JP2018077386A - Stabilizer for microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stabilizer for a microscope observation, capable of stably absorbing a moving observation object to enable a real-time observation.SOLUTION: A stabilizer for a microscope includes a base part 21, and at least one adsorption hole 41 provided at the base part 21 and having a first opening 42 opened at an observation object side and a second opening 43 opened at an opposite side to the first opening 42. The opening width of the first opening 42 is larger than the opening width of the second opening 42.SELECTED DRAWING: Figure 4

Description

  The present disclosure relates to a microscope stabilizer.

  In recent years, the emergence of a multiphoton excitation microscope capable of observing deep tissue in a minimally invasive manner has enabled “intravital imaging” for observing organs while keeping experimental animals alive under anesthesia. Intravital imaging is a technique for observing a tissue in a living state, and can therefore observe cell dynamics in a physiological tissue in which circulating blood flow is maintained.

  In recent years, observation of “moving” organs such as the heart has been attempted. However, for example, when observing the heart, it is necessary to keep the distance between the heart surface and the objective lens of the microscope constant during the pulsation of the heart, and it is difficult to observe in real time during the pulsation of the heart. Therefore, in previous studies, the heart removed from the living body was observed as a tissue specimen.

  On the other hand, there have been reported examples of attempts at intravital imaging of moving organs (Patent Document 1 and Non-Patent Document 1). In these documents, it is disclosed that a stabilizer is used to suppress the movement of the heart.

JP 2005-338631 A

Vinegoni, C. et al., “Imaging the beating heart in the mouse using intravital microscopy techniques”, Nat Protoc. 10, 1802-19, 2015

  In Non-Patent Document 1, an attempt is made to intracardial imaging of the heart using a stabilizer having a columnar suction hole. However, the stabilizer of Non-Patent Document 1 cannot suppress the movement of the heart. In Non-Patent Document 1, a plurality of images are acquired from a moving heart, and the images are reconstructed by connecting these images. Therefore, the technique of Non-Patent Document 1 cannot observe the moving heart in real time.

  In addition, regarding the technique of Patent Document 1, the inventors of the present application have attempted intratracheal imaging of the heart using the same configuration, but the heart tissue could not be sufficiently adsorbed. As a result, with the technique of Patent Document 1, the distance between the tissue and the objective lens fluctuates, and the moving heart cannot be observed in real time.

  The present disclosure provides a microscope observation stabilizer that stably adsorbs a moving observation object and enables real-time observation.

  The inventors of the present application have found that the above problems can be solved as a result of intensive studies on the opening shape of the adsorption holes of the stabilizer.

  The present application includes a plurality of means for solving the above-mentioned problems. To give an example, a microscope stabilizer, which is a base, a first opening provided on the base and opening on the observation object side, and And at least one suction hole having a second opening that opens on the opposite side of the first opening, and the opening width of the first opening is larger than the opening width of the second opening. A stabilizer is provided.

  According to the present disclosure, it is possible to stably adsorb a moving observation object and perform observation in real time. Further features related to the present disclosure will become apparent from the description of the present specification and the accompanying drawings. Further, problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

It is a whole lineblock diagram showing an example of a microscope concerning one embodiment of this indication. It is a schematic plan view and a sectional view of a stabilizer concerning one embodiment of this indication. It is a schematic sectional drawing of the stabilizer concerning one embodiment of this indication. FIG. 3 is an enlarged plan view and an enlarged sectional view of a dotted line portion A in FIG. 2. It is a figure explaining the adsorption state at the time of using the stabilizer concerning one embodiment of this indication. It is a figure explaining the adsorption | suction state at the time of using the stabilizer of a comparative example.

  Hereinafter, an embodiment of the present disclosure will be described with reference to the drawings. 1 to 5 are diagrams for explaining an embodiment according to the present disclosure. In the drawings attached to the present specification, for the sake of illustration and ease of understanding, the scale and the vertical / horizontal dimension ratio may be appropriately changed from those of the actual ones.

  In addition, as used in this specification, the shape and geometric conditions and the degree thereof are specified, for example, terms such as “parallel”, “orthogonal”, “vertical”, “same”, length and angle values, etc. Should be construed to include the extent to which a similar function can be expected without being bound to a strict meaning.

  In the present specification and the like, a numerical range expressed using “to” means a range including each of the numerical values described before and after “to” as a lower limit value and an upper limit value.

  FIG. 1 is an overall configuration diagram illustrating an example of a microscope according to an embodiment of the present disclosure. A multiphoton excitation microscope will be described as an example of a microscope, but the microscope observation stabilizer described below is applicable to other upright microscopes.

  The multiphoton excitation microscope 10 includes a stage 12 on which an observation object 11 is placed, a microscope unit 14 that includes an objective lens 13 that is arranged to face the observation object 11 on the stage 12, and the observation object 11 on the stage 12. The stabilizer 15 to hold | maintain is provided.

  The observation object 11 is, for example, a mammal. In particular, the stabilizer 15 is suitable for holding a moving organ (for example, a heart) of a mammal or a moving biological tissue. Mammals refer to warm-blooded vertebrates, for example, primates such as humans and monkeys, rodents such as mice, rats and rabbits, pets such as dogs and cats, and domestic animals such as cattle, horses and pigs. Is mentioned.

  The stage 12 may be a fixed stage or a movable stage. The stage 12 may be configured to move the observation object 11 in at least one direction among X, Y, and Z, for example.

  The microscope unit 14 irradiates the observation object 11 with excitation light and detects multiphoton fluorescence generated from the observation object 11. Since the general structure of a multiphoton excitation microscope is well-known, detailed description is abbreviate | omitted. For example, the microscope unit 14 includes a pulse laser light source capable of emitting pulse laser light, a first optical system for guiding the pulse laser light to the observation object 11, and pulse laser light at a predetermined position of the observation object 11. While collecting, the objective lens 13 which condenses the fluorescence emitted when the fluorescent substance is excited in the observation object 11, the second optical system for branching the emitted fluorescence for each wavelength, and the fluorescence are detected. And a photodetector. The photodetector may be, for example, a detector such as a CCD camera, a CMOS camera, or one or more photomultiplier tubes. Note that the fluorescence may be directly observed through an eyepiece or the like.

  The stabilizer 15 is connected to the suction pump 17 through the pipe 16. By operating the suction pump 17 in a state where the stabilizer 15 is in contact with the observation object 11, the space between the stabilizer 15 and the observation object 11 becomes a low-pressure state lower than the atmospheric pressure, and the stabilizer 15 and the observation object. 11 is maintained in the adsorption state.

  FIG. 2 is a schematic plan view and a cross-sectional view of the stabilizer 15 according to an embodiment of the present disclosure. The stabilizer 15 includes a base portion 21, a first support portion 22 and a second support portion 23 that extend in the horizontal direction from the base portion 21, and a protruding portion 24 that protrudes from the bottom surface of the base portion 21 toward the observation object 11. The base 21 includes a first recess 25 in which the objective lens 13 is disposed, and a second recess 26 that is provided on the bottom surface 25a of the first recess 25 and in which the transparent member 31 (see FIG. 3) is disposed.

  The first support portion 22 includes two arm portions 22a and 22b. As shown in FIGS. 1 and 2, a screw rod 18 is attached to the stage 12, and the screw rod 18 is disposed between the two arm portions 22a and 22b. Two nuts 19 and 20 are attached to the screw rod 18 and are arranged above and below the arm portions 22a and 22b. By sandwiching the arm portions 22a and 22b from above and below by the two nuts 19 and 20, the position of the stabilizer 15 in the X, Y, and Z directions can be fixed. Note that the second support portion 23 includes two arm portions 23 a and 23 b, similar to the first support portion 22. Since the fixing method using the two arm portions 23a and 23b is the same as that of the first support portion 22, the description thereof is omitted.

  FIG. 3 is a schematic cross-sectional view of the stabilizer 15 according to an embodiment of the present disclosure, and shows the state of the stabilizer 15 during observation. The objective lens 13 is disposed in the first recess 25. According to this configuration, the objective lens 13 can be disposed close to the observation object 11 by providing the first recess 25 in the base 21. A transparent member 31 is disposed in the second recess 26. The transparent member 31 is a glass substrate, for example. The height of the second recess 26 is substantially the same as the thickness of the transparent member 31. Therefore, when the transparent member 31 is disposed in the second recess 26, the surface of the transparent member 31 on the objective lens 13 side and the bottom surface 25a of the first recess 25 are substantially free of steps and are on the same plane.

  4 is an enlarged plan view and an enlarged sectional view of a dotted line portion A in FIG. The base 21 has an adsorption hole 41. The suction hole 41 is formed so as to penetrate the base portion 21 and the protruding portion 24. More specifically, the suction hole 41 is formed so as to penetrate the bottom surface 26 a of the second recess 26 of the base portion 21 and the protruding portion 24. The suction hole 41 has a first opening 42 that opens on the observation object 11 side, and a second opening 43 that opens on the opposite side (object lens 13 side) to the first opening 42. The first opening 42 is provided on the surface 24 a of the protrusion 24 on the observation object 11 side, and the second opening 43 is provided on the bottom surface 26 a of the second recess 26.

  The suction hole 41 forms a truncated cone space between the observation object 11 and the base 21. The protrusion 24 is provided with a pipe hole 24b, and the pipe 16 is connected to the suction hole 41 via the pipe hole 24b. A plurality of pipes may be connected to the suction hole 41 via the protrusion 24. According to this configuration, by operating the suction pump 17, the space surrounded by the transparent member 31, the suction hole 41, and the observation object 11 disposed in the second recess 26 is in a low pressure state lower than the atmospheric pressure. Can be. Thereby, the observation object 11 can be adsorbed to the adsorption hole 41.

  As shown in FIG. 4, it is preferable that the figure of the cross section of the adsorption hole 41 (the cross section cut in the height direction (Z direction) of the base portion 21) is a trapezoid. According to this configuration, the observation object 11 is easily attracted to the transparent member 31 side. In addition, the cross-sectional figure of the suction hole 41 is not limited to this. For example, the wall surface 41a of the suction hole 41 may have a curved cross section.

  As shown in FIG. 4, the figure formed by the outer edges of the first opening 42 and the second opening 43 (hereinafter referred to as “outer edge figure”) is circular. The outer edge figure of the first opening 42 and the second opening 43 is not particularly limited, and may be a circle (including a circle, a substantially circle, an ellipse, etc.), or a polygon such as a triangle or a rectangle. Good. The outer edge graphic is preferably circular. The outer edge graphic of the first opening 42 and the outer edge graphic of the second opening 43 may be different.

As a feature of the present embodiment, the opening width W ₁ of the first opening 42 of the suction hole 41 is larger than the opening width W ₂ of the second opening 43. When the outer edge graphics of the first opening 42 and the second opening 43 are circular, the “opening width” refers to the diameter of a circle. When the outer edge graphic of the first opening 42 and the second opening 43 is a polygon such as a triangle or a quadrangle, the “opening width” refers to the maximum distance between any two points on the outer edge of the graphic (for example, For rectangles, it is the length of the diagonal line).

  Further, as shown in FIG. 4, the area of the outer edge graphic of the first opening 42 is configured to be larger than the area of the outer edge graphic of the second opening 43 in the top view.

Opening width _{W 2} of the second opening 43, 0 preferably is 0.4 times the opening width _{W 1} of the first opening 42, and more preferably, the first opening 42 of the opening width _{W 1} .5 times or more.

  Further, when the angle formed by the wall surface 41a of the suction hole 41 shown in FIG. 4 and the opening surface of the first opening 42 (bottom surface of the truncated cone) is θ, tan θ is preferably 2 or less, more preferably Is 1.5 or less. Further, tan θ is preferably 0.5 or more, and more preferably 0.7 or more.

The height of the suction hole 41 (the height H ₁ of the bottom surface of the base 21 + the height H ₂ of the protrusion 24) is preferably as small as possible in order to make the observation object 11 easily attracted to the transparent member 31. The height of the suction hole 41 is preferably 5 mm or less, and more preferably 3 mm or less.

  The effect of this embodiment will be described. FIG. 5 is a diagram illustrating an adsorption state when the stabilizer 15 according to an embodiment of the present disclosure is used. In the case of the stabilizer 15 of the present embodiment, the moving observation object 11 (rat heart) is drawn to the position of the second opening 43 and is sufficiently adsorbed by the transparent member 31 (the state of FIG. 5B). . When the stabilizer 15 of the present embodiment is used, the distance between the heart tissue and the objective lens 13 can be kept constant, and the moving observation object 11 can be observed in real time.

FIG. 6 is a diagram illustrating an adsorption state when the stabilizer of the comparative example is used. The stabilizer 60 of the comparative example has a suction hole 61 that forms a cylindrical space. That is, in the stabilizer 60, the opening width D ₁ of the first opening 62 which opens to the observation object 11 side, the opening width D ₂ of the second opening 63 which opens into the objective lens 13 side is the same width. In the stabilizer 60 of the comparative example, since the hardness of the tissue is superior to the suction force, the observation object 11 cannot be drawn to the position of the second opening 63, and the adsorption to the transparent member 31 is insufficient (FIG. 6). (State of (b)). As a result, the distance between the heart tissue and the objective lens 13 fluctuated, and real-time observation was not possible.

[Experimental Example 1]
Next, Experimental Example 1 will be described. A GFP-rat under general anesthesia was tracheal intubated and thoracotomy was performed to expose the heart surface alive. In the stabilizer 15 used in this experimental example, W ₁ is 10 mm and W ₂ is 6 mm. Further, the height H ₁ of the bottom surface of the base portion 21 is 1.4 mm, and the height H ₂ of the protruding portion 24 is 1.6 mm. That is, the height (H ₁ + H ₂ ) of the suction hole 41 is 3 mm. there were. A 1 mm diameter brass pipe was connected to the suction hole 41 through the pipe hole 24b.

  The first opening 42 of the stabilizer 15 was fixed to the stage 12 while being pressed against the surface of the rat heart. Thereafter, by operating the suction pump 17, the transparent member 31 (cover glass) adsorbed the heart surface. In the adsorbed state, the heart was imaged in an ultrafast resonant mode using a two-photon microscope (A1R MP +, Nikon) (25 × immersion objective lens, CFI Apo 25 × W MP, Nikon). As a result, it was possible to observe in real time how the heart tissue having a sarcomere structure beats.

[Experiment 2]
Experimental example 2 will be described. The coronary artery (left anterior descending branch) was ligated to create an ischemic model rat. After 5 minutes, blood flow was resumed to obtain ischemia-reperfusion model rats. Using this rat, cells in the heart tissue were observed in real time using the same stabilizer 15 and method as in Experimental Example 1. As a result, it was observed that the cells in the heart tissue died over time, and the fluorescence intensity of GFP decreased. Furthermore, it was possible to observe how the leukocytes aggregated.

  According to the present embodiment, the observation area of the moving observation object 11 can be sufficiently adsorbed to the transparent member 31 side, and the adsorption state can be maintained, and the distance between the observation object 11 and the objective lens 13 at the time of observation. Can be kept constant. Thereby, it becomes possible to observe the behavior of the observation object 11 stably in real time. This would enable, for example, intra-cardiac imaging of cells in the heart, leading to pharmacokinetic analysis of new therapeutic agents and elucidation of the mechanism of arrhythmia.

  Note that the present disclosure is not limited to the above-described embodiment, and includes other various modifications. For example, the above-described embodiments have been described in detail in order to easily understand the present disclosure, and are not necessarily limited to those having all the configurations described. In addition, a part of the configuration of an embodiment may be replaced with the configuration of another embodiment, and the configuration of another embodiment may be added to the configuration of an embodiment. In addition, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

  In the above-described embodiment, the configuration in which the base 21 includes one suction hole 41 has been described. However, two or more suction holes may be provided in the base 21.

  In the above-described embodiment, the base 21 includes the first concave portion 25 in order to bring the objective lens 13 closer to the observation object 11, but is not limited thereto. The base 21 is not necessarily provided with the first recess 25, and the base 21 of the stabilizer 15 only needs to have at least an adsorption hole for adsorbing the observation object 11.

The numerical values of W ₁ , W ₂ , H ₁ , and H _{2 in} the above experimental example are examples, and the present invention is not limited to this. These numerical values may be set as appropriate according to the size of the observation object 11 and the like.

DESCRIPTION OF SYMBOLS 10 ... Multiphoton excitation microscope 11 ... Observation object 12 ... Stage 13 ... Objective lens 14 ... Microscope unit 15 ... Stabilizer 16 ... Pipe 17 ... Suction pump 18 ... Screw rod 19 ... Nut 21 ... Base 22 ... 1st support part 23 ... 2nd support part 24 ... protrusion 25 ... 1st recessed part 26 ... 2nd recessed part 31 ... transparent member 41 ... adsorption hole 42 ... 1st opening part 43 ... 2nd opening part

Claims (10)

A microscope stabilizer,
The base,
A first opening provided in the base and having an opening on the observation object side and at least one suction hole having a second opening opening on the opposite side of the first opening;
The microscope stabilizer, wherein an opening width of the first opening is larger than an opening width of the second opening.   The microscope stabilizer according to claim 1, wherein an area of a figure formed by an outer edge of the first opening is larger than an area of a figure formed by an outer edge of the second opening.   The microscope stabilizer according to claim 1, wherein the suction hole has a trapezoidal cross section.   4. The microscope stabilizer according to claim 1, wherein the opening width of the second opening is 0.5 times or more of the opening width of the first opening. 5.   5. The microscope stabilizer according to claim 1, wherein tan θ is 1.5 or less, where θ is an angle formed by the wall surface of the suction hole and the opening surface of the first opening. .   The microscope stabilizer according to claim 5, wherein the tan θ is 0.5 or more.   The microscope stabilizer according to claim 1, wherein a height of the suction hole is 3 mm or less.   The microscope stabilizer according to any one of claims 1 to 7, wherein the graphic formed by the outer edge of the first opening and the graphic formed by the outer edge of the second opening are circular. The base has a first recess in which the objective lens of the microscope is disposed, and a second recess in which a transparent member is disposed on the bottom surface of the first recess,
The microscope stabilizer according to any one of claims 1 to 8, wherein the second opening of the suction hole is provided in the second recess. Further comprising a protrusion protruding from the bottom surface of the base,
The microscope stabilizer according to any one of claims 1 to 9, wherein the suction hole is formed so as to penetrate the base and the protrusion.

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