JP2004070307A - Maceration medium supply apparatus, fluorescent analytic inspecting device and incubation microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a maceration medium supply apparatus, a fluorescent analytic inspecting device, and an incubation microscope which can stably perform temperature control over a sample and have simple constitution, and are reduced in cost. <P>SOLUTION: The maceration medium 13 charged between an observation sample and a maceration objective 12, a temperature controller 26 which controls the maceration medium 13 at specified temperature, and a maceration medium supply part (22 to 25) which supplies the maceration medium 13 whose temperature is controlled by the temperature controller 26 to the maceration objective 12, are included. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an immersion medium supply device that supplies an immersion medium to an immersion objective lens, a fluorescence spectroscopy device, and a culture microscope.
[0002]
[Prior art]
For example, it is known that the reaction of a biological specimen such as a protein is greatly affected by its temperature change. Therefore, when observing such a sample with a microscope, various devices have been devised to maintain the sample temperature at a predetermined temperature.
[0003]
Patent Literature 1 discloses a technique that realizes such an idea. A heating unit and an electric heating plate are provided on a stage, and the heating unit and the electric heating plate allow a sample in a glass dish placed on the stage to be sampled. By applying heat, the temperature of the sample is kept constant.
[0004]
Further, as disclosed in Patent Document 2, a pipe for supplying water controlled at an appropriate temperature is arranged from the periphery of the specimen to the periphery of the objective lens, and water of a predetermined temperature is flowed through the pipe to thereby provide a specimen. And a heating means provided in a heat retaining box of a solvent observation container mounted on a stage partially constituted by a heating unit as disclosed in Patent Document 3 There is also known a technique in which a solvent specimen in a solvent observation container is controlled to be kept at an appropriate temperature.
[0005]
On the other hand, recently, as disclosed in Patent Document 4, fluorescence microphotometry captures the movement of a fluorescently labeled biomolecule floating in a microscopic region in a liquid to specify the reaction of the biomolecule. Inspection methods are being considered.
[0006]
In this method, a sample container made of a resin called a microplate in which a large number of test wells are arranged or a bottom surface made of a cover glass is used. The sample is analyzed by storing the fluorescently labeled sample in the well of the microplate and monitoring the intensity of fluctuation emitted from the sample while detecting the fluorescence of the fluorescently labeled sample by a microplate reader. . In this case, the sample in the well of the microplate is in a state where large and small molecules are suspended in the liquid. Then, excitation light is condensed on the suspended molecules through a cover glass on the bottom surface of the microplate by an immersion objective lens, the excited fluorescence is detected at a molecular resolution, and the properties of the molecules are determined by calculation.
[0007]
[Patent Document 1] Japanese Utility Model Publication No. 60-156521
[0008]
[Patent Document 2] Japanese Utility Model Publication No. Hei 7-36118
[0009]
[Patent Document 3] Japanese Utility Model Publication No. 3-25598
[0010]
[Patent Document 4] Japanese Patent Publication No. 2001-502062
[0011]
[Problems to be solved by the invention]
An object of the present invention is to provide an immersion medium supply device, a fluorescence spectroscopy device, and a culture microscope that are capable of stably controlling the temperature of a specimen, have a simple configuration, and are inexpensive.
[0012]
[Means for Solving the Problems]
An immersion medium supply device according to a first aspect of the present invention includes an immersion medium filled between an observation sample and an immersion objective lens, and a temperature controller that controls the immersion medium to a predetermined temperature. An immersion medium supply unit that supplies the immersion medium, the temperature of which is controlled by the temperature controller, to the immersion objective lens.
[0013]
The fluorescence spectroscopy apparatus according to the second aspect of the present invention includes an immersion objective lens for condensing light from a specimen of a fluorescently labeled biomolecule floating in a liquid, the specimen and the immersion objective lens, An optical system including an immersion medium to be filled in between, an optical detector for detecting at least an enlarged image of the immersion objective lens, and fluorescent illumination; and a motion of the specimen is calculated from an output of the optical detector. A computing unit, a monitor that displays a computation result by the computing unit, a temperature controller that controls the immersion medium to a predetermined temperature, and the immersion medium that is temperature-controlled by the temperature controller. An immersion medium supply unit for supplying the liquid to the lens.
[0014]
A culture microscope according to a third aspect of the present invention includes a stage for mounting an observation sample, a microscope supporting the stage, a liquid immersion objective lens supported by the microscope, and an observation sample housed in the microscope. An incubator accommodating an imaging device and an optical system for capturing an enlarged image of an immersion medium filled between the observation sample and the immersion objective lens; and setting the immersion medium to a predetermined temperature. A temperature controller for controlling, an immersion medium supply unit that supplies the immersion medium, the temperature of which is controlled by the temperature controller, to the immersion medium lens, and a hot air blower that sends warm air to the incubator. It is characterized by doing.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0016]
(First Embodiment)
FIG. 1 shows a schematic configuration of an inspection apparatus to which the immersion medium supply device of the present invention is applied. In FIG. 1, an inverted microscope 2 is provided inside an apparatus main body 1.
[0017]
The inverted microscope 2 is provided with an XY stage 3. On the XY stage 3, a microplate 5 having a large number of wells 5a for holding a specimen 4 is placed.
[0018]
The microplate 5 is a sample container made of resin, and as shown in FIG. 3, a large number of wells 5a for receiving the sample 4 are arranged in an orderly manner. On the bottom surface of the microplate 5, a cover glass 5b having a thickness of, for example, about 0.17 mm is disposed as a transparent member. Thereby, as shown in FIG. 4, each well 5a of the microplate 5 has a structure in which a cover glass 5b is adhered to the bottom surface so as to be suitable for inverted observation from the lower surface. The specimen 4 placed in the well 5a is a specimen in which fluorescently labeled biomolecules become colloidal and float in the liquid.
[0019]
A microplate insertion window 1a is provided on the front of the apparatus main body 1, as shown in FIG. The microplate insertion window 1 a is a window for inserting and removing the microplate 5 into and out of the apparatus main body 1. When the microplate 5 is inserted into the apparatus main body 1 through the microplate insertion window 1a, the microplate 5 is placed on the XY stage 3, and a specimen test can be automatically performed in this state.
[0020]
Laser beams from the excitation laser light sources 6 and 7 having different wavelengths are selectively or simultaneously introduced into the inverted microscope 2 via the half mirror 8 and the mirror 9. Then, the laser light becomes parallel light by the collimating lens 10, is reflected by the dichroic mirror 11, and is introduced into the objective lens 12.
[0021]
In this case, as the objective lens 12, an immersion objective lens (a water immersion objective lens in the illustrated example) having an increased light-collecting ability so that light can be focused on a minute area of several micrometers in the liquid of the specimen 4 is used. (Hereinafter, this will be described as the water immersion objective lens 12). Further, as shown in FIG. 4, the water immersion objective lens 12 is arranged close to the cover glass 5b on the bottom surface of the microplate 5. An immersion medium (pure water) 13 is filled between the water immersion objective lens 12 and the cover glass 5b.
[0022]
The excitation light transmitted through the water immersion objective lens 12 passes through the immersion medium 13, passes through the cover glass 5 b, advances into the well 5 a of the microplate 5, and is focused on a minute area in the liquid of the specimen 4. Here, when a fluorescently labeled biomolecule floating in the liquid of the specimen 4 is present in the minute region where the excitation light is collected, it emits fluorescence at that moment.
[0023]
This fluorescence passes through the water immersion objective lens 12, passes through the dichroic mirror 11, passes through the excitation light barrier filter 14, and forms an enlarged image of the fluorescence by the water immersion objective lens 12 by the imaging lens 15 and the reflection mirror 16 , And reaches the photodetector 18 through the pinhole 17.
[0024]
In this case, the fluorescently labeled biomolecule floating in the liquid of the specimen 4 is a colloidal floating body, and thus vibrates due to Brownian motion and moves in and out of a minute region. As a result, the photodetector 18 detects the fluorescence of the biomolecules entering and exiting the minute area.
[0025]
The arithmetic unit 19 is connected to the light detector 18. The calculation device 19 calculates the fluctuation of the detection signal of the photodetector 18.
[0026]
A personal computer 20 provided as a central processing unit provided in the apparatus main body 1 is connected to the arithmetic unit 19. The personal computer 20 analyzes the properties of the biological molecule from the fluctuation calculated by the arithmetic unit 19 and displays the result on the display unit 21.
[0027]
On the other hand, the immersion objective lens 12 is provided with an immersion medium supply device as described below of the present invention.
[0028]
In this case, a gap between the water immersion objective lens 12 and the cover glass 5b on the bottom surface of the microplate 5 is filled with an immersion medium (pure water) 13. The immersion medium 13 is provided in a water supply bottle 22 as an immersion medium supply source. The water supply bottle 22 is provided with a water supply pipe 23a, a water supply pump 24, water supply pipes 23b and 23c, and a liquid supply nozzle (here, a water supply nozzle 25) constituting the immersion medium supply means. The immersion medium 13 in the water supply bottle 22 is sent to a water supply pump 24 via a water supply pipe 23a. The immersion medium 13 is supplied from a water supply nozzle 25 to a gap between the water immersion objective lens 12 and the cover glass 5b via water supply pipes 23b and 23c. In this case, as shown in FIG. 4, the distance 1 between the cover glass 5b and the tip of the water immersion objective lens 12 is extremely small, for example, 1 mm or less. Therefore, the immersion medium 13 supplied during this time is held in place by surface tension. A temperature control device 26 is provided between the water supply pipes 23b and 23c as temperature control means. The temperature control device 26 heats or cools the temperature of the immersion medium 13 to a predetermined temperature. The immersion medium 13 whose temperature has been adjusted by the temperature control device 26 is supplied to the water supply nozzle 25 side.
[0029]
A liquid receiving portion 27 is provided at the tip of the water immersion objective lens 12. The liquid receiving portion 27 is provided along the periphery of the water immersion objective lens 12 as shown in FIG. 4, and receives the liquid immersion medium 13 that cannot be held in the gap between the water immersion objective lens 12 and the cover glass 5b. Has become. The liquid receiving portion 27 is provided with a drain port 27a. The drain port 27 a is provided with a drain pipe 28, a drain pump 29, and a drain bottle 30 that constitute immersion medium discharging means, and drains the immersion medium 13 received by the liquid receiving unit 27 through the drain pipe 28. 29 and can be collected by a drainage bottle 30 by a drainage pump 29.
[0030]
A control device 32 is connected to the water supply pump 24, the temperature control device 26, and the drainage pump 29. The control device 32 controls operations of the water supply pump 24, the temperature control device 26, and the drainage pump 29 according to an instruction from the personal computer 20.
[0031]
The water supply bottle 22 and the drainage bottle 30 are arranged inside the apparatus main body 1, but they are arranged so that they can be easily replaced from the outside of the apparatus main body 1, or the immersion medium 13 can be supplied to the water supply bottle 22. It has a configuration. Of course, if the water supply bottle 22 and the drainage bottle 30 are arranged outside the apparatus main body 1, the replacement of the water supply bottle 22 and the drainage bottle 30 can be further started, and the supply of the immersion medium 13 to the water supply bottle 22 can be simplified.
[0032]
In such a configuration, first, a microplate 5 in which a large number of wells 5a accommodate fluorescently labeled specimens 4 is prepared. Then, the microplate 5 is inserted into the inside of the apparatus main body 1 through the microplate insertion window 1a, and is placed on the XY stage 3.
[0033]
In this state, when the XY stage 3 is moved and the first well 5a is positioned on the optical axis of the water immersion objective lens 12, the immersion medium 13 prepared in the water supply bottle 22 passes through the water supply pipe 23a. The water is supplied to the water supply pump 24 and supplied to the gap between the water immersion objective lens 12 and the cover glass 5b from the water supply nozzle 25 via the water supply pipes 23b and 23c. In this case, the immersion medium 13 is held in place by surface tension.
[0034]
At this time, the immersion medium 13 is controlled to a predetermined temperature by the temperature control device 26. As a result, when the immersion medium 13 comes into contact with the cover glass 5b, the sample 4 is subjected to heat exchange between the immersion medium 13 and the cover glass 5b and further heat exchange between the cover glass 5b and the sample 4. It is controlled to a predetermined temperature. In this case, the cover glass 5b having a thickness of about 0.17 mm is used, and its heat capacity is made smaller than that of the surrounding materials, so that it does not hinder heat exchange with the specimen 4, and conversely. Since heat conductivity is good, heat exchange can be advantageously performed.
[0035]
In this state, the fluorescence of the biomolecules floating in the liquid of the specimen 4 is detected by the photodetector 18 by performing the above-described fluorescence detection, and the personal computer 20 calculates the properties of the biomolecules based on the fluctuation calculated by the arithmetic unit 19. Is analyzed and displayed on the display unit 21.
[0036]
When the inspection of the first specimen 4 is completed, the XY stage 3 moves, the next well 5a is positioned on the optical axis of the water immersion objective lens 12, and the same fluorescence detection is performed.
[0037]
In this case, when the cover glass 5b moves with respect to the water immersion objective lens 12 with the movement of the XY stage 3, the immersion medium 13 that cannot be held between the water immersion objective lens 12 and the cover glass 5b falls. The liquid immersion medium 13 is received by a liquid receiving portion 27 provided around the water immersion objective lens 12. Then, the immersion medium 13 is sent from the drain port 27 a to the drain pipe 28, and is collected by the drain pump 29 in the drain bottle 30. At the same time, a certain amount of the immersion medium 13 is supplied from the water supply nozzle 25 to the gap between the water immersion objective lens 12 and the cover glass 5b.
[0038]
The replenishment of the liquid immersion medium 13 between the water immersion objective lens 12 and the cover glass 5b may be performed by a predetermined amount at regular time intervals.
[0039]
Hereinafter, similarly, fluorescence detection is performed for all the wells 5 a of the microplate 5, and the analysis result is displayed on the display unit 21 by the personal computer 20.
[0040]
Therefore, according to the above embodiment, the immersion medium 13 used for the immersion objective lens 12 is focused on, and the immersion medium 13 filled in the gap between the immersion objective lens 12 and the cover glass 5b is determined in advance. Therefore, the temperature of all the specimens 4 placed in the wells 5 a of the microplate 5 can be controlled to the predetermined temperature of the immersion medium 13. Thereby, the floating molecular motion in the liquid of the sample 4, that is, the Brownian motion in which the momentum changes due to the temperature change can be always kept constant, so that the sample 4 in all the wells 5a of the microplate 5 In contrast, stable fluorescence detection can be performed. That is, in the case of observing a biological specimen, an appropriate form can be observed, and stable data can be obtained in fluorescence photometry that captures molecular motion, and correct determination can be made.
[0041]
As an example of the inspection method, there is a fluorescence correlation spectroscopy, and information on the diffusion intensity and information on the number of molecules can be obtained by analyzing the fluctuation of the fluorescence intensity. This device is used for a fluorescent molecule inspection device such as fluorescence correlation spectroscopy.
[0042]
Since the temperature control range is pure, the maximum range is 0 to 100 ° C. To give an example of the temperature, in the case of seawater using seawater, about 25 ° C, biological cells such as animal proteins remain alive. In some cases, the temperature may be 37 ° C., and in the case of DNA, 80 ° C. It is desirable that the temperature range in which the living cells remain alive is 37 ° C. ± 1 ° C. or less. The accuracy of the temperature is appropriately selected depending on the purpose of the inspection and the temperature sensitivity of the object.
[0043]
Further, by setting a large amount of the immersion medium 13 supplied to the gap between the water immersion objective lens 12 and the cover glass 5b, the temperature of the specimen 4 can be stably controlled, and the immersion medium can be controlled. Since the supply and discharge of the sample 13 are frequently repeated, the temperature control of the sample 4 can be easily changed, so that accurate temperature control can be performed.
[0044]
In addition, since the temperature of the immersion medium 13 is controlled using the immersion medium 13 used for the water immersion objective lens 12, the configuration of the entire apparatus can be simplified and the cost can be reduced. .
[0045]
In the above-described embodiment, the temperature control device 26 is disposed between the water supply pipes 23b and 23c. Alternatively, or simultaneously, the temperature control device 26 is brought close to the water supply bottle 22 so that the temperature of the entire water supply bottle 22 is controlled. A bottle temperature controller 31 may be provided.
[0046]
Further, a sensor S which constantly detects the temperature of the immersion medium 13 held in the gap between the water immersion objective lens 12 and the cover glass 5b. _T And this sensor S _T Of the immersion medium 13 between the water immersion objective lens 12 and the cover glass 5b may be controlled to be always at a constant temperature by controlling the temperature control device 26 with the output of.
[0047]
Further, in the above-described embodiment, the immersion medium 13 of the water supply bottle 22 is supplied to the gap between the immersion objective lens 12 and the cover glass 5b, and the excess immersion medium 13 is collected in the drainage bottle 30. However, the liquid immersion medium 13 supplied between the water immersion objective lens 12 and the cover glass 5b may be circulated. In this case, foreign matter and the like may be mixed into the immersion medium 13 by the circulation of the immersion medium 13, and thus it is necessary to surely remove the foreign matter and the like using a filter or the like.
[0048]
Further, noting that the temperature inside the apparatus main body 1 is several degrees higher than the outside temperature, a heat exchanger is provided in place of the temperature control device 26, and the immersion medium 13 is set to the same temperature as the inside of the apparatus by the heat exchanger. May be supplied to the gap between the water immersion objective lens 12 and the cover glass 5b.
[0049]
Furthermore, in the above-described embodiment, an example of the water immersion method using pure water for the liquid immersion medium 13 has been described, but an oil immersion method using oil can be similarly applied.
[0050]
The water supply nozzle 25 supplies the immersion medium 13 to the gap between the water immersion objective lens 12 and the cover glass 5b. The water supply nozzle 25 is detachably held outside the water immersion objective lens 12. . The water supply nozzle 25 may be embedded in the outer frame of the water immersion objective lens 12. The water supply nozzle 25 may be held by the inverted microscope 2, or may be held by a liquid receiving section 27 installed around the distal end of the water immersion objective lens 1212. Although the number of water supply nozzles 25 is one in the figure, it may be plural, for example, three or five.
[0051]
(Modification of First Embodiment)
Next, a modification of the first embodiment of the present invention will be described.
[0052]
This modification shows an example in which the immersion medium supply device according to the first embodiment of the present invention is applied to an inverted microscope.
[0053]
FIG. 5 shows a schematic configuration of a modified example of the first embodiment.
[0054]
In FIG. 5, a stage 42 is disposed on the front fixed portion 41a and the rear fixed portion 41b of the microscope main body 41.
[0055]
A petri dish 43 is placed on the stage 42. The Petri dish 43 has a cover glass on the bottom surface, and a biological specimen is placed in the Petri dish.
[0056]
Then, a light beam from an unillustrated epi-illumination light source is applied to the biological specimen from the bottom (cover glass) side of the petri dish 43 via the water immersion objective lens 44. The light from the petri dish 43 is relayed by a relay optical system (not shown) via a water immersion objective lens 44, then enters an eyepiece 46 attached to an observation lens barrel 45, and is visually observed.
[0057]
Also in this case, an immersion medium (pure water) 47 is filled in a gap between the water immersion objective lens 44 and the cover glass on the bottom surface of the petri dish 43. The immersion medium 47 is controlled to a predetermined temperature by a temperature control device (not shown) in the same manner as described with reference to FIG. And is held in place by surface tension. The liquid immersion medium 47 that cannot be held between the water immersion objective lens 44 and the cover glass is discharged from the drain pipe 51 via the liquid receiver 50.
[0058]
In this case, the supply of the immersion medium 47 may be electric or manual using a manual pump.
[0059]
In FIG. 5, a transmitted illumination light source 52 and a condenser lens 53 are also shown.
[0060]
According to this modification, the immersion medium 47 filled in the gap between the water immersion objective lens 44 and the cover glass on the bottom surface of the petri dish 43 can be adjusted at a predetermined temperature and supplied in advance. In other words, the temperature of the biological specimen placed in the petri dish can be controlled to a predetermined constant temperature of the immersion medium 47, and a high-quality biological specimen can be observed.
[0061]
(Second embodiment)
Next, a second embodiment of the present invention will be described.
[0062]
FIG. 6 shows a schematic configuration of a culture microscope to which the immersion medium supply device of the present invention is applied. 6, the same components as those in the first embodiment are denoted by the same reference numerals.
[0063]
In FIG. 6, an inverted microscope 2 is provided inside the apparatus main body 1. The inverted microscope 2 is provided with an XY stage 3 via a stage receiver 2 '. The XY stage 3 has a rack R _XY , Pinion P _XY And drive motor M _XY Is provided. On the XY stage 3, a microplate 5 having a large number of wells 5a for holding a specimen 4 is placed.
[0064]
The microplate 5 is a sample container made of resin, and as shown in FIG. 3, a large number of wells 5a for receiving the sample 4 are arranged in an orderly manner. On the bottom surface of the microplate 5, a cover glass 5b having a thickness of, for example, about 0.17 mm is disposed as a transparent member. Thereby, as shown in FIG. 4, each well 5a of the microplate 5 has a structure in which a cover glass 5b is adhered to the bottom surface so as to be suitable for inverted observation from the lower surface. The specimen 4 placed in each of the wells 5a is in a state where the fluorescently labeled biological cells are alive in the culture solution.
[0065]
A microplate insertion window 1a is provided on the front of the apparatus main body 1, as shown in FIG. The microplate insertion window 1 a is a window for inserting and removing the microplate 5 into and out of the apparatus main body 1. When the microplate 5 is inserted into the apparatus main body 1 through the microplate insertion window 1a, the microplate 5 is placed on the XY stage 3, and a specimen test can be automatically performed in this state.
[0066]
The illumination light from the light source 6 ′ is introduced into the inverted microscope 2. The illumination light passes through an excitation light exciter filter 14 ′ and a lens 10 ′, is reflected by a dichroic mirror 11, and is introduced into an objective lens 12.
[0067]
In this case, as the objective lens 12, an immersion objective lens (a water immersion objective lens in the illustrated example) having an increased light-collecting ability so that light can be collected on biological cells in the liquid of the specimen 4 is used (hereinafter, referred to as an objective lens). This will be described as the water immersion objective lens 12). Further, as shown in FIG. 4, the water immersion objective lens 12 is arranged close to the cover glass 5b on the bottom surface of the microplate 5. An immersion medium (pure water) 13 is filled between the water immersion objective lens 12 and the cover glass 5b.
[0068]
The excitation light transmitted through the water immersion objective lens 12 passes through the immersion medium 13, passes through the cover glass 5b, enters the well 5a of the microplate 5, and is focused on biological cells in the liquid of the specimen 4. Here, when the fluorescently labeled biological cells in the liquid of the specimen 4 exist in the region of the excitation light, they emit fluorescence.
[0069]
This fluorescence passes through the water immersion objective lens 12, passes through the dichroic mirror 11, passes through the excitation light barrier filter 14, and forms an enlarged image of the fluorescence by the water immersion objective lens 12 by the imaging lens 15 and the reflection mirror 16 To reach an image sensor 18 'such as a solid-state image sensor (CCD). Since the water immersion objective lens 12 moves up and down with the inverted microscope 2, a rack R is provided on the water immersion objective lens 12 side. _Z Is provided, and pinion P is attached to the inverted microscope. _Z And motor M _Z Is provided.
[0070]
In this case, the fluorescently labeled biological cells in the liquid of the specimen 4 are magnified by the water immersion objective lens 12, and the imaging device 18 'captures a fluorescent image of the biological cells.
[0071]
A personal computer 20 provided as a central processing unit provided in the apparatus main body 1 is connected to the imaging element 18 '. A display unit 21 such as a monitor and an input device 33 such as a keyboard are connected to the personal computer 20.
[0072]
On the other hand, the immersion objective lens 12 is provided with an immersion medium supply device as described below of the present invention.
[0073]
In this case, a gap between the water immersion objective lens 12 and the cover glass 5b on the bottom surface of the microplate 5 is filled with an immersion medium (pure water) 13. The immersion medium 13 is provided in a water supply bottle 22 as an immersion medium supply source. The water supply bottle 22 is provided with a water supply pipe 23a, a water supply pump 24, water supply pipes 23b and 23c, and a liquid supply nozzle (here, a water supply nozzle 25) constituting the immersion medium supply means. The immersion medium 13 in the water supply bottle 22 is sent to a water supply pump 24 via a water supply pipe 23a. The immersion medium 13 is supplied from a water supply nozzle 25 to a gap between the water immersion objective lens 12 and the cover glass 5b via water supply pipes 23b and 23c. In this case, as shown in FIG. 4, the distance 1 between the cover glass 5b and the tip of the water immersion objective lens 12 is extremely small, for example, 1 mm or less. Therefore, the immersion medium 13 supplied during this time is held in place by surface tension. A temperature control device 26 is provided between the water supply pipes 23b and 23c as temperature control means. The temperature control device 26 heats or cools the temperature of the immersion medium 13 to a predetermined temperature. The immersion medium 13 whose temperature has been adjusted by the temperature control device 26 is supplied to the water supply nozzle 25 side.
[0074]
A liquid receiving portion 27 is provided at the tip of the water immersion objective lens 12. The liquid receiving portion 27 is provided along the periphery of the water immersion objective lens 12 as shown in FIG. 4, and receives the liquid immersion medium 13 that cannot be held in the gap between the water immersion objective lens 12 and the cover glass 5b. Has become. The liquid receiving portion 27 is provided with a drain port 27a. The drain port 27 a is provided with a drain pipe 28, a drain pump 29, and a drain bottle 30 that constitute immersion medium discharging means, and drains the immersion medium 13 received by the liquid receiving unit 27 through the drain pipe 28. 29 and can be collected by a drainage bottle 30 by a drainage pump 29.
[0075]
A control device 32 is connected to the water supply pump 24, the temperature control device 26, and the drainage pump 29. The control device 32 controls operations of the water supply pump 24, the temperature control device 26, and the drainage pump 29 according to an instruction from the personal computer 20. Further, a thermocouple type medium temperature sensor S provided in the immersion medium 13 and fixed to the water immersion objective lens 12 is provided. _T However, the control device 32 is connected. This medium temperature sensor S _T Is fed back to the control device 32 to control the temperature of the temperature control device 26.
[0076]
The water supply bottle 22 and the drainage bottle 30 are arranged inside the apparatus main body 1, but they are arranged so that they can be easily replaced from the outside of the apparatus main body 1, or the immersion medium 13 can be supplied to the water supply bottle 22. It has a configuration. Of course, if the water supply bottle 22 and the drainage bottle 30 are arranged outside the apparatus main body 1, the replacement of the water supply bottle 22 and the drainage bottle 30 can be further started, and the supply of the immersion medium 13 to the water supply bottle 22 can be simplified.
[0077]
Further, a hot air blower 34 and carbon dioxide (CO 2) ₂ A humidifying water 37 and a heater 36 for heating the humidifying water 37 are provided outside the supply device and the device main body 1. The control device 32 is connected to the hot air blower 34 and the heater 36. Thermocouple type temperature / humidity sensor S provided inside the apparatus main body 1 _TH Are connected to the control device 32.
[0078]
In such a configuration, first, a microplate 5 in which a large number of wells 5a accommodate fluorescently labeled specimens 4 is prepared. Then, the microplate 5 is inserted into the inside of the apparatus main body 1 through the microplate insertion window 1a, and is placed on the XY stage 3.
[0079]
In this state, when the XY stage 3 is moved and the first well 5a is positioned on the optical axis of the water immersion objective lens 12, the immersion medium 13 prepared in the water supply bottle 22 passes through the water supply pipe 23a. The water is supplied to the water supply pump 24 and supplied to the gap between the water immersion objective lens 12 and the cover glass 5b from the water supply nozzle 25 via the water supply pipes 23b and 23c. In this case, the immersion medium 13 is held in place by surface tension.
[0080]
At this time, the immersion medium 13 is controlled to a predetermined temperature by the temperature control device 26. As a result, when the immersion medium 13 comes into contact with the cover glass 5b, the sample 4 is subjected to heat exchange between the immersion medium 13 and the cover glass 5b and further heat exchange between the cover glass 5b and the sample 4. It is controlled to a predetermined temperature. In this case, the cover glass 5b having a thickness of about 0.17 mm is used, and its heat capacity is made smaller than that of the surrounding materials, so that it does not hinder heat exchange with the specimen 4, and conversely. Since heat conductivity is good, heat exchange can be advantageously performed.
[0081]
Carbon dioxide (CO ₂ ) The PH concentration in the liquid of the specimen 4 is adjusted by the supply device. This is CO ₂ Is supplied to the inside of the main body 1, as a result, the PH of the specimen can be adjusted. The humidification water 37 heated by the heater 36 is heated and cooled by the temperature / humidity sensor _TH Is controlled by feeding back the value of 標本, and the sample 4 is prevented from drying.
[0082]
Temperature / humidity sensor S by hot air blower 34 _TH And the temperature of the inverted microscope 2, the stage receiver 2 ', and the XY stage 3 are controlled to be substantially the same as the temperature of the immersion medium 13. In addition, in an example of a biological cell, the temperature is maintained between ± 1 ° C. around 37 ° C.
[0083]
In this state, by performing the above-described fluorescence observation, the fluorescence of the biological cells in the liquid of the specimen 4 is captured by the imaging device 18 ′, and the biological cells are displayed on the display unit 21 via the personal computer 20.
[0084]
When the inspection of the first specimen 4 is completed, the XY stage 3 moves, the next well 5a is positioned on the optical axis of the water immersion objective lens 12, and the same fluorescence observation is performed.
[0085]
In this case, when the cover glass 5b moves with respect to the water immersion objective lens 12 with the movement of the XY stage 3, the immersion medium 13 that cannot be held between the water immersion objective lens 12 and the cover glass 5b falls. The liquid immersion medium 13 is received by a liquid receiving portion 27 provided around the water immersion objective lens 12. Then, the immersion medium 13 is sent from the drain port 27 a to the drain pipe 28, and is collected by the drain pump 29 in the drain bottle 30. At the same time, a certain amount of the immersion medium 13 is supplied from the water supply nozzle 25 to the gap between the water immersion objective lens 12 and the cover glass 5b.
[0086]
Hereinafter, fluorescence observation is performed for all the wells 5a of the microplate 5 in the same manner, and the biological cells are displayed on the display unit 21 by the personal computer 20.
[0087]
The XY movement and focusing of the microplate 5 are performed by the control device 32 via the personal computer 20 in accordance with an instruction from the input device 33. _XY And motor M for focusing _Z Done by The instruction from the input device 33 may be sent directly to the control device 32.
[0088]
Therefore, according to the above embodiment, the immersion medium 13 used for the immersion objective lens 12 is focused on, and the immersion medium 13 filled in the gap between the immersion objective lens 12 and the cover glass 5b is determined in advance. Therefore, the temperature of all the specimens 4 placed in the wells 5 a of the microplate 5 can be controlled to the predetermined temperature of the immersion medium 13. In addition, the immersion medium 13 causes the water immersion objective lens 12 to have substantially the same temperature as the predetermined temperature. The temperature of the inverted microscope 2 and the specimen 4 is also maintained at a predetermined temperature by the warm air blower 34, and the temperature of the water immersion objective lens 12 is also substantially the same as the predetermined temperature.
[0089]
Thus, the biological cells in the liquid of the specimen 4 can be constantly maintained at a constant temperature, so that stable fluorescence observation can be performed on the specimen 4 in all the wells 5a of the microplate 5.
[0090]
Further, by setting a large amount of the immersion medium 13 supplied to the gap between the water immersion objective lens 12 and the cover glass 5b, the temperature of the specimen 4 can be stably controlled, and the immersion medium can be controlled. Since the supply and discharge of the sample 13 are frequently repeated, the temperature control of the sample 4 can be easily changed, so that accurate temperature control can be performed.
[0091]
In the above-described embodiment, the temperature control device 26 is disposed between the water supply pipes 23b and 23c, but instead or simultaneously, a bottle temperature controller 31 that controls the temperature of the entire water supply bottle 22 is provided. You may do so.
[0092]
Further, in the above-described embodiment, the immersion medium 13 of the water supply bottle 22 is supplied to the gap between the immersion objective lens 12 and the cover glass 5b, and the excess immersion medium 13 is collected in the drainage bottle 30. However, the liquid immersion medium 13 supplied between the water immersion objective lens 12 and the cover glass 5b may be circulated. In this case, foreign matter and the like may be mixed into the immersion medium 13 by the circulation of the immersion medium 13, and thus it is necessary to surely remove the foreign matter and the like using a filter or the like.
[0093]
Further, noting that the temperature inside the apparatus main body 1 is several degrees higher than the outside temperature, a heat exchanger is provided in place of the temperature control device 26, and the immersion medium 13 is set to the same temperature as the inside of the apparatus by the heat exchanger. May be supplied to the gap between the water immersion objective lens 12 and the cover glass 5b.
[0094]
Furthermore, in the above-described embodiment, an example of the water immersion method using pure water for the liquid immersion medium 13 has been described, but an oil immersion method using oil can be similarly applied.
[0095]
In the above-described embodiment, the case of the inverted microscope has been described, but the present invention can be similarly applied to an upright microscope. Although the example of the water immersion method using pure water for the liquid immersion medium 47 has been described, the oil immersion method using oil can be similarly applied.
[0096]
As described above, according to the embodiment of the present invention, by using the immersion medium supplied to the immersion objective lens, the temperature of the specimen can be stably controlled, the configuration is simple, An immersion medium supply device, a fluorescence spectroscopy device, and a culture microscope that are inexpensive can be provided.
[0097]
The present invention is not limited to the above embodiments, and various other modifications can be made in the implementation stage without departing from the spirit of the invention. Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements.
[0098]
Further, for example, even if some components are deleted from all the components shown in each embodiment, the problems described in the section of the problem to be solved by the invention can be solved, and the effects described in the effects of the invention can be solved. Is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0099]
From the above embodiments, for example, the following inventions can be extracted. The following inventions may be applied independently, or may be applied in appropriate combinations.
[0100]
An immersion medium supply device according to a first aspect of the present invention includes an immersion medium filled between an observation sample and an immersion objective lens, and a temperature controller that controls the immersion medium to a predetermined temperature. An immersion medium supply unit that supplies the immersion medium, the temperature of which is controlled by the temperature controller, to the immersion objective lens. By adjusting the immersion medium filled between the immersion objective lens and the observation sample to a predetermined temperature in advance and supplying it, the sample can be controlled to a predetermined temperature. Thus, for example, in the case of observing a biological specimen, an appropriate form can be observed, and stable data can be obtained in fluorescence photometry that captures molecular motion, and correct judgment can be made.
Further, since the temperature of the immersion medium is controlled by using the immersion medium used for the immersion objective lens, the configuration of the entire apparatus can be simplified and the cost can be reduced.
[0101]
In the first aspect, preferred embodiments are as follows.
(1) The immersion medium supply unit includes an immersion medium supply source and a liquid supply nozzle for supplying the immersion medium to the immersion objective lens, and the temperature controller includes an immersion medium supply source and a liquid supply nozzle. Be provided between.
(2) The water supply nozzle of the liquid immersion medium supply unit is at least one or more and is detachably held by the liquid immersion objective lens.
(3) The water supply nozzle is embedded in an outer frame of the immersion objective lens.
(4) The water supply nozzle is buried in a liquid receiving portion provided around a distal end of the liquid immersion objective lens.
[0102]
The fluorescence spectroscopy apparatus according to the second aspect of the present invention includes an immersion objective lens for condensing light from a specimen of a fluorescently labeled biomolecule floating in a liquid, the specimen and the immersion objective lens, An optical system including an immersion medium to be filled in between, an optical detector for detecting at least an enlarged image of the immersion objective lens, and fluorescent illumination; and a motion of the specimen is calculated from an output of the optical detector. A computing unit, a monitor that displays a computation result by the computing unit, a temperature controller that controls the immersion medium to a predetermined temperature, and the immersion medium that is temperature-controlled by the temperature controller. An immersion medium supply unit for supplying the liquid to the lens. Here, it is preferable that the calculation unit performs calculation by fluorescence correlation spectroscopy. When analyzing the fluctuations in fluorescence intensity to obtain information about the diffusion rate and information about the number of molecules, the temperature of the sample is kept constant, so that the motion of suspended molecules in the liquid of the sample is kept constant. And stable fluorescence detection can be performed on the specimens in all the wells of the microplate. In fluorescence measurement that captures molecular motion, stable data can be obtained, and correct judgment can be made.
[0103]
In the first and second aspects, it is preferable to further include a sensor for measuring a temperature of the immersion medium. Here, it is preferable that the sensor is arranged near the immersion objective lens.
[0104]
A culture microscope according to a third aspect of the present invention includes a stage for mounting an observation sample, a microscope supporting the stage, a liquid immersion objective lens supported by the microscope, and an observation sample housed in the microscope. An incubator accommodating an imaging device and an optical system for capturing an enlarged image of an immersion medium filled between the observation sample and the immersion objective lens; and setting the immersion medium to a predetermined temperature. A temperature controller for controlling, an immersion medium supply unit that supplies the immersion medium, the temperature of which is controlled by the temperature controller, to the immersion medium lens, and a hot air blower that sends warm air to the incubator. It is characterized by doing. Here, it is preferable that a sensor for measuring the temperature of the immersion medium and the humidity in the incubator is further provided, and the sensor is disposed near the immersion objective lens. The observation sample and the immersion objective can be maintained at a predetermined temperature with the immersion medium, and the microscope and the immersion objective can be maintained at the predetermined temperature by maintaining the inside of the culture device at a predetermined temperature with a warm air blower. Become. When the immersion objective lens reaches a predetermined temperature, the observation sample via the immersion medium becomes more stable.
[0105]
In the first to third aspects, it is preferable that the apparatus further includes an immersion medium discharge unit that discharges the immersion medium supplied from the immersion medium supply unit to the immersion objective lens.
Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent features. For example, even if some components are deleted from all the components shown in the embodiments, the problem described in the column of the problem to be solved by the invention can be solved, and the problem described in the column of the effect of the invention can be solved. In the case where the effect described above is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention. For example, although not specifically stated in the embodiment, as described in the second embodiment, the method of adjusting the internal environment by covering the entire microscope with a box-shaped object is described in the first embodiment. The present invention can also be applied to a fluorescence measuring device of the form (1).
[0106]
【The invention's effect】
According to the present invention, it is possible to provide an immersion medium supply device, a fluorescence spectroscopic inspection device, and a culture microscope that can stably control the temperature of a specimen, have a simple configuration, and are inexpensive.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.
FIG. 2 is a diagram illustrating an appearance of an apparatus main body according to the first embodiment.
FIG. 3 is a diagram showing a schematic configuration of a microplate used in the first embodiment.
FIG. 4 is an enlarged view showing a configuration between a water immersion objective lens according to the first embodiment and a cover glass of a microplate.
FIG. 5 is a diagram showing a schematic configuration of a modified example of the first embodiment of the present invention.
FIG. 6 is a diagram showing a schematic configuration of a second embodiment of the present invention.
[Explanation of symbols]
R _XY …rack
P _XY … Pinion
M _XY ... Drive motor
R _Z …rack
P _Z … Pinion
M _Z …motor
S _T … Temperature sensor
S _TH … Temperature and humidity sensor
1. The device body
1a: Microplate insertion window
2. Inverted microscope
3. XY stage
4 ... Specimen
5a ... Well
5. Microplate
5b ... Cover glass
6.7 ... Laser light source for excitation
6 ... Light source
8 Half mirror
9 ... Mirror
10 ... Collimating lens
11 ... Dichroic mirror
12 Objective lens
13 ... Immersion medium
14. Excitation light barrier filter
15 ... imaging lens
16 ... Reflection mirror
17 ... Pinhole
18. Photodetector (imaging device)
19 arithmetic unit
20 ... PC
21 Display unit
22 ... water bottle
23a, 23b, 23c ... water supply pipe
24 ... Water supply pump
25 ... water supply nozzle
26 ... Temperature control device
27a ... drain
28 ... Drainage pipe
29… Drain pump
30 ... drainage bottle
31… Bottle temperature controller
32 ... Control device
33 ... Input device
34 ... Hot air blower
36 ... heater
37 ... Humidifying water
41 ... Microscope body
41a: Front fixed part
41b ... rear fixed part
42 ... Stage
43: Petri dish specimen
44 ... Water immersion objective
45 ... Observation lens barrel
46 ... Eyepiece
47 ... Immersion medium
48… water supply pipe
49… Water supply nozzle
51 ... drainage pipe
52 ... Transmission illumination light source
53… Condenser lens

Claims (3)

An immersion medium filled between the observation sample and the immersion objective,
A temperature controller for controlling the immersion medium to a predetermined temperature,
An immersion medium supply unit for supplying the immersion medium whose temperature is controlled by the temperature controller to the immersion objective lens. An immersion objective that collects light from a specimen of fluorescently labeled biomolecules floating in the liquid,
An immersion medium filled between the specimen and the immersion objective,
An optical system including a photodetector and fluorescent illumination for detecting at least an enlarged image of the immersion objective lens,
A calculation unit that calculates the movement of the sample from the output of the photodetector;
A monitor for displaying a calculation result by the calculation unit,
A temperature controller for controlling the immersion medium to a predetermined temperature,
An immersion medium supply unit for supplying the immersion medium, the temperature of which is controlled by the temperature controller, to the immersion medium lens. A stage for mounting the observation sample, a microscope supporting the stage, an immersion objective lens supported by the microscope, an imaging device and an optical system that capture an enlarged image of the observation sample housed in the microscope, And an incubator for storing
An immersion medium filled between the observation sample and the immersion objective,
A temperature controller for controlling the immersion medium to a predetermined temperature,
An immersion medium supply unit that supplies the immersion medium, the temperature of which is controlled by the temperature controller, to the immersion medium lens;
And a warm air blower for feeding warm air to the incubator.

Images