and together with the first transparent member and the second transparent member, forms a culture space capable of accommodating the well plate. The sealing member seals the liquid injected into the culture space between the first transparent member and the housing member.

  Patent Document 2 discloses a floating cell culture method that prevents floating cells from being removed at the time of exchanging the culture solution, and enables observation of floating cells without removing the floating cells from the culture vessel. In this culturing method, a pair of permeation plates are arranged opposite to each other with a spacer interposed therebetween, whereby a flat chamber is formed between the permeation plates, and a guide tube for introducing a culture medium into the chamber and a chamber around the chamber Is a method for culturing floating cells using a cell provided with a second needle that is provided with a guide tube for discharging the culture medium from the tube and that can be inserted into the guide tube with its tip protruding into the chamber. The culture method includes a capturing step of discharging the culture solution while introducing the culture solution containing floating cells into the chamber, and capturing the suspension cells in the chamber; and culturing while introducing the culture solution into the chamber. A culture medium exchange step of exchanging the culture medium in the chamber by sequentially discharging the liquid.

  Patent Document 3 discloses a permeability test apparatus that can vary the number of cells according to the number of samples. In this permeability test apparatus, a permeable membrane is accommodated in the interior, the interior is divided into an upper space and a lower space by the permeable membrane, a donor liquid is accommodated in the upper space, and a receiver liquid is accommodated in the lower space. And a cell base configured to detachably hold a plurality of preset cell units. This permeability test apparatus can mount an arbitrary number of cell units within a preset number range on the cell base.

Japanese Patent Application Laid-Open No. 2014-064507 JP 2011-135826 A JP-A-2015-219114

However, in the conventional observation apparatus, when the objective lens of the microscope is observed close to the observation apparatus, if the objective lens comes into contact with a member corresponding to the cover glass and is pressed even a little, the member is damaged. there were. Moreover, the conventional observation apparatus has no apparatus that can observe both the observation of the culture environment and the observation of the permeability test, and each observation apparatus has a single function.
Therefore, the present invention provides an observation apparatus in which even if an objective lens contacts a member corresponding to a cover glass and is pressed slightly, the member is not damaged. Furthermore, the present invention provides an observation apparatus that can be used not only for observation of culture environments but also for real-time observation of permeability tests.

In order to solve the above-described problem, a plate having a through-hole, a support portion having a diameter larger than the diameter of the through-hole at both ends of the through-hole, and a peripheral portion supported by at least one support portion and transmitting to the center portion A flexible connection member having a hole, a transmission member having an outer edge bonded around the transmission hole of the connection member, and a step portion formed by the support portion being recessed from the surface of the plate, An observation device is provided in which the height of the step portion is higher than the height of one or both of the connection member and the transmission member from the support portion.
According to this, the transmissive member is provided in the portion recessed by the step portion, and the transmissive member is attached to the plate by the flexible connecting member, so that even if the objective lens presses the transmissive member, the transmissive member Can be provided.

Furthermore, the diameter of the transmissive member may be equal to or smaller than the diameter of the through hole.
According to this, since the diameter of the transmissive member is equal to or smaller than the diameter of the through hole, the transmissive member can be provided on the back side of the connection member with respect to the objective lens, and the objective lens does not directly contact the transmissive member. It is possible to prevent the transmission member from being damaged. Further, even when the transmissive member is provided on the front side of the connection member, it is possible to prevent the transmission member from being damaged by utilizing the flexibility of the connection member when the diameter of the transmission member is equal to or less than the diameter of the through hole. it can.

Furthermore, the connection member may be bonded to both sides of the outer edge of the transmission member.
According to this, since the connection member is bonded to both surfaces of the outer edge of the transmission member, even if the objective lens strongly presses the connection member or the transmission member, the transmission member is prevented from falling off the connection member. Can do.

Further, the support portion supports the connection member at both ends of the through hole, and introduces a liquid into a chamber formed by the inner surface of the through hole and the connection member and / or the transmission member, and discharges the liquid to discharge the liquid. A road may be provided.
According to this, even if it is an inverted microscope or an erecting microscope, the observation apparatus which can prevent a permeable member from being damaged and can observe a culture environment can be provided.

Furthermore, the supporting portion that does not support the connecting member includes a sealing member that is disposed through the film to be observed disposed across the supporting portion, and a pressing member that presses the sealing member against the supporting portion. This may be a feature.
According to this, it is possible to provide an observation apparatus that uses an inverted microscope and can observe the permeability test while preventing the transmission member from being damaged.

Furthermore, the outer diameter of the sealing member may coincide with the outer diameter of the support portion, and the inner diameter of the sealing member may coincide with the inner diameter of the support portion.
According to this, an accurate permeability test can be performed.

  ADVANTAGE OF THE INVENTION According to this invention, even if an objective lens contacts the member corresponded to a cover glass and presses a little, the said observation apparatus with which the said member is not damaged can be provided. Furthermore, according to the present invention, it is possible to provide an observation apparatus that can be used not only for observation of the culture environment but also for real-time observation of the permeability test.

(A) Plan view of plate, (B) Cross section view of plate in AA section, (C) Plan view of connecting member and transmission member, (D) B of the observation apparatus of the first embodiment according to the present invention. Sectional drawing of the connection member in the B cross section, and a permeation | transmission member. BRIEF DESCRIPTION OF THE DRAWINGS (A) Top view of the observation apparatus of 1st Example which concerns on this invention, (B) Sectional drawing in CC cross section. (A) Plan view of connection member and transmission member, (B) Cross section of connection member and transmission member in cross section DD, (C) Cross section of observation device of modification of first embodiment according to present invention . (A) Plan view of plate, (B) Cross section in EE cross section, (C) Plan view of observation apparatus of second embodiment according to the present invention. Explanatory drawing when the objective lens of the inverted microscope approaches in the observation apparatus of 2nd Example which concerns on this invention. (A) Plan view of plate, (B) Cross section in FF cross section, (C) Plan view of observation apparatus of modification of second embodiment according to the present invention.

  The observation apparatus according to the present invention is an apparatus that can be used for both observation of floating cells in a culture environment and observation of a permeability test. An apparatus that can be used for observation of floating cells in a culture environment maintains an environment suitable for culturing living cells floating in a culture solution, and floats without removing floating cells to be observed from the culture vessel. It is an apparatus that makes it possible to observe cells under a microscope. An apparatus that can be used for observation of a permeability test is an apparatus that enables a sample to observe under a microscope a state in which a sample permeates and diffuses through a permeable membrane in the permeability test. In the permeability test, a permeable membrane such as skin is sandwiched between an upper container and a lower container, a sample such as a drug as a measurement target substance is supplied to the upper container, and a receptor liquid is supplied to the lower container. In this test, the diffusion rate is measured by measuring the concentration of the receptor fluid sample after a predetermined time.

Each example will be described below with reference to the drawings.
<First Example>
With reference to FIG. 1 and FIG. 2, the observation apparatus 100 in a present Example is demonstrated. The observation apparatus 100 includes a flat plate 10, two connection members 20 attached to the plate 10, and a transmission member 30 connected to each connection member 20. The plate 10 has a flat plate shape that can be easily attached to the stage of the microscope, and has a thickness that can include a chamber that can contain a culture environment.

  As shown in FIGS. 1A and 1B, the plate 10 includes a through hole 11, a support 12 having a diameter larger than the diameter R 1 of the through hole 11 at both ends BT of the through hole 11, and the surface of the plate 10. 14, and a step portion 13 formed by the depression of the support portion 12. The through hole 11 is a hole penetrating between the two surfaces 14 having the largest area of the plate 10. In the present embodiment, the through hole 11 has a circular shape with a diameter R1 in plan view, but is not limited thereto, and may be a rectangle with a side length R1.

  The support portion 12 is formed in a portion that is larger than the diameter R1 of the through hole 11 at both ends BT in the vicinity of the surface 14 of the through hole 11 and is recessed from the surface 14 in a plan view. The depth in which the support portion 12 is recessed is H1. That is, the step portion 13 is formed by the depression of the support portion 12 from the surface 14 of the plate 10, and the height of the step portion 13 is H1. The width of the support portion 12 in plan view, that is, how much the support portion 12 is larger than the diameter R <b> 1 of the through hole 11, is appropriately determined in relation to the connection strength with the connection member 20. The material of the plate 10 is not particularly limited, and may be metal, glass, hard synthetic resin, or the like.

  As shown in FIG. 1 (C), the connecting member 20 is composed of an annular peripheral portion 21 having a transmission hole 22 at the center, and is a member having water resistance, chemical resistance, flexibility, etc. It is made from a polymer component such as a resin molded into a thin film (film). The connecting member 20 is supported by the peripheral portion 21, more specifically, the outer edge portion of the peripheral portion 21 by the support portion 12, and is bonded and connected to the support portion 12 by an adhesive tape or the like. In addition, since the connection member 20 has flexibility, it is preferable that the adhesive tape is flexible to some extent even after being bonded in order to make it difficult to break at the bonding boundary.

  As shown in FIG. 1D, the transmission member 30 is a flat plate having a constant thickness and a circular shape in plan view, and is formed with a diameter larger than the diameter of the transmission hole 22 of the connection member 20. The peripheral edge portion 21 and the outer edge 31 thereof are bonded. The transmissive member 30 is made of a material that allows passage of a wavelength band used when observing floating cells. For example, when the transmission member 30 is used for observation by Raman spectroscopy, it is necessary that the transmission member 30 transmits a wavelength region used during observation and that the transmission member 30 does not generate fluorescence or Raman scattered light. For this reason, it is preferable that the transmissive member 30 is made of quartz. Moreover, when the transmissive member 30 is used for observation with visible light, the transmissive member 30 is preferably made of glass having transparency in the visible range. A material suitable for floating cells may be selected. In general, the plate thickness of the transmissive member 30 is 0.12 to 0.17 mm.

  As shown in FIGS. 1C and 1D, the observation apparatus 100 is first connected by bonding the outer edge 31 of the transmission member 30 to the inner edge of the peripheral portion 21 of the connection member 20 with an adhesive or the like. As shown, the outer edge portion of the peripheral edge portion 21 of the connecting member 20 integrated with the transmitting member 30 is bonded and connected to the two support portions 12 of the plate 10. The diameter of the transmissive member 30 is smaller than the diameter R1 of the through-hole 11 but has substantially the same diameter, and thus is disposed so as to be located on the inner side (center side of the plate 10) than the support portion 12. A cylindrical sealed space whose side surface is the inner side surface of the through-hole 11 of the plate 10 and whose upper and lower surfaces are constituted by the transmission member 30 constitutes a chamber C that can include a culture environment filled with the culture medium.

  The height H <b> 1 of the stepped portion 13 is higher than the height of the peripheral edge portion 21 of the connecting member 20 from the support portion 12. In other words, the height H <b> 1 of the step portion 13 is longer than the thickness of the peripheral portion 21 from the support portion 12, and the peripheral portion 21 is disposed at a position deeper than the surface 14 of the plate 10. When observing floating cells in the culture environment in the chamber C of the observation apparatus 100 with a microscope, the objective lens of the microscope is brought close to the surface 14 of the plate 10. At that time, depending on the operation of the microscope, the objective lens may come into contact with the surface 14 of the plate 10. In the conventional observation apparatus, the transmissive member may be damaged because the objective lens directly contacts the transmissive member corresponding to the cover glass. However, in the observation apparatus 100 according to the present invention, the transmissive member 30 is damaged. Since the peripheral portion 21 of the connecting member 20 is provided in a portion recessed from the surface 14 by the step portion 13, the objective lens does not come into contact therewith. Even if the objective lens has a small diameter so as to come into direct contact with the transmission member 30, the transmission member 30 is attached to the plate 10 by the flexible connection member 20. Even if the transmission member 30 is pressed, the pressure pressed by the connection member 20 being bent can be absorbed, and the transmission member 30 can be prevented from being damaged.

  Moreover, when the diameter of the transmissive member 30 is equal to or smaller than the diameter R1 of the through hole 11 as in this embodiment, the transmissive member 30 is provided on the back side of the connection member 20 (the center side of the plate 10) with respect to the objective lens. Therefore, the possibility that the objective lens directly contacts the transmission member 30 is reduced, and the transmission member 30 can be prevented from being damaged. Further, even when the transmission member 30 is provided on the front side of the connection member 20, the pressure pressed by the connection member 20 being bent is absorbed and transmitted by the diameter of the transmission member 30 being equal to or less than the diameter of the through hole 11. It is possible to prevent the member 30 from being damaged. When the transmission member is provided on the front side of the connection member 20 (the surface side of the plate 10), the height H1 of the step portion 13 is equal to the height of the connection member 20 from the support portion 12 and the height of the transmission member 30. It is preferably higher than the sum. Even if the transmissive member 30 is on the front side, it is provided in a portion that is recessed from the surface 14 by the step portion 13, so that the objective lens can be prevented from contacting.

  As described above, the support portion 12 in the observation apparatus 100 according to the present embodiment includes the connection member 20 supported by both ends BT of the through hole 11. Then, as shown in FIGS. 1B and 2B, the observation apparatus 100 is configured such that a liquid such as a culture solution is contained in a chamber C formed by the inner surface of the through hole 11 and the connection member 20 or the transmission member 30. An introduction path 15 for introducing the liquid and a discharge path 16 for discharging the liquid are provided. In such an observation apparatus 100, since the transmission member 30 is provided on both the upper surface side (upper side in the drawing) and the lower surface side (lower side in the drawing) of the plate 10, either an inverted microscope or an upright microscope can be used. The culture environment can be observed while preventing the permeable member 30 from being damaged.

  In addition, although the press member attachment hole 17 is shown in FIG. 1 and FIG. 2, when observing the culture environment which is the usage method of a present Example, the press member attachment hole 17 is not used.

<Modification of the first embodiment>
With reference to FIG. 3, an observation apparatus 100 ′ according to a modification of the present embodiment will be described. In addition, in order to avoid duplication description, the same code | symbol is attached | subjected to the same component and description is abbreviate | omitted. The observation apparatus 100 ′ includes a plate 10, two connection members 20 ′ attached to the plate 10, and a transmission member 30 connected to each connection member 20 ′. The connecting member 20 ′ is bonded to both surfaces of the outer edge 31 of the transmissive member 30.

  More specifically, the connection member 20 ′ includes a clamping part 23 ′ that protrudes from the peripheral part 21 ′ and sandwiches the outer edge 31 of the transmission member 30. The sandwiching portion 23 ′ is preferably made of the same member having water resistance, chemical resistance, flexibility and the like as the peripheral portion 21. Thus, even if the objective lens strongly presses the connection member 20 ′ and the transmission member 30 because the connection member 20 ′ is bonded to both surfaces of the outer edge 31 of the transmission member 30, the transmission member 30 is connected to the connection member 20. Can be prevented from falling off, and liquid leakage from the chamber C hardly occurs.

<Second Example>
With reference to FIG. 4, the observation apparatus 100A in the present embodiment will be described. In addition, in order to avoid duplication description, the same code | symbol is attached | subjected to the same component and description is abbreviate | omitted. The observation apparatus 100 </ b> A includes a flat plate 10, one connection member 20 attached to the lower end of the through hole 11 of the plate 10, a transmission member 30 connected to the connection member 20, and the connection member 20. An unsupported upper support portion 12 in the figure includes a sealing member 40 disposed through the permeable membrane MB to be observed disposed across the support portion 12 and the through hole 11. The permeable membrane MB is a membrane such as skin that is used in the permeability test and is an observation target. The permeable membrane MB is disposed over the entire support portion 12 having a diameter larger than that of the through hole 11 and is disposed so as to close the through hole 11 from the upper surface.

  The sealing member 40 is disposed on the support portion 12 via the permeable membrane MB, and presses and fixes the permeable membrane MB to the support portion 12. The sealing member 40 has a cylindrical side surface shape, and stores a sample such as a drug on the inner side surface of the cylinder. That is, the sealing member 40 stores a sample such as a medicine in a space (upper container) formed by a permeable membrane MB serving as a cylindrical bottom surface and an inner surface of the cylinder, and in that state, transmits the transmissive member with an inverted microscope from below. The permeability test can be observed by observing the receptor liquid in the lower surface of the permeable membrane MB and the chamber C (lower container) through 30.

  The thickness of the cylindrical side surface of the sealing member 40 is preferably equal to the radial width of the support portion 12. That is, it is preferable that the outer diameter of the sealing member 40 matches the outer diameter of the support portion 12, and the inner diameter of the sealing member 40 matches the inner diameter of the support portion 12. When the outer diameter of the sealing member 40 does not coincide with the outer diameter of the support portion 12, the sealing member 40 may shift within the diameter of the support portion 12, and the permeable membrane MB can be firmly fixed. It is not possible to conduct an accurate permeability test. In addition, when the inner diameter of the sealing member 40 that defines the transmission portion on the upper surface of the permeable membrane MB and the inner diameter of the support portion 12 that defines the transmission portion on the lower surface of the permeable membrane MB do not match, the transmission path in the permeable membrane MB is uniform. Therefore, an accurate permeability test cannot be performed. Therefore, the outer diameter of the sealing member 40 matches the outer diameter of the support portion 12, and the inner diameter of the sealing member 40 matches the inner diameter of the support portion 12, so that an accurate permeability test can be performed. It becomes.

  As shown in FIG. 5, the state in which a sample such as a medicine stored inside the sealing member 40 permeates the permeable membrane MB and diffuses into the receptor liquid in the chamber C is inverted through the lower permeable member 30. It can be observed with a microscope MC. Also in the observation apparatus 100A of the present embodiment, since the transmission member 30 is disposed at a position recessed from the surface 14, the possibility that the inverted microscope MC directly contacts the transmission member 30 is reduced, and the transmission member 30 is damaged. This can be prevented. Therefore, it is possible to provide an observation apparatus 100A that can use an inverted microscope to prevent the transmission member 30 from being damaged and perform observation of the permeability test in real time.

  As shown in this figure, the liquid FD is held between the transmission member 30 and the connection member 20 disposed at a position recessed from the surface 14 and the objective lens of the adjacent inverted microscope MC by utilizing capillary action. be able to. The liquid FD does not flow out due to surface tension unless the distance between the objective lens and the plate 10 is so large. Since the refractive index of the liquid FD is larger than the refractive index of air, the liquid FD is observed in a state where the liquid FD is held between the transmission member 30 and the like disposed at a position recessed from the surface 14 and the objective lens of the inverted microscope MC. By doing so, it becomes possible to observe with higher resolution.

<Modification of the second embodiment>
With reference to FIG. 6, the observation apparatus 100A ′ in the present embodiment will be described. In addition, in order to avoid duplication description, the same code | symbol is attached | subjected to the same component and description is abbreviate | omitted. The observation apparatus 100A ′ includes a flat plate 10, one connection member 20 attached to the lower end of the through hole 11 of the plate 10, a transmission member 30 connected to the connection member 20, and the connection member 20. In the upper support portion 12 in the figure that does not support the sealing member 40 disposed through the permeable membrane MB to be observed disposed across the support portion 12 and the through hole 11, and the sealing member 40 is supported. A pressing member 50 that presses against the portion 12, and a pressing member mounting rod 18 that is inserted into the pressing member mounting hole 17 and fixes the pressing member 50.

  The pressing member 50 includes an opening 51 having an inner diameter that is substantially the same as the inner diameter of the sealing member 40 at a position that matches the inner diameter of the sealing member 40. The sealing member 40 is pressed by the surface of the pressing member 50 on the plate 10 side, and the sealing member 40 is fixed. In the observation apparatus 100A of the second embodiment, the permeable membrane MB is pressed against the support portion 12 by the dead weight of the sealing member 40, but is more stable by pressing the permeable membrane MB via the sealing member 40 with the pressing member 50. Can be made. It is preferable that the pressing member mounting hole 17 has a female screw and the pressing member mounting rod 18 has a male screw and is fixed with a screw. In this way, it is possible to provide an observation apparatus that uses an inverted microscope and can observe the permeability test while preventing the transmission member 30 from being damaged.

  In addition, this invention is not limited to the illustrated Example, The implementation by the structure of the range which does not deviate from the content described in each item of a claim is possible. That is, although the present invention has been particularly illustrated and described with respect to particular embodiments, it should be understood that the present invention has been described in terms of quantity, quantity, and amount without departing from the scope and spirit of the present invention. In other detailed configurations, various modifications can be made by those skilled in the art.

DESCRIPTION OF SYMBOLS 100 Observation apparatus 10 Plate 11 Through-hole 12 Support part 13 Step part 14 Surface 15 Introduction path 16 Discharge path 17 Press member attachment hole 18 Press member attachment rod R1 Diameter of through hole BT Both ends of through hole H1 Height of step part C Chamber 20 connecting member 21 peripheral edge portion 22 permeation hole 23 clamping portion 30 permeation member 31 outer edge 40 sealing member 50 pressing member 51 opening MB film MC inverted microscope FD liquid

Claims (6)

A plate comprising a through hole and a support portion having a diameter larger than the diameter of the through hole at both ends of the through hole;
A flexible connecting member having a peripheral edge supported by at least one of the supporting parts and having a transmission hole in the central part;
A transmission member having an outer edge bonded around the transmission hole of the connection member;
A step formed by the support portion being recessed from the surface of the plate;
With
The height of the stepped portion is higher than the height of one or both of the connecting member and the transmitting member from the support portion,
Observation device.   The observation apparatus according to claim 1, wherein a diameter of the transmission member is equal to or less than a diameter of the through hole.   The observation apparatus according to claim 1, wherein the connection member is bonded to both surfaces of the outer edge of the transmission member. The support portion supports the connection member at both ends of the through hole,
2. An introduction path for introducing a liquid into a chamber formed by an inner surface of the through-hole and the connection member and / or the transmission member, and a discharge path for discharging the liquid are provided. 4. The observation device according to any one of 3. In a support portion that does not support the connection member, a sealing member that is disposed through a film to be observed disposed over the support portion;
A pressing member that presses the sealing member against the support;
The observation apparatus according to claim 1, further comprising: The outer diameter of the sealing member matches the outer diameter of the support portion,
The observation apparatus according to claim 5, wherein an inner diameter of the sealing member coincides with an inner diameter of the support portion.