JP2005338631A - Microscopic observation system and microscopic observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a clear image by suppressing the dynamic motion of a sample such as the cell of mammals including experimental small animals, organic tissue such as muscles or various internal organs such as the heart and the liver. <P>SOLUTION: The microscopic observation system is provided with the microscopic observation apparatus equipped with an objective unit 3 arranged adjacent to the sample A, and also, provided with a stabilizer 5 which is brought in tight contact with the sample A on the circumference B of an observation area with the microscopic observation apparatus. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microscope observation system and a microscope observation method.

In recent years, visualization of ion concentration, membrane potential, and the like with a fluorescent probe has been performed using an optical microscope. For example, biological function observation of nerve cells, organs, etc., in particular, dynamic behavior as a specimen is performed. It is like that.
As a device for observing such a dynamic behavior, a microphotographing device is known (for example, see Patent Document 1).
JP 2000-275539 A

  However, such a conventional microphotographing apparatus takes a photograph in accordance with the dynamic behavior of the specimen. However, while keeping the focusing distance of the camera constant, the dynamic behavior of the specimen is not limited. However, since the in-focus still state is selectively photographed, the obtained image is fragmented, and in particular, there is a problem that the state of the moving sample cannot be observed.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a microscopic image capturing apparatus that can obtain a clear image from a living body exhibiting dynamic behavior.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a microscope observation system that includes a microscope observation apparatus including an objective unit that is disposed close to a sample, and a stabilizer that is brought into close contact with the sample at least around an observation range of the microscope observation apparatus.
According to the present invention, since the behavior of the observation range disposed inside the stabilizer is suppressed even if the sample dynamically behaves by closely attaching the stabilizer to the sample, the relative distance between the objective unit and the sample Change is reduced, and a clear image without blurring can be obtained by the microscope observation apparatus.

In the said invention, it is preferable that the range where the said stabilizer is closely_contact | adhered is arrange | positioned in the radial direction outer side of the said objective unit.
By doing in this way, it can avoid that a stabilizer enters into the observation range of an objective unit, and can narrow the observation range.

Moreover, in the said invention, it is preferable to provide the adsorption | suction means to adsorb | suck the said stabilizer to the surface of a sample.
By doing so, the stabilizer is adsorbed on the surface of the sample by the operation of the adsorption means, and the dynamic behavior of the sample surface near the adsorption site is suppressed. Since the behavior of the sample surface can be suppressed without increasing the pressing pressure applied to the sample, the burden on the sample can be reduced.

Moreover, in the said invention, it is good also as a transparent member closely_contact | adhering to the sample distribute | arranged to the said observation range being provided in the said stabilizer.
By closely contacting the transparent member, the behavior of the sample surface arranged in the observation range can be directly suppressed, and the surface of the sample whose behavior is suppressed can be observed by the objective unit through the transparent member.

Moreover, in the said invention, the said stabilizer is good also as being formed in the ring shape surrounding the perimeter of an observation range.
The periphery of the observation range can be suppressed over the entire circumference by the stabilizer, and the dynamic behavior of the observation range can be effectively suppressed.

Moreover, in the said invention, the said stabilizer is good also as being formed in the shape surrounding the perimeter except for a part of periphery of an observation range.
The stabilizer can suppress the periphery of the observation range over almost the entire circumference, and can perform various treatments on the observation range from the outside using a treatment tool or the like through a portion that is not suppressed.

Furthermore, in the said invention, the said stabilizer may be a different body from the said microscope observation apparatus, and may be provided integrally with the said microscope observation apparatus.
By setting the stabilizer and the microscope observation device as separate bodies, the setting operation of the stabilizer on the sample surface can be performed separately from the setting of the observation conditions by the microscope observation device. That is, after performing various treatments on the sample such as injection of a fluorescent material in a state where the dynamic behavior of the sample surface is suppressed by the stabilizer, the focus adjustment with respect to the observation range of the microscope observation apparatus can be performed.
In addition, by integrating the stabilizer and the microscope observation apparatus, adjustments that are made each time are unnecessary, and observation can be performed quickly.

In the above invention, the stabilizer may be provided so that the relative position in the optical axis direction can be adjusted with respect to the objective unit, and may include a fixing means for fixing the relative position between the stabilizer and the objective unit.
Adjust the relative position of the stabilizer and the objective unit in the optical axis direction according to the type of the objective unit to which the stabilizer is attached, and operate the fixing means in this state to fix them together so that the stabilizer adheres to the sample. And the focus of the objective unit can be set to match the sample. As a result, the stabilizer can be integrated with the microscope observation apparatus, and quick observation can be performed.

Furthermore, in the above invention, the stabilizer is a first stabilizer provided integrally with the microscope observation device, and a second stabilizer provided around the first stabilizer and provided separately from the microscope observation device. And a stabilizing means for adsorbing the first and second stabilizers on the surface of the sample, and the second stabilizer has a lower adsorption force than that of the first stabilizer. Good.
By operating the adsorption means of the first and second stabilizers to adsorb both stabilizers to the sample, the dynamic behavior of the sample is suppressed, and a clear image without blurring can be obtained. In this case, since the second stabilizer disposed around the first stabilizer is adsorbed to the sample with a lower adsorption force than the first stabilizer, the binding force on the sample at the adsorption site by the second stabilizer is the first stabilizer. Set smaller. As a result, the restraining force increases sequentially from the outside toward the observation range, and the dynamic behavior of the observation range where fluctuations should be suppressed is sufficiently reduced, while avoiding excessive sample restraint in the vicinity. The burden on the sample can be reduced.

Moreover, in the said invention, the said stabilizer is arrange | positioned around the 1st stabilizer provided integrally with the said microscope observation apparatus, and the circumference | surroundings of this 1st stabilizer, and the 2nd provided separately from a microscope observation apparatus. A stabilizer, and the second stabilizer may be supported with lower rigidity than the first stabilizer.
In this way, as in the case of the above-described invention, the restraining force increases sequentially from the outside toward the observation range, and the dynamic behavior of the observation range where the fluctuation should be suppressed is sufficiently reduced, while the surrounding area is excessive. Therefore, it is possible to reduce the burden on the sample.

Furthermore, in the above invention, the stabilizer is a first stabilizer provided integrally with the microscope observation device, and a second stabilizer provided around the first stabilizer and provided separately from the microscope observation device. A stabilizer, and the second stabilizer may be made of an elastic material having rigidity lower than that of the first stabilizer.
In this way, as in the case of the above-described invention, the restraining force increases sequentially from the outside toward the observation range, and the dynamic behavior of the observation range where the fluctuation should be suppressed is sufficiently reduced, while the surrounding area is excessive. Therefore, it is possible to reduce the burden on the sample.

The present invention also provides a microscope observation method in which a stabilizer is brought into close contact with a sample at least around the observation range when performing microscope observation with an objective unit arranged close to the sample.
According to the present invention, since the microscope observation is performed in a state where the periphery of the observation range of the sample is suppressed by the stabilizer, it is possible to obtain a clear image in which the blur of the sample is suppressed.

  According to the present invention, since the microscope observation is performed in a state where the periphery of the observation range of the sample is suppressed by the stabilizer, it is possible to obtain a clear image in which the blur of the sample is suppressed. In addition, there is an effect that observation can be performed during dynamic behavior, instead of selecting and observing a stationary state.

Hereinafter, a microscope observation system and a microscope observation method according to an embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, the microscope observation system 1 according to the present embodiment is provided with a sample A of mammalian cells including experimental small animals, biological tissues such as muscles, or various organs such as heart and liver. A stage 2 to be placed, a microscope observation device 4 provided on the stage 2 and provided with an objective unit 3 arranged to face the sample A on the stage 2, and a stabilizer 5 arranged in the vicinity of the microscope observation device 4 It has.

The stage 2 includes an adjustment dial 6, and by operating the adjustment dial 6, the sample can be moved in two horizontal directions, for example, X and Y directions.
The microscope observation device 4 is attached to a support column 8 extending vertically from a base 7 by an elevating mechanism 9 so as to be movable up and down. By disposing the objective unit 3 vertically downward, the sample A on the stage 2 can be observed. Further, by operating the elevating mechanism 9, the objective unit 3 can be moved closer to and away from the sample A and the focus can be adjusted.

  The stabilizer 5 is also connected to the arm 5a, an arm 5a attached to a support column 10 extending vertically from the base 7 so as to be movable up and down by an elevating mechanism 11, a tip 5b disposed at the tip of the arm 5a, and the arm 5a. And a suction pump 13 for sucking air through the tube 12. As shown in FIG. 2, the distal end portion 5 b includes two finger portions 14 that are divided into two forks from the arm 5 a and are formed in a substantially U shape. The two finger portions 14 are arranged with a larger interval than the objective unit 3, and an observation range B (dashed line in the figure) by the objective unit 3 is arranged between the two finger portions 14. It has become.

  The arm 5a and the finger part 14 have a hollow structure. As shown in FIG. 3, a plurality of suction holes 14 a are provided on the lower surface of the finger portion 14. When the suction pump 13 is operated, the air sucked from the suction hole 14 a is sucked through the finger portion 14, the arm 5 a and the tube 12. That is, by operating the suction pump 13 in a state where the tip portion 5b is in contact with the sample A so as to block all the suction holes 14a on the lower surface of the finger portion 14, the inside of the finger portion 14, the arm 5a, and the tube 12 is large. The low pressure state is lower than the atmospheric pressure, and the tip 5b and the sample A are maintained in the adsorbed state.

The operation of the microscope observation system 1 according to this embodiment configured as described above will be described below.
In order to observe the sample A such as an experimental small animal using the microscope observation system 1 according to the present embodiment, first, as shown in FIG. After exposing A, the elevating mechanism 11 is operated to lower the stabilizer 5, and the tip portion 5 b of the stabilizer 5 is brought into close contact with the surface of the sample A. Next, the suction pump 13 is operated, the air in the tube 12 and the arm 5a is sucked, and the sample A is adsorbed by the suction hole 14a. In this state, the objective lens 3 of the microscope observation device 4 is brought close to the observation range B arranged between the two finger portions 14 of the stabilizer 5 to perform focusing, and the microscope observation device 4 performs observation.

  According to the microscope observation system 1 according to the present embodiment, the dynamic behavior of the surface of the sample A adsorbed on the stabilizer 5 is suppressed by the stabilizer 5. Therefore, the observation range B between the finger portions 14 in which the dynamic behavior is suppressed by the stabilizer 5 can be observed by the microscope observation device 4, and a clear image without blur can be acquired.

  In this case, in the microscope observation system 1 according to the present embodiment, the sample A is adsorbed by the suction hole 14a provided in the finger portion 14 of the tip portion 5b of the stabilizer 5 and its dynamic behavior is suppressed. On the other hand, there is an advantage that an excessive burden is not required. That is, as shown in FIG. 4 (a), when the sample A behaves dynamically within the range of fluctuation d up and down, the finger portion 14 of the stabilizer 5 is simply pressed against the surface of the sample A. In order to suppress the dynamic behavior of the sample A, it is necessary to push down the stabilizer 5 until the surface of the sample A is lowest due to the dynamic behavior, as shown in FIG. In this case, when the surface of the sample A rises to the highest position, it is necessary to suppress the total variation d, and an excessive burden is placed on the sample A. On the other hand, when the sample A is adsorbed by the stabilizer 5 as in the present invention, it is sufficient to hold the sample A by a half of the total fluctuation d of the sample A, as shown in FIG. Can be greatly reduced.

  Further, according to the microscope observation system 1 according to the present embodiment, the finger portion 14 of the stabilizer 5 is disposed on the sample A with a slightly larger interval than the objective unit 3, so that the finger portion 14 is disposed on the objective unit 3. Thus, it is possible to ensure a sufficient observation range B. In addition, since the fluctuation of the sample A is suppressed at a position sufficiently close to the observation range B, it is possible to effectively prevent image blurring.

  Further, according to the microscope observation system 1 according to the present embodiment, the distal end portion 5b is formed in a U shape and is disposed so as to surround the entire circumference except for a part of the observation range B. Various treatment tools such as a scalpel and an injection needle can be inserted from the gap provided between the two finger portions 14 while suppressing the dynamic behavior of the sample A. That is, the stabilizer 5 can be used not only for microscopic observation but also for suppressing dynamic behavior during treatment.

  Furthermore, according to the microscope observation system 1 according to the present embodiment, the stabilizer 5 that suppresses the dynamic behavior of the sample A is provided separately from the objective unit 3 of the microscope observation apparatus 4. The preparatory work for contact arrangement and the observation work by the microscope observation device 4 can be performed separately. Therefore, in the preparatory work, various treatments can be performed without the microscope observation device 4 being in the way.

In addition, the microscope observation system 1 which concerns on this invention is not limited to the said embodiment, The following structures are employable.
That is, in the above-described embodiment, the suction hole 14a is provided in the tip 5b of the stabilizer 5 so that the sample A is sucked and fixed to reduce the burden on the sample A. Instead, the movement of the sample A is changed. When the fluctuation d of the dynamic behavior is relatively small, the dynamic behavior of the sample A may be suppressed only by the pressing pressure without providing the suction hole 14a.

  Moreover, in the said embodiment, although the front-end | tip part 5b of the stabilizer 5 was formed in U shape, it replaces with this, and as shown in FIG. 5, the ring-shaped front-end | tip which surrounds the observation range B over a perimeter The part 5b may be adopted. Thereby, the dynamic behavior of the sample A in the observation range B can be more reliably suppressed.

  Further, as shown in FIG. 6, a glass plate 16 that is brought into close contact with the sample A in the observation range B may be arranged in the opening 15 arranged in the observation range B of the tip 5 b of the stabilizer 5. . By doing in this way, since the contact area of the stabilizer 5 and the sample A can be increased and the pressure added to the sample A can be reduced, the burden concerning the sample A can be reduced. Further, by closely contacting the glass plate 16 in the observation range B, it is possible to suppress fine dynamic behavior in the observation range B.

  In the above embodiment, the microscope observation device 4 and the stabilizer 5 are separately mounted on the separate support columns 8 and 10 so as to be movable up and down. However, as shown in FIGS. May be configured integrally with the objective unit 3. 7 shows a case where the glass plate 19 is not provided in the opening 18 of the distal end portion 17a, and FIG. 8 shows a case where the glass plate 19 is provided.

  In these cases, the stabilizer 17 is fitted to a cylindrical surface constituting the outer surface of the objective unit 3 so as to be slidable along the axial direction, and a distal end portion having a suction hole 20 disposed at the distal end thereof. The cylindrical portion 17b is slid in the axial direction with respect to the objective unit 3 according to the focal position of the objective unit 3, and is fixed to the objective unit 3 by fixing the set screw 21. It is preferable. The code | symbol 22 is a tube connected to the suction pump for attracting | sucking the inside of the front-end | tip part 17a to negative pressure.

  With this configuration, the positional relationship between the objective unit 3 and the stabilizer 17 can be adjusted in advance in accordance with the focal position of the objective unit 3, and the sample A is adsorbed on the tip 17 a of the stabilizer 17. At the same time, since the focal position of the objective unit 3 is made to coincide with the sample A, it is possible to save the trouble of performing the focal position adjustment every observation.

Next, a microscope observation system 30 according to the second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation system 1 according to the first embodiment described above, and the description thereof is omitted.

  As shown in FIG. 9, the microscope observation system 30 according to the present embodiment includes a stabilizer 31 that is fixed to the objective unit 3 of the microscope observation apparatus 4, and is slidable in the axial direction on the outer surface of the objective unit 3. A first bracket 32 having a cylindrical portion 32a fitted to the first bracket 32; a second bracket 33 having a cylindrical portion 33a fitted to the outer surface of the first bracket 32 so as to be slidable in the axial direction; And a compression spring 34 disposed between the second brackets 32 and 33.

  The tip portions 32b and 33b of the first and second brackets 32 and 33 are each formed in a ring shape, and suction holes 32c and 33c are provided on the tip surfaces brought into contact with the sample A, respectively. The negative pressure is supplied to the suction holes 32c and 33c by the same suction pump 13. That is, the adsorption force with respect to the sample A by the distal end portion 32b of the first bracket 32 and the distal end portion 33b of the second bracket 33 is set to be substantially the same.

The first bracket 32 has a set screw 35 inserted in a screw hole penetrating in the radial direction. The tip of the set screw 35 is arranged so as to be able to press the outer surface of the objective unit 3, and the first bracket 32 is fixed to an arbitrary position in the axial direction of the objective unit 3 by fastening the set screw 35. Can be done.
The compression spring 34 is elastically deformed to the extent that a predetermined pressing force is generated when the front end surfaces of the front end portions 32b and 33b of the first bracket 32 and the second bracket 33 are substantially flush with each other. It has become.

The operation of the microscope observation system 30 according to this embodiment configured as described above will be described below.
In order to observe the sample A by the microscope observation system 30 according to the present embodiment, first, the first bracket 32 is fitted to the outer surface of the objective unit 3, and the first bracket is matched with the focal position of the objective unit 3. 32 is slid in the axial direction, and the first bracket 32 is integrally fixed to the objective unit 3 by a set screw 35.

  Next, the front end portions 32 b and 33 b of the first bracket 32 and the second bracket 33 thus attached to the objective unit 3 are brought into close contact with the surface of the sample A. In this state, the suction pump 13 is operated to adsorb the surface of the sample A by the tip portions 32b and 33b of the first and second brackets 32 and 33. Since the tip 32b of the first bracket 32 is set in accordance with the focal position of the objective unit 3, when the first bracket 32 is brought into close contact with the surface of the sample A, the focal position of the objective unit 3 is set to the sample A. Can be matched with high accuracy.

Since the surface of the sample A is adsorbed by the first bracket 32 fixed to the objective unit 3, even if the dynamic behavior of the sample A occurs, the image of the sample A is blurred in the microscope observation device 4. It will be acquired without.
In this case, according to the microscope observation system 30 according to the present embodiment, the first bracket 32 and the second bracket 33 are in close contact with the sample A by substantially the same adsorption force. The bracket 32 is fixed to the objective unit 3, while the second bracket 33 disposed around the bracket 32 is compressed by a compression spring 34 disposed between the first bracket 32 and the first bracket 32. Supported by a rigidity lower than 32. Therefore, when the dynamic behavior occurs in the sample A, the first bracket 32 almost completely suppresses the dynamic behavior of the sample A by the tip portion 32b, whereas the second bracket 33 has the compression spring 34. Is elastically deformed to allow some dynamic behavior. As a result, the dynamic behavior of the sample A is constrained by the restraining force that gradually increases from the outside to the inside of the observation range B, and suddenly by the single tip 5b as in the first embodiment. There is an advantage that the burden on the sample A can be reduced as compared with the case of restraining to.

  In the present embodiment, the first bracket 32 and the second bracket 33 are connected by the compression spring 34, and the sample A is adsorbed by substantially the same adsorbing force. As shown in FIG. 10, ring-shaped tip portions 36a and 36b are integrally formed and fixedly supported on the outer surface of the objective unit 3, while the regulators 37 and 38 vary the suction force at the tip portions 36a and 36b. You may decide. In other words, it is preferable to set the suction force at the first tip portion 36a disposed on the inside large and the suction force at the second tip portion 36b disposed on the outside small.

  With this configuration, the restraining force of the dynamic behavior of the sample A by the inner first tip portion 36a is increased, while the restraining force of the dynamic behavior of the sample A by the outer second tip portion 36b is increased. By making it relatively small and slightly allowing dynamic behavior, the restraining force can be increased sequentially toward the inner observation range B as described above. As a result, there is an effect that the stress applied to the sample A due to a sudden change in the binding force can be reduced and the burden on the sample A can be reduced.

  Further, as shown in FIG. 10, the inner first tip portion 39a is hard, and the outer second tip portion 39b is made of a relatively flexible material, for example, rubber, and the sample is obtained with substantially the same adsorption force. A may be adsorbed. In this way, the second tip portion 39b can be elastically deformed following the dynamic behavior of the sample A, and the second tip portion 39b allows a slight amount of dynamic behavior while being more inward. In the first tip portion 39a arranged in the, the same effect as described above can be obtained by firmly suppressing the dynamic behavior.

  Note that in the microscope observation system 1 according to the first embodiment, the sample A is adsorbed by the stabilizer 5 and the objective unit 3 is arranged at a predetermined interval from the sample A. Instead of this, the stabilizer 5 is used. While the sample A is sucked, the tip surface of the objective unit 3 may be pressed against the sample A. If the suction force by the stabilizer 5 is adjusted, the restraining force can be sequentially increased from the outside toward the observation range B as described above.

1 is an overall configuration diagram showing a microscope observation system according to a first embodiment of the present invention. It is a perspective view which shows the relationship between the objective unit and stabilizer in the microscope observation system of FIG. It is a bottom view which shows the structure of the front-end | tip part of the stabilizer of FIG. It is a figure explaining the suppression state of the dynamic behavior of the sample by the stabilizer of FIG. It is a bottom view which shows the 1st modification of the stabilizer of FIG. It is a longitudinal cross-sectional view which shows the 2nd modification of the stabilizer of FIG. It is a longitudinal cross-sectional view which shows the 3rd modification of the stabilizer of FIG. It is a longitudinal cross-sectional view which shows the 4th modification of the stabilizer of FIG. It is a longitudinal cross-sectional view which shows the relationship between the objective unit of the microscope observation system which concerns on the 2nd Embodiment of this invention, and a stabilizer. It is a longitudinal cross-sectional view which shows the 1st modification of the stabilizer of FIG. It is a longitudinal cross-sectional view which shows the 2nd modification of the stabilizer of FIG.

Explanation of symbols

A Sample B Observation range 1,30 Microscope observation system 3 Objective unit 4 Microscope observation device 5, 17, 31, 36, 39 Stabilizer 12 Tube (adsorption means)
13 Suction pump (adsorption means)
14a, 32c, 33c Suction hole (adsorption means)
16, 19 Glass plate (transparent member)
21, 35 Set screw (fixing means)
32b, 36a, 39a Tip (first stabilizer)
33b, 36b, 39b Tip (second stabilizer)

Claims (13)

A microscope observation apparatus including an objective unit arranged close to the sample;
A microscope observation system comprising: a stabilizer that is brought into close contact with a sample at least around an observation range of the microscope observation apparatus.   The microscope observation system according to claim 1, wherein a range in which the stabilizer is in close contact is disposed radially outward of the objective unit.   The microscope observation system according to claim 1, further comprising adsorption means for adsorbing the stabilizer to a surface of a sample.   The microscope observation system according to any one of claims 1 to 3, wherein the stabilizer is provided with a transparent member that is in close contact with a sample disposed in the observation range.   The microscope observation system according to any one of claims 1 to 4, wherein the stabilizer is formed in a ring shape surrounding the entire circumference of the observation range.   The microscope observation system according to any one of claims 1 to 4, wherein the stabilizer is formed in a shape surrounding an entire circumference except a part of the periphery of the observation range.   The microscope observation system according to any one of claims 1 to 6, wherein the stabilizer is separate from the microscope observation apparatus.   The microscope observation system according to any one of claims 1 to 6, wherein the stabilizer is provided integrally with the microscope observation apparatus. The stabilizer is provided so that the relative position in the optical axis direction can be adjusted with respect to the objective unit, and
The microscope observation system according to claim 8, further comprising a fixing unit that fixes a relative position between the stabilizer and the objective unit. The stabilizer includes a first stabilizer provided integrally with the microscope observation device, and a second stabilizer provided around the first stabilizer and provided separately from the microscope observation device,
With an adsorbing means for adsorbing the first and second stabilizers on the surface of the sample,
The microscope observation system according to claim 1, wherein an adsorption force of the second stabilizer is lower than an adsorption force of the first stabilizer. The stabilizer includes a first stabilizer provided integrally with the microscope observation device, and a second stabilizer provided around the first stabilizer and provided separately from the microscope observation device,
The microscope observation system according to claim 1, wherein the second stabilizer is supported with lower rigidity than the first stabilizer. The stabilizer includes a first stabilizer provided integrally with the microscope observation device, and a second stabilizer provided around the first stabilizer and provided separately from the microscope observation device,
The microscope observation system according to claim 1, wherein the second stabilizer is made of an elastic material having rigidity lower than that of the first stabilizer.   A microscope observation method in which a stabilizer is brought into close contact with a sample at least around the observation range when the microscope is observed with an objective unit arranged close to the sample.

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