JP2010026218A - Liquid supply device and microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To allow a user to observe an object for many hours without deteriorating optical performance of liquid immersion objective lens. <P>SOLUTION: A detection part composed of an optical fiber 81 and a light-receiving sensor 82 detects amount of remaining liquid immersion water 71 filled between the objective lens 22A for liquid immersion and a bottom part transparent member 2A. A reservoir 83 supplies the water 72 collected for supply between the objective lens 22A for liquid immersion and the bottom part transparent member 2A when amount of remaining liquid immersion water 71 is insufficient. Consequently, a liquid immersion part at a tip of the objective lens 22A for liquid immersion is filled with sufficient amount of liquid immersion water 71 always so that optical performance of the objective lens 22A for liquid immersion is not deteriorated even when the user observes an object for many hours by this microscope. This invention can be applied to the liquid supply device used in the microscope having the liquid immersion objective lens. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a liquid supply apparatus and a microscope, and more particularly to a liquid supply apparatus and a microscope that are suitable for long-time observation.

  Conventionally, observation with a microscope using an immersion objective lens has been performed. When this immersion objective lens is used, by filling the space between the immersion objective lens and the specimen with liquid (immersion), the numerical aperture of the optical system is increased, and high resolution and brightness are improved.

This type of immersion objective lens is disclosed in, for example, Patent Documents 1 and 2.
Utility model registration No. 2603486 JP 2004-70307 A

  However, in Patent Document 1, the temperature of the specimen is controlled by circulating heat-retaining water. However, when an immersion objective lens is used in time-lapse image capturing of live cells over several hours, this immersion objective lens is used. Since there is only a distance of about 0.2 mm between the cover glass and the cover glass, the immersion water evaporates in about one hour in a working environment at room temperature of about 25 ° C., and the optical performance of the objective lens is significantly deteriorated.

  In Patent Document 2, a fixed amount of temperature-controlled immersion medium is supplied to the tip of the immersion objective lens, but the supplied immersion spills from the tip of the immersion objective lens. Thus, there is a possibility that the image will be disturbed due to the refractive reflection of the illumination light to the water droplet, which may be an obstacle to stable image recording.

  The present invention has been made in view of such circumstances, and makes it possible to perform observation for a long time without deteriorating the optical performance of the immersion objective lens.

  The liquid supply apparatus of the present invention is a liquid supply apparatus used in a microscope having an immersion objective lens, and detects the remaining amount of immersion liquid filled between the immersion objective lens and the transparent member. When the detection means and the detection means detect that the remaining amount of the liquid for immersion is below a predetermined amount, the immersion liquid stored for replenishment is transferred to the immersion objective lens. And a liquid supply means for supplying between the transparent member and the transparent member.

  A microscope according to the present invention includes the above-described liquid supply device, and an immersion objective lens that is filled with a transparent member by the immersion liquid supplied from the liquid supply device.

  According to the present invention, long-time observation can be performed without deteriorating the optical performance of the immersion objective lens.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a diagram showing an embodiment of a microscope to which the present invention is applied.

  The main body 11 of the inverted microscope 1 is provided with a transmission illumination device 12 and an epi-illumination device 13, and the specimen in the Petri dish 2 on the stage 21 is observed using transmission illumination or epi-illumination. Can do. Further, a CCD (Charge Coupled Device) camera 14 is attached to the main body 11 of the microscope 1, and it is possible to take an image of a specimen by transmitted illumination or epi-illumination.

  A focusing unit 25 is provided on the lower surface of the stage 21. The focusing unit 25 moves the focusing movable unit 24, which supports the revolver 23, on which a plurality of objective lenses (only two objective lenses 22A and 22B are shown in FIG. 1) is mounted. Supports to move in the direction. Hereinafter, the objective lenses 22 </ b> A and 22 </ b> B are simply referred to as the objective lens 22 when it is not necessary to distinguish between them.

  In addition, a plurality of filter blocks each including a combination of two types of filters and a dichroic mirror are provided below the focusing movable unit 24 (in FIG. 1, two filter blocks 16A and 16B are illustrated. ) A filter turret 15 is provided. Note that the filter blocks 16A and 16B are simply referred to as the filter block 16 when it is not necessary to distinguish between them.

  When the specimen is observed with the microscope 1 using the transmission illumination, the transmission illumination light emitted from the light source 31 of the transmission illumination device 12 is transmitted through the collector lens 32 and then reflected toward the condenser lens 34 by the reflection mirror 33. Then, the sample in the Petri dish 2 is irradiated through the condenser lens 34. When the optical path conversion prism 52 is removed from the optical path, the transmitted illumination light transmitted through the specimen is transmitted through the objective lens 22, the filter block 16, and the second objective lens 51, and an image of the specimen by the transmitted illumination light is obtained by the CCD camera 14. Taken. When the optical path conversion prism 52 is inserted in the optical path, the transmitted illumination light that has passed through the sample passes through the objective lens 22, the filter block 16, and the second objective lens 51, and is directed in the direction of the prism 53 by the optical path conversion prism 52. The reflected light is reflected in the direction of the eyepiece lens 55 by the prism 53 and the reflection boundary 54, and the user observes the image of the specimen by the transmitted illumination light through the eyepiece lens 55.

  In addition, when the specimen is observed using the epi-illumination with the microscope 1, the epi-illumination light emitted from the light source 41 of the epi-illumination device 13 passes through the relay lenses 42 and 43 and is then transmitted to a predetermined wavelength by the filter block 16. Only is transmitted, is reflected in the direction of the objective lens 22, and is irradiated to the specimen in the Petri dish 2 through the objective lens 22. When the optical path conversion prism 52 is removed from the optical path, the fluorescence emitted from the specimen by irradiating the epi-illumination light passes through the objective lens 22, and then only a predetermined wavelength is extracted by the filter block 16, and the CCD camera 14 An image of the fluorescence emitted from the specimen is taken. Further, when the optical path conversion prism 52 is put in the optical path, the fluorescence emitted from the specimen by irradiating the incident illumination light passes through the objective lens 22, and then only a predetermined wavelength is extracted by the filter block 16, and the optical path Reflected in the direction of the prism 53 by the conversion prism 52 and reflected in the direction of the eyepiece lens 55 by the prism 53 and the reflecting mirror 54, the user observes an image of the fluorescence emitted from the specimen through the eyepiece lens 55. .

  FIG. 2 is a diagram showing in detail the configuration around the stage 21 and the objective lens 22 of the microscope 1 of FIG. FIG. 2 shows an example in which the Petri dish 2 is filled with the culture solution 102, and the specimen 101 which is a cell to be observed is placed on the bottom transparent member 2A constituting the bottom of the Petri dish 2. It is shown. The bottom transparent member 2A is made of a material such as glass or plastic. In the microscope 1, by providing a heat plate on the stage 21, the temperature of the culture solution 102 can be kept substantially constant (for example, 37 ° C.), and the specimen 101 can be observed alive.

  An immersion water 71 is provided between the objective lens 22A for immersion and the bottom transparent member 2A to increase the numerical aperture of the optical system and achieve high resolution and brightness (for example, immersion water). 71 has a refractive index of about 1.3). That is, the immersion water 71 fills the tip of the immersion objective lens 22A. Moreover, the thickness of the immersion water 71 is about 0.2 mm, and the layer of the water is distributed circularly by the surface tension by atmospheric pressure. Hereinafter, a region between the immersion objective lens 22 </ b> A filled with the immersion water 71 and the bottom transparent member 2 </ b> A is also referred to as an immersion part.

  By the way, the immersion water 71 diverges into the surrounding air and evaporates after a certain period of time. Such a phenomenon appears remarkably with the passage of time, particularly when long-time observation is performed. When the immersion water 71 evaporates and an air layer enters the immersion water 71, this becomes a noise component, and the optical performance of the immersion objective lens 22A is reduced. Therefore, in the present embodiment, water is replenished by a necessary amount according to the remaining amount of the immersion water 71. In order to detect the remaining amount of the immersion water 71, in the example of FIG. A detection unit including 81 and a light receiving sensor 82 is provided.

  Here, FIG. 3 shows an arrow view when the detector is viewed from the direction of arrow A in FIG. In FIG. 3, the lower view is a view as viewed from A, and the upper view is a top view as viewed from A.

  As shown in FIG. 3, the optical fiber 81 is embedded in the tip of the immersion objective lens 22A, and the region 81a in contact with the immersion water 71 has a structure in which there is no cladding layer and the core is exposed. . A light source 111 such as an LED (Light Emitting Diode) light source is provided on the incident end side of the optical fiber 81. Light collected from the light 112 and incident on the optical fiber 81 is incident on the optical fiber 81. Is received by the light receiving sensor 82 via the lens 112 provided on the output end side.

  That is, as shown in FIGS. 4 and 5, the optical fiber 81 is composed of a central core 121 and a clad 122 having a different refractive index, and when light enters the core 121, the light is at the interface with the clad 122. Proceed while repeating total reflection. However, when the immersion water 71 is in the immersion state between the objective lens 22A of FIG. 4 and the bottom transparent member 2A, that is, when the immersion water 71 exists around the optical fiber 81, the immersion water. Since refraction occurs at the boundary between 71 and the core 121, a part of the light propagating through the core 121 of the optical fiber 81 is transmitted to the outside as transmitted light from the region 81a. Loss occurs. As a result, the amount of light received by the light receiving sensor 82 is reduced.

  Conversely, when there is no immersion water 71 between the objective lens 22A of FIG. 5 and the bottom transparent member 2A, that is, when there is no immersion water 71 around the optical fiber 81, the immersion water 71 exists. Compared to the case, the amount of light received by the light receiving sensor 82 increases. The light receiving sensor 82 detects these light amounts and outputs them to the optical fiber sensor driving unit 94. Information about the detected light quantity is notified to the control unit 91 from the optical fiber sensor driving unit 94 that drives the light receiving sensor 82.

  Note that the detection method using the optical fiber 81 and the light receiving sensor 82 in the present embodiment is an example of a detection unit that detects the remaining amount of the immersion water 71 in the immersion unit. It is possible to employ other immersion detection methods, such as a method of detecting the remaining amount of immersion water 71 using the

  When the information on the amount of light is supplied from the optical fiber sensor driving unit 94, the control unit 91 compares the value corresponding to the amount of received light with a predetermined threshold value, and the immersion water in the immersion unit. It is determined whether the remaining amount of 71 is equal to or less than a predetermined amount. When it is determined that the remaining amount of the immersion water 71 in the immersion part is equal to or less than the predetermined amount, the control unit 91 controls the driving of the pump drive unit 95 so that a necessary amount of water is supplied to the immersion part. Let them replenish.

  Specifically, the reservoir 83 that stores water 72 for replenishing the liquid immersion unit pushes out the stored water 72 by the pump 84. The pump 84 is driven by a pump drive unit 95 controlled by the control unit 91 and replenishes the liquid immersion unit with water 72 stored in the reservoir 83 by the nozzle 85. That is, when the control unit 91 is notified from the optical fiber sensor driving unit 94 that the remaining amount of the immersion water 71 in the immersion unit has become a predetermined amount or less, the control unit 91 is insufficient for the pump driving unit 95. An instruction is given to replenish the water 72 stored in the reservoir 83 so that the immersion water 71 is filled. Then, the water 72 stored in the reservoir 83 reaches the liquid immersion part through the nozzle 85 and is replenished as the immersion water 71.

  Here, the outer frame of the immersion objective lens 22A is heated and cooled by, for example, a heating / cooling unit 86 formed of a heater, a Peltier element, or the like. Based on the temperature of the tip of the immersion objective lens 22 </ b> A measured by the temperature sensor 92, the control unit 91 instructs the heat-retaining heating / cooling unit 93 to perform heating / cooling, whereby the immersion objective is used. The heating / cooling unit 86 is controlled so that the lens 22A and the specimen 101 have substantially the same temperature (for example, 37 ° C.).

  At this time, the nozzle 85 for replenishing the water 72 is disposed in the vicinity of the heating / cooling unit 86 (in FIG. 2, the space between the immersion objective lens 22A and the heating / cooling unit 86). The heating and cooling unit 86 that is heated and cooled in accordance with the control of the heat-retaining heating and cooling unit 93 maintains the temperature substantially the same as that of the immersion objective lens 22A. That is, the water 72 supplied to the liquid immersion portion as the immersion water 71 is not only stored in the reservoir 83 but also stored in the nozzle 85 (because it is in a standby state). The water 72 accumulated in the water is maintained at substantially the same temperature as the immersion objective lens 22A by the heating and cooling unit 86. When the immersion water 71 is replenished by driving the pump 84 by the pump drive unit 95, first, the immersion objective lens 22A is almost the same as the water 72 stored in the reservoir 83. The water 72 accumulated in the nozzle 85 maintained at the same temperature reaches the liquid immersion part and is replenished as the immersion water 71.

  As described above, in the present embodiment, the nozzle 85 is disposed between the immersion objective lens 22A and the heating / cooling unit 86, so that the immersion objective lens 22A is provided for heating and cooling. By simultaneously heating and cooling the nozzle 85 using the heating / cooling unit 86, water 72 having a temperature close to the immersion objective lens 22A (substantially the same temperature) can be supplied to the immersion unit.

  In addition, the supply speed or supply amount of the water 72 to the liquid immersion unit may be set in advance. Therefore, the control unit 91 supplies the water 72 to be replenished to the liquid immersion unit by controlling the pump driving unit 95 so that the supply speed or the supply amount set in advance by the user or at the time of factory shipment, for example. Make fine adjustments. For example, by adjusting the supply speed to a lower speed, the water 72 can be replenished with an image in which a small amount of water is added with a dropper. Thereby, for example, it is possible to prevent water 72 supplied to the liquid immersion part from spilling out from the tip of the immersion objective lens 22A.

  In addition, when the control unit 91 determines that the remaining amount of the immersion water 71 in the immersion unit is equal to or less than a predetermined amount, the control unit 91 outputs a warning sound, a warning screen, or the like to the warning notification unit 96, for example. Notify that it is missing. The timing for outputting the warning sound or the like may be before the water 72 is replenished to the liquid immersion part or after it is replenished, and the immersion water 71 is insufficient for the user. It is only necessary to be able to notify that it is. In addition, the warning notification method is not limited to the warning sound or the warning screen, and may be any method that can call attention to the user, for example, another sound or blinking of an LED.

  By the way, when the water 72 is replenished to the immersion part, the illumination light becomes unstable due to a change in the state of the illumination optical system (transmission illumination or epi-illumination in FIG. 1) generated by the supply of the water 72. There is a risk of affecting the specimen photographed by the CCD camera 14. Therefore, in the present embodiment, when the specimen 101 is photographed by the CCD camera 14, the control unit 91 prevents the water 72 collected for replenishment from being replenished in the liquid immersion part, and then the photographing is performed. After the completion, control is performed so that the water 72 is replenished in the liquid immersion unit.

  The microscope 1 is configured as described above.

  In the present embodiment, the optical fiber 81 to the nozzle 85, the control unit 91, and the optical fiber sensor driving unit 94 to the warning notification unit, which are provided to supply the water 72 to the liquid immersion unit shown in FIG. Reference numeral 96 is configured as a liquid supply apparatus, and the liquid supply apparatus can be regarded as being attached to the microscope 1.

  Next, the immersion water supply control process executed by the controller 91 will be described with reference to the flowchart of FIG.

  In step S11, the control unit 91 determines the liquid in the liquid immersion unit based on the detection result supplied from the optical fiber sensor driving unit 94 (in FIG. 2, the detection result by the detection unit including the optical fiber 81 and the light receiving sensor 82). It is determined whether or not the remaining amount of the flooded 71 is equal to or less than a predetermined amount.

  If it is determined in step S11 that the remaining amount of the immersion water 71 exceeds the predetermined amount, the immersion part is filled with a sufficient amount of the immersion water 71, and it is necessary to replenish the water 72 stored for replenishment. Therefore, the process of step S11 is repeated until it is determined by the determination process that the remaining amount of the immersion water 71 is equal to or less than a predetermined amount.

  On the other hand, when it is determined in step S11 that the remaining amount of the immersion water 71 is equal to or less than the predetermined amount, in step S12, the control unit 91 notifies the warning notification unit 96 of a warning sound or the like, for example, to the user. To notify that the immersion water 71 is insufficient. Thereby, the user who is observing with the microscope 1 can recognize that the remaining amount of the immersion water 71 in the immersion part is insufficient.

  In step S <b> 13, the control unit 91 determines whether it is the timing when the specimen 101 is captured by the CCD camera 14.

  If it is determined in step S13 that the timing of photographing the sample 101 is reached, the processing in step S13 is repeated until the photographing of the sample 101 by the CCD camera 14 is completed.

  Thereafter, when the imaging by the CCD camera 14 is completed and it is determined in step S13 that it is not the timing for imaging the specimen 101, in step S14, the control unit 91 controls the pump driving unit 95 to store in the reservoir 83. The accumulated water 72, that is, the water 72 accumulated in the nozzle 85 maintained at substantially the same temperature as the immersion objective lens 22A by the heating / cooling unit 86, is used as the immersion water 71 for the shortage. To supply. As a result, the liquid immersion portion at the tip of the immersion objective lens 22A is replenished with the necessary amount of immersion water evaporated in the air and filled with the immersion water 71.

  Subsequently, the process returns to the process of determining the remaining amount of the immersion water 71 in step S11, and the processes of step S11 to step S14 are repeated until the observation with the microscope 1 is completed. That is, during the observation, the immersion water supply control process of FIG. 6 is continuously executed by the control unit 91, so that the immersion part at the tip of the immersion objective lens 22A is immersed in a sufficient amount of immersion water for a long time. 71, the original optical performance of the immersion objective lens 22A can be maintained, and as a result, the state of the illumination light can be stabilized. This appears as a more prominent effect when performing time-lapse image capturing of live cells for several hours such as 2 hours, 3 hours,..., 24 hours.

  In addition, it is conceivable that the heat of the Petri dish 2 escapes due to heat conduction from the liquid immersion part, but in the present embodiment, heating for making the temperature of the immersion objective lens 22A substantially constant. By simultaneously heating and cooling the nozzle 85 by the cooling unit 86, the water 72 stored in the nozzle 85 can be made substantially the same as the temperature of the objective lens 22A for immersion. As a result, the water 72 to be replenished in the immersion part is maintained at substantially the same temperature as the immersion objective lens 22A, so that biological changes due to temperature are applied to the cells through the bottom transparent member 2A. You can avoid giving.

  Note that the timing for replenishing the immersion water 71 may be determined using the contrast of an image taken by the CCD camera 14. For example, when the control unit 91 monitors the difference between the highest luminance signal and the lowest luminance signal from the image photographed by the CCD camera 14, and the difference becomes lower than a predetermined threshold value. The water immersion unit may be supplemented with water 72.

  The series of processes described above can be executed by hardware or can be executed by software. When a series of processing is executed by software, a program constituting the software executes various functions by installing a computer incorporated in dedicated hardware or various programs. For example, it is installed from a program recording medium in a general-purpose personal computer or the like.

  In the present specification, the step of describing the program stored in the recording medium is not limited to the processing performed in chronological order according to the described order, but is not necessarily performed in chronological order. It also includes processes that are executed individually.

  Furthermore, the embodiments of the present invention are not limited to the above-described embodiments, and various modifications can be made without departing from the gist of the present invention.

It is a figure which shows the structure of one Embodiment of the microscope to which this invention is applied. It is the figure which showed the structure of the periphery of the stage and objective lens of a microscope in detail. It is a figure which shows the example of a detailed structure of an optical fiber and a light reception sensor. It is a figure which shows the example of the light which propagates the inside of the core of an optical fiber in case there is immersion water between an objective lens and a bottom transparent member. It is a figure which shows the example of the light which propagates the inside of the core of an optical fiber in case there is no immersion water between an objective lens and a bottom transparent member. It is a flowchart explaining an immersion water supply control process.

Explanation of symbols

  1 microscope, 2 petri dish, 14 CCD camera, 22, 22B objective lens, 23 revolver, 24 focusing movable part, 25 focusing part, 71 immersion water, 72 water, 81 optical fiber, 82 light receiving sensor, 83 reservoir, 84 Pump, 85 nozzle, 86 Heating / cooling unit, 91 Control unit, 92 Temperature sensor, 93 Heating / cooling unit for heat insulation, 94 Optical fiber sensor driving unit, 95 Pump driving unit, 96 Warning notification unit

Claims (7)

In a liquid supply apparatus used in a microscope having an immersion objective lens,
Detecting means for detecting a remaining amount of liquid for immersion filled between the immersion objective lens and the transparent member;
When it is detected by the detecting means that the remaining amount of the liquid for immersion is below a predetermined amount, the liquid for immersion stored for replenishment is transferred to the objective lens, the transparent member, and the immersion objective lens. And a liquid supply means for supplying between the liquid supply device and the liquid supply device. The liquid supply means supplies the immersion liquid stored for replenishment by a nozzle disposed in the vicinity of a heating / cooling unit provided in the microscope for heating and cooling the immersion objective lens. ,
The liquid supply apparatus according to claim 1, wherein the nozzle is heated and cooled by the heating and cooling unit so that the nozzle has substantially the same temperature as the immersion objective lens. The detection means comprises an optical fiber and a sensor for detecting light propagating through the core at the center thereof, and according to the change in the amount of light obtained from the light refracted by the immersion liquid around the optical fiber, The liquid supply device according to claim 1, wherein a remaining amount of the liquid for immersion is detected. The liquid supply apparatus according to claim 1, wherein the liquid supply unit supplies the liquid for immersion stored for replenishment according to a preset supply speed or supply amount. 2. The liquid according to claim 1, further comprising notification means for notifying that when the detection means detects that the remaining amount of the liquid for immersion is below a predetermined amount. Feeding device. A liquid supply device according to any one of claims 1 to 5,
A microscope comprising: an immersion objective lens that is filled with a transparent member by the immersion liquid supplied from the liquid supply device. An illumination optical system for illuminating the specimen;
An imaging means for imaging the specimen illuminated by the illumination optical system;
The liquid supply device supplies the immersion liquid stored for replenishment between the immersion objective lens and the transparent member when the specimen is not photographed by the imaging means. The microscope according to claim 6.

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