Microscopy and observation methods

Abstract

The microscope (1) of the present embodiment has an objective lens (11) that is irradiated with light from a sample (200), and a sample freezing unit that freezes the sample (200) by supplying a freezing agent (160) to the sample (200) in the field of view of the objective lens (11) or bringing the freezing agent (160) into contact with the sample (200). By using the microscope (1) of this structure, it is possible to freeze and observe the sample in the field of view of the objective lens (11).

Description

Technical Field

[0001] This invention relates to microscopes and observation methods, and more particularly to techniques for observing frozen samples. Background Technology

[0002] In optical microscopy of biological samples, samples are sometimes pre-fixed to avoid changes caused by observation or pre-treatment. In such cases, chemical fixation using aldehydes based on cross-linked proteins or dehydration fixation using organic solvents are employed. These fixation methods are sufficient to maintain morphological information at the optical microscopy observation level. However, on the other hand, depending on the molecules, they may sometimes be oxidized by the aldehyde or dissolved in the organic solvent. Furthermore, the fixation reaction takes several minutes, and even longer is required for thick samples to fix deeper layers. There is also the problem that not all molecules may be fixed.

[0003] Existing technical documents

[0004] Patent documents

[0005] Non-patent literature 1: Fuest, et al., J. Microsc. 272, 87 (2018). Summary of the Invention

[0006] Against this backdrop, freeze fixation, as a novel method for fixing biological samples, has recently gained attention. Freeze fixation physically stops the movement of molecules and ions in the sample by freezing water. As long as freezing is performed within a short period of time, biological samples can be fixed in their original state.

[0007] Non-Patent Document 1 discloses a method using a heat sink and a heater. In this method, a thin NiCr heater is disposed on a heat sink cooled by liquid nitrogen. By controlling the start and stop of the heater, the sample placed on the heater can be frozen at any time. By allowing the sample to enter a microchannel, the sample volume can be reduced. In this method, nematodes were successfully frozen in tens of microseconds. However, in this prior art, the freezing rate is limited by the thickness of the heater.

[0008] The microscope of this embodiment includes: an objective lens that is illuminated by light from a sample; and a sample freezing unit that freezes the sample by supplying a cryopressor to the sample within the field of view of the objective lens or by contacting the sample.

[0009] In the microscope described above, it is also possible to observe the sample while the cryoprotectant is being supplied.

[0010] In the microscope described above, the microscope may also be equipped with an introduction mechanism that introduces the cryoprotectant into the sample based on a signal from the sample or a signal used to stimulate the sample.

[0011] In the microscope described above, the sample freezing unit may also have a supply pipe through which the cryo-ant flows, and the valve provided in the supply pipe of the cryo-ant becomes the inlet mechanism.

[0012] In the aforementioned microscope, the microscope may also have a pressurizing unit for pressurizing the sample, thereby freezing the sample while pressurizing it.

[0013] In the microscope described above, a pressurizing unit can also be used to pressurize the sample while observing it.

[0014] In the aforementioned microscope, the microscope may also be equipped with a control unit that controls the timing of pressurizing the sample and the timing of introducing the cryoprotectant.

[0015] In the microscope described above, the liquid cryoprotectant can also be supplied to the sample from above.

[0016] In the aforementioned microscope, the microscope may also have a sample holder for holding the sample, and the sample holder may be provided with a refrigerant storage tank for storing refrigerant or a flow channel for refrigerant flow.

[0017] In the microscope described above, the objective lens may be positioned above the sample, the sample freezing unit may have a nozzle that sprays the cryoprotectant onto the sample from above, and a protective shell may be provided below the objective lens.

[0018] The microscope described above may also have the function of melting at least a portion of the frozen sample by irradiating it with light.

[0019] The observation method of this embodiment includes the step of placing the sample within the field of view of the objective lens;

[0020] The steps of freezing the sample by supplying or moving a cryoprotectant into the field of view of the objective lens; and the steps of observing the frozen sample.

[0021] In the above observation method, the sample can also be observed while the refrigerant is being supplied.

[0022] In the above observation method, the cryoprotectant may also be introduced into the sample based on a signal from the sample or a signal used to stimulate the sample.

[0023] In the above observation method, a supply pipe through which the refrigerant flows can be provided, and the timing of introducing the refrigerant can be adjusted by a valve provided in the refrigerant supply pipe.

[0024] In the above observation method, at least a portion of the frozen sample can also be melted by irradiating it with light.

[0025] According to the present invention, a microscope and observation method capable of freezing and observing samples can be provided. Attached Figure Description

[0026] Figure 1 This is a perspective view showing the structure of the sample holder.

[0027] Figure 2 This is a perspective view showing the structure of the sample holder.

[0028] Figure 3 This is a diagram showing the structure of the optical microscope according to Embodiment 1.

[0029] Figure 4 It is a cross-sectional view schematically showing the structure of the specimen.

[0030] Figure 5 This is a schematic diagram illustrating an example of a pressurizing unit that applies pressure to a sample.

[0031] Figure 6 These are photographic images of the sample before and after freezing.

[0032] Figure 7 This is a diagram showing the structure of the inverted microscope according to Embodiment 2.

[0033] Figure 8 This is a cross-sectional view showing the detailed structure of the refrigerant supply section.

[0034] Figure 9 This is a perspective view showing the detailed structure of the refrigerant supply section.

[0035] Figure 10 This is a diagram showing the structure of the upright microscope according to Embodiment 3.

[0036] Figure 11 This is a side view showing the detailed structure of the refrigerant supply section.

[0037] Figure 12 It is a photographic image showing the sample as it is frozen.

[0038] Figure 13 This is a perspective view showing the specimen holder of modified Example 1.

[0039] Figure 14 This is a perspective view showing the specimen holder of modified Example 1.

[0040] Figure 15 This is a perspective view showing the specimen holder of modified example 2.

[0041] Figure 16 This is a perspective view showing the specimen holder of modified example 2.

[0042] Figure 17 This is a schematic diagram showing the structure of the microscope according to Embodiment 4.

[0043] Figure 18 It is a waveform diagram showing the timing of refrigerant administration corresponding to electrocardiogram and electrical stimulation.

[0044] Figure 19 This is a schematic diagram showing the structure of the microscope according to Embodiment 5.

[0045] Figure 20 This is a schematic diagram showing the structure of the microscope according to Embodiment 6. Detailed Implementation

[0046] Hereinafter, embodiments to which the present invention can be applied will be described. In the following description, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments. For clarity, appropriate omissions and simplifications are sometimes made in the following description. Furthermore, those skilled in the art can easily modify, add to, or transform the elements of the following embodiments within the scope of the present invention. In addition, in the figures, elements labeled with the same reference numerals represent the same elements, and descriptions are appropriately omitted.

[0047] Implementation Method 1

[0048] The optical microscope (hereinafter, also simply referred to as the microscope) of this embodiment uses a cryostat to freeze the sample. The microscope has a sample freezing unit that freezes the sample by supplying a cryostat. The sample freezing unit supplies a cryostat to the sample within the objective lens's field of view. The cryostat supplied from the sample freezing unit freezes the sample. The sample within the objective lens's field of view can be frozen. Therefore, the frozen sample can be easily observed.

[0049] The structure of the sample holder 100 with a sample freezing unit will be described. Furthermore, the XYZ three-dimensional Cartesian coordinate system is appropriately shown in the following figures. The Z direction is parallel to the optical axis. The XY plane is a plane orthogonal to the Z direction and serves as the focal plane for arranging the sample.

[0050] Figure 1and Figure 2 This is a perspective view showing the structure of the sample holder 100. The sample holder 100 has a first plate 110, a second plate 120, and a refrigerant supply unit 130. The refrigerant supply unit 130 serves as a freezing unit for freezing the sample by supplying refrigerant to the sample.

[0051] The sample holder 100 is used in an inverted microscope where the objective lens is positioned directly below the sample. Liquid or solid cryogenic agents can be used as the cryogenic agent. Figure 1 This indicates that the refrigerant supply unit 130 is in the off state. Figure 2 This shows the refrigerant supply unit 130 in the open position. Figure 1 The specimen was observed in the state shown. Figure 2 The sample is positioned in the sample holder 100 as shown in the diagram.

[0052] The first plate 110 and the second plate 120 are disc-shaped components. The first plate 110 and the second plate 120 are, for example, metal plates such as stainless steel. The first plate 110 and the second plate 120 are not limited to stainless steel and can be formed from metal plates such as titanium. Alternatively, the first plate 110 and the second plate 120 can be formed from polymer plates with low thermal conductivity that are not easily cracked even at low temperatures. Polymer plates can be made of materials such as Teflon (registered trademark), polycarbonate, and polylactic acid. Furthermore, the first plate 110 and the second plate 120 can also be ceramic plates used for thermal insulation materials.

[0053] The first plate 110 serves as the base for the microscope stage. A disc-shaped second plate 120 is fixed to the first plate 110. In the XY plane, the first plate 110 and the second plate 120 are approximately identical in shape.

[0054] An opening 113 is provided at the center of the first plate 110 in the XY plane. The opening 113 extends through in the Z direction. The opening 113 and its periphery form a sample setting section 117. A sample is set in the sample setting section 117. Therefore, the sample setting section 117 becomes the focal plane of the objective lens.

[0055] The refrigerant supply section 130 is connected to the second plate 120 via a hinge 125. The refrigerant supply section 130 serves as a cover over the opening 113. The refrigerant supply section 130 is opened and closed by rotating about the axis of the hinge 125. By closing the refrigerant supply section 130, it is positioned above the opening 113. Furthermore, by opening the refrigerant supply section 130, the upper side of the opening 113 is opened. This allows the sample in the sample setting section 117 to be replaced.

[0056] A cylindrical refrigerant storage section 133 is provided at the upper part of the refrigerant supply section 130. With the refrigerant supply section 130 closed, the refrigerant storage section 133 is located directly above the opening 113. A refrigerant inlet 134 is provided on the upper side of the refrigerant storage section 133. By allowing liquid refrigerant to flow in through the refrigerant inlet 134, refrigerant is stored in the refrigerant storage section 133. The refrigerant comes into contact with the sample in the sample placement section 117, cooling the sample. As a result, the sample freezes. For example, to supply refrigerant, the user simply moves the refrigerant from the container to the refrigerant storage section 133.

[0057] A gas supply port 111 is provided on the side of the first plate 110. The gas supply port 111 is connected to the opening 113. Anti-condensation gas sprayed onto the objective lens is supplied through the gas supply port 111.

[0058] A refrigerant outlet 127 is provided on the upper surface of the second plate 120. The refrigerant outlet 127 communicates with the opening 113. Refrigerant outlets 127 are arranged on both sides of the refrigerant supply section 130 in the X direction. The refrigerant supplied by the refrigerant supply section 130 is discharged from the refrigerant outlet 127. That is, the refrigerant used to cool the sample is discharged from the refrigerant outlet 127. Therefore, since a continuous supply of fresh refrigerant is possible, the cooling rate can be increased. Of course, the refrigerant outlet 127 can also be omitted.

[0059] Figure 3 This is a side cross-sectional view showing the sample holder 100, which has a refrigerant supply section 130, positioned on the stage 10 of the microscope 1. Additionally, Figure 3 The sample holder 100 shown is not related to Figure 1 , Figure 2 The sample holder 100 shown is strictly consistent. For example, in Figure 1 , Figure 2 In the middle, the axis of hinge 125 is parallel to the X direction, but... Figure 3 In this configuration, the axis of hinge 125 is parallel to the Y-direction. Furthermore, O-rings O1 to O4 are provided to prevent leakage of the liquid refrigerant 160. By preventing the refrigerant 160 from leaking from the sample holder 100, operation can be easily performed.

[0060] The sample holder 100 is fixed on the stage 10. An objective lens 11, which receives light from the sample 200, is disposed within the opening 113 of the sample holder 100. The objective lens 11 is inserted into the opening 113 from below. The sample 200 is positioned directly above the objective lens 11. The sample 200 is positioned within the field of view of the objective lens 11. This allows for simultaneous observation of the sample 200 and freezing. The sample 200 is sealed to the first plate 110 by an O-ring O1. A microscope body 12 is disposed below the objective lens 11. The microscope body 12 includes an optical system comprising a light source, a camera, a mirror, and an imaging lens. Light from the objective lens 11 propagates within the microscope body 12. The camera captures an image of the sample 200 imaged by the imaging lens. Alternatively, the microscope body 12 may also have an eyepiece for direct observation by the user.

[0061] A refrigerant supply section 130 is disposed on the first plate 110. An O-ring O2 seals the first plate 110 and the refrigerant supply section 130. The refrigerant supply section 130 has a refrigerant storage section 133 at its upper part for storing refrigerant. The refrigerant storage section 133 is disposed directly above the sample 200. The refrigerant storage section 133 and the sample setting section (in...) Figure 3 The sample 200 (not shown in the figure) is connected. Refrigerant 160 is stored in the refrigerant storage section 133 of the refrigerant supply section 130. Refrigerant 160 flows into the refrigerant storage section 133 from the refrigerant inlet 134. The sample 200 is frozen by bringing the refrigerant 160 into contact with it.

[0062] A sample clamping member 135 is provided inside the refrigerant storage section 133. The sample clamping member 135 clamps the sample 200 from above, thereby fixing the sample 200. The sample clamping member 135 is configured to be detachable from the refrigerant supply section 130. The sample clamping member 135 can be replaced according to the thickness, size, etc. of the sample 200. The sample clamping member 135 and the sample 200 are sealed by an O-ring O4. In addition, the sample clamping member 135 is sealed to the inner wall of the refrigerant storage section 133 by an O-ring O3.

[0063] A gas supply port 111 leading to the opening 113 is provided on the side wall of the first plate 110. Dried nitrogen gas is supplied through the gas supply port 111. The nitrogen gas from the gas supply port 111 flows in the space between the objective lens 11 and the first plate 110. The nitrogen gas from the gas supply port 111 is sprayed onto the objective lens 11. By spraying nitrogen gas to prevent condensation onto the objective lens 11, condensation on the objective lens 11 and the sample 200 can be prevented. Furthermore, a thermocouple 17 for temperature measurement is provided on the wall surface of the first plate 110 on the side of the opening 113 or on the lower surface of the sample 200. This allows for the evaluation of the cooling temperature of the refrigerant 160.

[0064] The gas supply port 111 can also be used as a gas suction port to draw gas from the periphery of the objective lens 11. That is, a vacuum pump or the like draws gas through the gas supply port 111. This vents the space between the objective lens 11 and the first plate 110. This allows the periphery of the objective lens 11 to be in a depressurized or vacuum state. In this way, condensation on the objective lens 11 can be prevented. Furthermore, by being in a depressurized or vacuum state, the heat transferred from the objective lens 11 to the sample 200 can be reduced. This improves thermal insulation.

[0065] Figure 4 This is a detailed cross-sectional view showing an example of a frozen specimen 200. The specimen 200 has a substrate 201, a gasket 202, a cover glass 203, and an object of observation 204.

[0066] The substrate 201 is, for example, a circular copper plate with a thickness of 0.2 mm and a diameter of 25 mm. Of course, the material of the substrate 201 is not limited to copper. For example, the material of the substrate 201 can also be diamond, SiC (silicon carbide), SiN (silicon nitride), stainless steel, etc. The cover glass 203 is a transparent glass substrate with a thickness of 0.17 mm. The spacer 202 has a thickness of 0.02 mm.

[0067] The substrate 201 and the cover glass 203 are arranged facing each other with a spacer 202 between them. That is, a gap corresponding to the thickness of the spacer 202 is formed between the substrate 201 and the cover glass 203. The object of observation 204 is arranged in the gap between the substrate 201 and the cover glass 203.

[0068] Excitation light is incident on the object 204 from the coverslip 203 side. Then, fluorescence emitted from the coverslip 203 side enters the objective lens. A liquid cryoprotectant is supplied from the substrate 201 side. The liquid cryoprotectant is a mixture of liquid propane and isopentane in a ratio of 2:1 or 3:1.

[0069] In this way, a refrigerant supply unit 130 is provided to supply refrigerant to the sample 200. Therefore, the object 204 in the field of view of the objective lens can be frozen quickly, and the frozen sample 200 can be observed.

[0070] In this embodiment, the cryoprotectant is supplied directly to the sample 200. That is, the cryoprotectant comes into contact with the substrate 201 of the sample 200, thus freezing the sample 200 in a short time. Biological samples can be fixed in their original state. For cells or small biological tissue sections, millisecond-level freezing can be achieved. By directly supplying liquid cryoprotectant to the internal organs of a biological organism, the original state of the organism can be fixed.

[0071] Because the refrigerant is supplied directly, even large samples 200 can be frozen in a short time. The limitations on the thickness and size of the sample 200 are reduced. Therefore, a wide variety of samples 200 can be frozen and observed. In particular, since only a thin substrate 201 is placed between the object of observation 204 and the refrigerant, rapid cooling and freezing are possible. Rapid freezing can be performed even if the sample 200 is at room temperature before being added to the refrigerant.

[0072] Alternatively, the substrate 201 disposed between the objective lens 11 and the object of observation 204 can be made into a transparent substrate. In this way, illumination light and excitation light can be irradiated onto the object of observation from the substrate 201 side. By making the substrate 201 a transparent substrate, observation can be performed from the substrate 201 side.

[0073] The cryoprotectant is not limited to a liquid; it can also be a solid such as a powder. For example, cooled metal can be used as a cryoprotectant. Then, by moving the metal to contact the sample 200, the sample 200 can be frozen. The cryoprotectant supply unit 130 can freeze the sample 200 by moving the cryoprotectant from a position away from the sample 200 toward the sample 200. The sample freezing unit for freezing the sample by cryoprotectant is not limited to the above example. The sample freezing unit only needs to be able to freeze the sample by supplying or moving the cryoprotectant into or toward the sample within the field of view of the objective lens 11. The cryoprotectant only needs to be able to cool to a temperature lower than the freezing temperature of the sample.

[0074] The sample freezing unit is not limited to a structure that supplies the refrigerant in contact with the sample 200. That is, the sample freezing unit can also be designed to supply the refrigerant in a manner that prevents the refrigerant from contacting the sample 200. For example, a structure in which the refrigerant cools the sample through a substrate, film, sheet, or similar material can also be used. In other words, the sample freezing unit supplies the refrigerant to the sample, bringing the refrigerant into contact with a substance other than the sample. The sample is frozen by cooling it through another substance. Thus, the sample freezing unit can supply the refrigerant while the sample 200 is separated from the refrigerant by another substance.

[0075] Furthermore, two or more refrigerants can be used. For example, a first refrigerant for freezing the sample 200 and a second refrigerant for maintaining the frozen state of the sample 200 can be used separately. First, the first refrigerant is supplied to the sample 200 at room temperature. After the sample 200 freezes, the supply of the first refrigerant is stopped, and the second refrigerant is supplied. The second refrigerant maintains the frozen state of the sample 200. Thus, the frozen state of the sample 200 can be observed. The first and second refrigerants can be supplied from the same refrigerant supply unit or from different refrigerant supply units. Furthermore, a cooling unit other than a refrigerant can also be used. For example, electronic cooling such as a Peltier element can be used.

[0076] It is also possible to observe the sample 200 while supplying refrigerant. That is, refrigerant can be continuously supplied from the refrigerant supply unit 130 while observing the sample 200. For example, by providing a refrigerant outlet 127, etc., new refrigerant can be continuously supplied. As a result, cooling can be continuously performed to prevent the sample 200 from melting.

[0077] Alternatively, a refrigerant can be supplied to the sample 200 while pressure is being applied to it. This freezes the sample under pressure. By pressurizing the sample, the formation of ice crystals can be suppressed. For example, the pressurizing unit applies a pressure of 210 MPa or less to the sample 200. Even with a thick sample 200, it is possible to freeze the sample 200 without forming ice crystals.

[0078] As a pressurizing unit for pressurizing the sample, pressure jetting can be used. For example, nozzles for ejecting gas can be provided on the upper and lower sides of the sample 200. Then, pressure is applied to the sample by ejecting high-pressure gas from above and below. In this way, freezing can be performed under pressure.

[0079] Alternatively, a pressure-applying mechanism such as a piston can be used as the unit for applying pressure. Figure 5 This diagram schematically illustrates the structure using piston 300 as the pressurization unit. Figure 5 In this design, a piston 300 is positioned on the upper side of the sample 200, and an objective lens 11 is positioned on the lower side. A refrigerant supply section 301 is provided inside the piston 300. Specifically, a through hole extending from the side to the bottom of the piston 300 serves as the refrigerant supply section 301. In this way, the mechanism for applying pressure and the mechanism for freezing are provided separately.

[0080] Two openings 302 are provided on the side of the piston 300, and an opening 303 is provided in the center of the bottom surface. A refrigerant supply section 301 is formed by the passage from one opening 302 through the opening 303 to the other opening 302. One opening 302 serves as the refrigerant supply port, and the other opening 302 serves as the refrigerant discharge port. The refrigerant supplied from one opening 302 flows to the opening 303. Thus, the sample 200 facing the opening 303 is frozen by the refrigerant. Then, the frozen refrigerant is discharged from the other opening 303. In this way, freezing can be performed while pressure is applied to the sample 200. Furthermore, observation can be performed while pressure is being applied.

[0081] The timing of pressure application can also be synchronized with the timing of refrigerant introduction and stimulation of sample 200. The control of the timing of refrigerant introduction, pressure application, and stimulation of sample 200 will be described later.

[0082] Figure 6 This image shows microscopic images of the sample at room temperature and after freezing. The sample was composed of Fluo-4-labeled HeLa cells. The objective lens was 60x with a numerical aperture of 0.95, and the excitation source was a mercury lamp. The excitation light intensity in the sample was 1.04 mW.

[0083] For samples at room temperature, the microscope camera exposure time was set to 30 ms for imaging. For frozen samples, the microscope camera exposure time was set to 5 sec. This allows for instantaneous freezing of the sample 200 by supplying cryoprotectant. The sample can be frozen while maintaining cell shape. Because the camera exposure time can be extended, the sample can be imaged with a high S / N ratio.

[0084] By simply introducing cryoprotectant at any time, the sample 200 under microscope observation can be fixed in a state close to its original shape. Furthermore, since it is independent of the type of optical microscope and the optical response used for observation, it can be applied to any optical microscope. With the microscope of this embodiment, dynamic snapshots of biological samples, which are difficult to accumulate signals over long periods, can be captured. The microscope 1 described above can be used in research observing biological samples. Furthermore, the microscope 1 described above can also be used clinically.

[0085] Implementation Method 2

[0086] In embodiment 2, the refrigerant supply unit includes a supply pipe and a valve. (Usage) Figures 7-9 The microscope 4 of Embodiment 2 will be described. Figure 7 This is a schematic diagram of the overall structure of microscope 4. Figure 8 This is a cross-sectional view showing the structure of the sample holder 401. Figure 9 This is a perspective view of the sample holder 401. Here, microscope 4 is an inverted microscope. Furthermore, liquid nitrogen is used as the cryoprotectant.

[0087] like Figure 7 As shown, a sample holder 401 is disposed on the microscope body 412. The refrigerant supply unit 460 includes a Dewar flask 450, a supply tube 461, and a valve 462. The supply tube 461 of the refrigerant supply unit 460 is connected to the sample holder 401. One end of the supply tube 461 is connected to the sample holder 401, and the other end is disposed inside the Dewar flask 450. The Dewar flask 450 and the sample holder 401 are connected via the supply tube 461. A valve 462 is disposed midway through the supply tube 461. The supply tube 461 is preferably covered with insulating material.

[0088] A refrigerant 451 is stored in a Dewar flask 450. A pressurized gas supply line 453 is connected to the Dewar flask 450 via a valve 454. The pressurized gas supply line 453 supplies nitrogen gas into the Dewar flask 450. The nitrogen gas from the pressurized gas supply line 453 makes the pressure inside the Dewar flask 450 higher than 1 atmosphere. In addition, the pressure inside the sample holder 401 is 1 atmosphere. The refrigerant is filled at the other end of the supply line 461 to the valve 462. When the valve 462 is opened, the refrigerant 451 in the supply line 461 is discharged into the sample holder 401. The refrigerant 451 passes through the supply line 461 to reach the sample.

[0089] A discharge pipe 473 for discharging refrigerant is connected to the sample holder 401. The discharge pipe 473 and the supply pipe 461 are connected via a flow channel 403. The refrigerant supplied to the sample holder 401 by the supply pipe 461 is discharged from the discharge pipe 473. One end of the discharge pipe 473 is connected to the sample holder 401, and the other end is disposed in a refrigerant recovery tank 470. The refrigerant recovery tank 470 is at a pressure of 1 atmosphere. The refrigerant used in cooling the sample is stored in the refrigerant recovery tank 470. In this way, the used refrigerant 471 can be recovered. Preferably, the discharge pipe 473 is covered with an insulating material.

[0090] Preferably, the supply pipe 461, valve 462, and discharge pipe 473 are pre-cooled. For example, the refrigerant is pre-flowed before the sample is placed in the sample holder 401. This allows the sample holder 401, supply pipe 461, valve 462, discharge pipe 473, etc., to be cooled. Then, after heating the sample holder 401, the sample is placed in the sample holder 401. Afterward, the sample 200 is frozen by opening valve 462 and allowing refrigerant to flow out from the supply pipe 461.

[0091] like Figure 8 and Figure 9 As shown, the sample holder 401 has a base 402 and a sample clamping member 405. Furthermore, as... Figure 8 As shown, the sample holder 401 has a cover 407. Additionally, in Figure 9 In the diagram, cover 407 is omitted. A flow channel 403 for refrigerant flow is formed in the base 402 or the sample clamping member 405. Furthermore, cover 407 covers the sample clamping member 405. Additionally, the base 402 has an opening 404. The opening 404 forms a through hole penetrating the base 402 in the Z direction. Figure 8 As shown, objective lens 11 is disposed in opening 404.

[0092] like Figure 8As shown, a flow channel 403 is provided within the sample holder 401. The flow channel 403 is arranged along the X direction. A supply pipe 461 and a discharge pipe 473 are connected via the flow channel 403. A sample 200 is positioned directly below the flow channel 403. A refrigerant flows directly above the sample 200. The refrigerant flowing within the flow channel 403 contacts the sample 200. This allows the sample 200 to be cooled.

[0093] The sample clamping member 405 is detachably mounted to the base 402. With the sample clamping member 405 removed from the base 402, the sample 200 is positioned at the opening 404 of the base 402. The sample 200 is positioned to cover the opening 404. Then, by mounting the sample clamping member 405 to the base 402 from above the sample 200, the sample 200 can be fixed to the sample holder 401.

[0094] Thus, by providing valve 462 to the supply pipe 461 of the refrigerant supply section 460, the sample 200 can be frozen at an appropriate time. The measurement results after measuring the temperature change over time using a thermocouple provided in the flow channel 403 are explained. The temperature immediately drops to -189.5°C after valve 462 is opened. Therefore, the refrigerant can be brought into contact with the sample 200 immediately after valve 462 is opened, freezing the sample 200. Thus, freezing can be achieved in a short time.

[0095] Valve 462 can also be opened and closed by a user operating a switch or similar device. Alternatively, a control unit 465 can be provided to control the opening and closing of valve 462. The control unit 465 can control the timing of opening and closing of valve 462. Valve 462 is opened and closed based on the control signal output from the control unit 465.

[0096] Regarding the signal used to open and close the trigger valve 462, various signals can be used. For example, the control unit 465 can control the valve based on a signal from the sample 200. Specifically, the control unit 465 outputs a control signal based on a light signal or an electrical signal from the sample 200. As a light signal from the sample 200, the intensity of scattered light or fluorescence intensity can be used. Furthermore, the sample 200 can be photographed, and a control signal corresponding to its shape, size, etc., can be used. For example, if the sample 200 is a pulsating internal organ, the control unit 465 outputs a control signal based on the morphological changes of the sample 200. Thus, it is possible to freeze the organ in time with the pulsation.

[0097] Based on the photographic image obtained by photographing the sample 200, the control unit 465 controls the opening and closing timing of the valve 462. Furthermore, in the case of fluorescence observation, the sample can be frozen in conjunction with the timing of changes in fluorescence intensity. If the sample 200 is a heart, the control unit 465 can also control the valve 462 based on the electrocardiogram signal. Thus, the control unit 465 can control the freezing timing based on signals from the sample 200. Therefore, the sample 200 can be frozen at the desired time.

[0098] Alternatively, based on a signal used to stimulate the sample 200, for example, when light stimulation, electrical stimulation, electromagnetic stimulation, magnetic stimulation, etc., are applied to the sample 200, the control unit 465 opens and closes the valve 462 according to the signal used to apply the stimulation. Thus, the sample 200 can be frozen in conjunction with the timing of stimulation application. Furthermore, the stimulation of the sample 200 is not limited to light stimulation, electrical stimulation, electromagnetic stimulation, or magnetic stimulation. Stimulation of the sample 200 can be applied through pharmaceuticals, temperature, mechanical stimulation, electromagnetic waves, electron beams, radiation, optogenetics, photodissociation of cage-like compounds, etc. By using a signal used to stimulate the sample 200, the sample can be frozen at an appropriate time.

[0099] In this way, the control unit 465 can adjust the timing of refrigerant introduction by controlling the opening and closing of the control valve 462. Of course, two or more signals can also be used for the control unit 465 to control the timing of refrigerant introduction. Furthermore, the refrigerant introduction mechanism for introducing refrigerant into the refrigerant supply unit and flow channel 403 is not limited to valve 462. For example, in... Figures 1-3 In structures such as these, a mechanism that drives the container of refrigerant by injecting refrigerant into the refrigerant inlet 134 can also be used. A container for storing refrigerant is positioned above the refrigerant inlet 134. Then, the container can be tilted according to a control signal. By tilting the container, refrigerant can be introduced into the refrigerant inlet 134. Alternatively, refrigerant can be introduced into the flow channel 403 by controlling a valve 454 for pressurizing the Dewar flask 450. For example, valve 462 is opened when refrigerant is introduced. This pressurizes the space inside the Dewar flask 450, and refrigerant 451 flows in the flow channel 403. Furthermore, the refrigerant can be supplied to the sample by switching the path of the refrigerant flowing in the flow channel near the sample. For example, two flow channels are prepared, one in contact with the sample and the other not, and a flow channel switching solenoid valve is positioned near the sample. First, the refrigerant flows in the non-contact flow channel, and the solenoid valve is controlled by an external signal to switch the path, introducing refrigerant into the sample. In this way, a lower temperature refrigerant can be introduced into the sample at less time intervals.

[0100] Furthermore, the timing of refrigerant introduction can also be synchronized with the pressurization timing of Embodiment 1. For example, the control unit 465 controls the timing of valve opening and closing in a manner that synchronizes with the pressurization timing. Similarly, the timing of stimulating the sample can also be synchronized with the pressurization timing of Embodiment 1. The control unit 465 controls the pressurization timing based on a signal from the sample 200. Alternatively, the control unit 465 controls the pressurization timing based on a signal that applies stimulation to the sample 200.

[0101] Implementation Method 3

[0102] use Figure 10 , Figure 11 The microscope of Embodiment 3 will be described. Figure 10 This is a schematic diagram showing the overall structure of microscope 5. Figure 11 This is a magnified view of the objective lens 11 and the periphery of the sample 200. In Embodiment 3, the microscope 5 is an upright microscope. Furthermore, similar to Embodiment 2, the freezing timing is controlled by opening and closing the valve 462.

[0103] A sample 200 is positioned directly below the objective lens 11. The sample 200 is positioned on the stage 10. The sample 200 is positioned within the field of view of the objective lens 11. The sample 200 has a substrate 201 and an object 204 disposed on the substrate 201. A microscope body 412 is positioned above the objective lens 11. The microscope body 412 has an optical system including a light source, a camera, an imaging lens, and a mirror. Light from the objective lens 11 propagates within the microscope body 412. The camera captures an image of the sample 200 formed by the imaging lens. Alternatively, the microscope body 412 may also have an eyepiece for direct observation by the user.

[0104] The refrigerant supply unit 460 includes a Dewar flask 450, a supply tube 461, and a valve 462. One end of the supply tube 461 is located near the sample 200, and the other end is located inside the Dewar flask 450. The valve 462 is located midway through the supply tube 461. Preferably, the valve 462 is located near the objective lens 11.

[0105] Similar to Embodiment 2, a refrigerant 451 is stored in a Dewar flask 450. A pressurized gas supply line 453 is connected to the Dewar flask 450 via a valve 454. The pressurized gas supply line 453 supplies nitrogen gas into the Dewar flask 450. The nitrogen gas from the pressurized gas supply line 453 causes the pressure inside the Dewar flask 450 to exceed one atmosphere. The refrigerant is filled at the other end of the supply line 461 to the valve 462. When the valve 462 is opened, the refrigerant 451 in the supply line 461 is discharged to the sample holder 401. The refrigerant 451 passes through the supply line 461 to reach the sample.

[0106] like Figure 11As shown, the top end of the supply tube 461 is a nozzle 464 for spraying refrigerant. The inner diameter of the nozzle 464 is 3 mm. The objective lens 11 is a long working distance objective lens. The top end of the nozzle 464 is positioned between the objective lens 11 and the sample 200. The nozzle 464 sprays refrigerant onto the sample 200. The top end of the nozzle 464 can also be positioned directly above the sample 200.

[0107] A valve 462 is disposed to the side of the objective lens 11. Before observation, the sample 200 and the objective lens 11 are preheated. Preferably, the valve 462 is positioned near the sample 200. When the valve 462 is opened, refrigerant is ejected from the nozzle 464 in the direction of the arrow. The refrigerant is sprayed from an upward angle towards the object of observation 204. The refrigerant sprayed onto the sample 200 freezes the object of observation 204 of the sample 200. Therefore, the sample 200 can be frozen in a short time.

[0108] Furthermore, a protective device 19 for protecting the lens is provided around the top periphery of the objective lens 11. The protective device 19 then holds the transparent protective glass 18. The protective glass 18 is positioned between the objective lens 11 and the sample 200. This prevents coolant from being sprayed onto the objective lens 11.

[0109] Furthermore, dry gas can be allowed to flow in the space between the protective device 19 and the objective lens 11. The space between the protective device 19 and the objective lens 11 can also be vented to create a vacuum or reduced pressure state. This prevents condensation on the objective lens 11 and allows for cooling. Similarly, dry gas can be allowed to flow in the space between the protective glass 18 and the objective lens 11. Furthermore, the space between the protective device 19 and the objective lens 11 can be vented. This prevents condensation on the surface of the objective lens 11 and maintains its lens function.

[0110] Figure 12 These are microscope photographs showing the appearance of frozen samples. Figure 12 The image shown is a microscopic image of a rat perfused heart taken from its upper surface using a stereomicroscope. The nozzle tip is positioned on the left side of the sample. The moment the cryoprotectant is ejected from the nozzle is set to 00'00". Microscopic images taken every 0.03 seconds or 0.04 seconds are shown. This ensures the sample is immediately frozen after the cryoprotectant is ejected from the nozzle. This allows for separate observation of the heart's diastolic and systolic phases.

[0111] Variation Example 1

[0112] use Figure 13 and Figure 14 A modified example of the specimen holder, which serves as the specimen freezing unit, will be described. Figure 13 and Figure 14This is a perspective view showing the sample holder 600 of modified example 1. The sample holder 600 is used for an upright microscope. Therefore, the objective lens is positioned on the upper side of the sample holder 600.

[0113] The sample holder 600 has a first plate 610, a second plate 620, and a refrigerant reservoir 630. The refrigerant reservoir 630 is connected to the second plate 620 via a hinge 625. Figure 13 and Figure 14 In the middle, the angle of the refrigerant storage section 630 around the hinge 625 is different. (The remaining text appears to be incomplete and requires further context.) Figure 13 The specimen was observed in the state shown. Furthermore, it was possible to observe the specimen in the following manner. Figure 14 The sample is removed from the sample holder 600 in the state shown.

[0114] The first plate 610 and the second plate 620 are disc-shaped components. The first plate 610 and the second plate 620 are, for example, metal plates such as stainless steel. They are not limited to stainless steel and can be formed from metal plates such as titanium. Alternatively, the first plate 610 and the second plate 620 can be formed from polymer plates with low thermal conductivity that are not easily cracked even at low temperatures. Polymer plates can be made of materials such as Teflon, polycarbonate, and polylactic acid. Furthermore, the first plate 610 and the second plate 620 can also be ceramic plates used as insulation materials.

[0115] The first plate 610 is the base of the microscope stage. A disc-shaped second plate 620 is fixed on the first plate 610. In the XY plane, the first plate 610 and the second plate 620 are roughly the same in shape.

[0116] like Figure 14 As shown, an opening 613 is provided at the center of the first plate 610 in the XY plane. The opening 613 extends through in the Z direction. The opening 613 and its periphery form a sample placement section 617. A sample is placed in the sample placement section 617. Therefore, the sample placement section 617 becomes the focal plane of the objective lens.

[0117] The refrigerant reservoir 630 serves as a cover for holding the sample. For example, the sample 200 is positioned between the refrigerant reservoir 630 and the first plate 610. The refrigerant reservoir 630 rotates about the axis of the hinge 625. By rotating the refrigerant reservoir 630 closer to the first plate 610, the sample 200 is clamped between the refrigerant reservoir 630 and the first plate 610. Thus, the sample holder 600 holds the sample, allowing it to be observed. Furthermore, by rotating the refrigerant reservoir 630 away from the first plate 610, the upper side of the sample setting section 617 is opened. This allows the sample to be placed in the sample setting section 617. Alternatively, the sample can be removed from the sample setting section 617.

[0118] A lens mounting section 636 is provided in the refrigerant storage section 630, and an objective lens is mounted thereon. The lens mounting section 636 includes an opening extending through in the Z direction. The lens mounting section 636 is positioned directly above the opening 613. Here, corresponding to the shape of the objective lens, the lens mounting section 636 has a tapered bevel 637.

[0119] A supply pipe 633, functioning as a refrigerant supply section, is obliquely disposed in the refrigerant storage section 630. When the sample 200 is sandwiched between the refrigerant storage section 630 and the first plate 610, the supply pipe 633 is located obliquely above the lens arrangement section 636. Furthermore, the arrangement of the sample 200 is not limited to... Figure 4 The structure is as follows. For example, the sample 200 can also be an internal organ itself. A refrigerant inlet 634 is provided on the upper side of the supply pipe 633. A pipe for flowing refrigerant can also be connected to the refrigerant inlet 634. By allowing liquid refrigerant to flow in from the refrigerant inlet 634, the refrigerant flows down from the supply pipe 633 to the inclined surface 637. The refrigerant comes into contact with the sample in the sample setting part 617, and the refrigerant cools the sample. Thus, the sample is frozen. For example, in order to supply refrigerant, the user only needs to move the refrigerant placed in the container to the supply pipe. Alternatively, the timing of refrigerant supply can be controlled by opening and closing a valve, as in Embodiment 2. With this structure, the sample in the field of view of the objective lens can be frozen with a simple structure and method.

[0120] A refrigerant outlet 627 is provided on the upper surface of the second plate 620. The refrigerant outlet 627 communicates with the opening 613. Refrigerant outlets 627 are arranged on both sides of the refrigerant storage section 630 in the X direction. The refrigerant supplied by the supply pipe 633 is discharged from the refrigerant outlet 627. That is, the refrigerant used in cooling the sample is discharged from the refrigerant outlet 627. This allows for a continuous supply of fresh refrigerant, thus increasing the cooling rate. Of course, the refrigerant outlet 627 can be omitted.

[0121] Variation Example 2

[0122] use Figure 15 and Figure 16 A modified example 2 of the specimen holder, which serves as the specimen freezing unit, will be described. Figure 15 and Figure 16 This is a perspective view showing the structure of the sample holder 800. The sample holder 800 of Modified Example 2 can be used in a stereomicroscope. More specifically, the sample holder 800 can be used in a stereomicroscope for observing perfused hearts. The sample holder 800 can be used in an upright microscope. Furthermore, the sample holder 800 can also be used in an inverted microscope.

[0123] The sample holder 800 has a base 810 and sidewalls 820. The base 810 is a flat plate along the XY plane. Because the base 810 is formed of a transparent member, illumination or observation from below is possible. Regarding the base 810, a refrigerant storage tank 833 is provided at the center of the base 810 viewed from above in the XY plane. The refrigerant storage tank 833 is a circular recess. Steps surrounding the refrigerant storage tank 833 form a sample placement section 817. A sample (not shown) is placed above the sample placement section 817. An objective lens (not shown) is positioned above or below the sample placement section 817. Therefore, the sample is positioned within the field of view of the objective lens.

[0124] A drain port 813 is provided on the -Y side of the base 810. The drain port 813 is connected to the sample placement section 817 and the cryoprotectant storage tank 833. The perfusion fluid is discharged from the drain port 813. Alternatively, the cryoprotectant can also be discharged from the drain port 813. The drain port 813 is provided along the Y direction. The drain port 813 extends through the interior of the base 810 until it is connected to the sample placement section 817 or the cryoprotectant storage tank 833. A groove 814 for arranging electrodes and perfusion tubes for the heart is formed on the upper surface of the base 810. The groove 814 is provided along the Y direction. The groove 814 is connected to the cryoprotectant storage tank 833. The groove 814 extends from both sides of the cryoprotectant storage tank 833.

[0125] A sidewall 820 is provided at the +X side end of the base 810. The sidewall 820 is erected vertically from the upper surface of the base 810 toward the +Z side. A refrigerant supply port 830 is provided in the sidewall 820. The refrigerant supply port 830 obliquely extends through the sidewall 820. Specifically, the refrigerant supply port 830 descends from the +X side toward the -X side. Therefore, when refrigerant is injected into the refrigerant supply port 830 from the +X side, the refrigerant flows down from the -X side of the refrigerant supply port 830 into the refrigerant storage tank 833. Thus, the refrigerant comes into contact with the sample, and the sample can be frozen. With this structure, the sample in the field of view of the objective lens can be frozen with a simple structure and method.

[0126] This embodiment is not limited to inverted microscopes, upright microscopes, or stereomicroscopes, but can also be applied to other types of microscopes. For example, it can also be applied to microscopes that provide side illumination, where the illumination light enters the sample from the side of the objective lens. The microscope is suitable for observing biological samples such as cultured cells and cardiomyocytes.

[0127] The light observed under a microscope can respond to any of the following: scattering, absorption, emission, or reflection. Microscopes are also suitable for fluorescence observation and Raman scattering observation. Samples under microscope observation can be frozen using a cryostat, and the frozen samples can be observed directly and continuously at low temperatures.

[0128] While observing the fluorescence of calcium ions in an isolated perfused rat heart housed in a sample holder 600 using an upright microscope or a stereomicroscope, a liquid propane-isopentane cryopropellant mixture was introduced into the storage compartment at any time for freeze-fixation. By performing freeze-fixation during the diastolic and systolic phases of the heart separately, the signal accumulation time, currently limited by the rapid changes in phenomena, could be extended, and the diastolic and systolic phases could be observed separately.

[0129] By observing a frozen sample 200, various applications are possible. For example, fluorescence lifetime can be measured to infer quantum yield. Furthermore, cell membrane potential can be measured via membrane potential imaging. It is possible to simultaneously measure cell membrane potential and the surrounding ion distribution.

[0130] The sample 200 can be observed in relation to its redox potential and redox state. In the case that the sample 200 is a biological sample, the sample 200 can be observed in relation to the results of blood flow and oxygen concentration measurements.

[0131] The sample 200 can also be melted by irradiating it with light after it has been frozen. For example, the sample 200 can be melted by irradiating it with an infrared laser. The laser can be irradiated onto the sample 200 through the objective lens 11 or from the opposite side of the objective lens 11. Furthermore, by adjusting the irradiation area of ​​the sample 200, it is possible to adjust the melting to a localized manner. For example, the laser can be irradiated only onto the portion of the sample 200 that is desired to be melted.

[0132] Furthermore, after melting the sample 200 using laser irradiation, the laser irradiation can be stopped, thereby refreezing the sample 200. This allows for repeated freezing and thawing of the sample while simultaneously observing it. Alternatively, the melted portion can be observed by melting the sample using laser irradiation. Or, the sample can be observed while it has been thawed once and then refrozen.

[0133] Furthermore, the specimens observed under a microscope are not limited to those mounted on a specimen substrate. For example, specimens flowing in channels such as microchannels can also be observed. That is, the specimen includes the fluid flowing within the channel. A coolant is introduced into the channel within the objective lens's field of view. For example, the coolant is brought into contact with a substrate having the channel. This allows the specimen within the channel to be frozen instantly.

[0134] Implementation Method 4

[0135] In embodiment 4, the cryocoolant is supplied based on a signal from the sample or a signal used to stimulate the sample. Figure 17 The structure for supplying refrigerant based on signals from the sample or signals used to stimulate the sample is described. Figure 17 This is a diagram showing the structure of the microscope according to Embodiment 4.

[0136] The microscope 500 includes a camera 501, an imaging lens 502, a housing 503, an objective lens 510, a reflecting mirror 511, a support 521, a raising stage 522, a stage 523, a lifter 524, and a sample freezing unit 550. Furthermore, the microscope 500 includes a stimulation electrode 553 for applying stimulation to the sample 200, a function generator 552, and an electrocardiogram measuring device 530, etc.

[0137] The specimen 200 is held in the specimen chamber 540. The specimen 200 is an ex vivo heart. The specimen chamber 540 is fixed above a height-adjustable lifter 524. A specimen freezing unit 550 is positioned directly above the specimen 200. The specimen 200 is illuminated using indoor lighting.

[0138] A reflector 511 is disposed to the side of the sample 200. The reflector 511 is supported by a support column 521, a raised platform 522, and a stage 523. Above the reflector 511 are an objective lens 510, a housing 503, an imaging lens 502, and a camera 501. Light incident from the sample 200 onto the reflector 511 is reflected by the reflector 511 and enters the objective lens 510. Light incident onto the objective lens 510 is refracted by the objective lens 510 and enters the housing 503.

[0139] Light from objective lens 510 enters imaging lens 502 via housing 503. Imaging lens 502 images the sample 200 onto the light-receiving surface of camera 501. Thus, camera 501 can capture an image of sample 200. Known techniques can be used for the optical systems used to guide light from the light source to sample 200 and for guiding light from sample 200 to camera 501, therefore descriptions are omitted.

[0140] The sample freezing unit 550 has a function generator 552. The sample freezing unit 550 has a pressurized gas inlet 561, a pressure gauge 562, a safety valve 563, a sealing cover 564, an insulated container 565, a refrigerant channel 571, a solenoid valve 573, and a precooling chamber 574.

[0141] Liquid refrigerant 570 is stored in an insulated container 565. The upper side of the insulated container 565 is sealed by a sealing cap 564. Furthermore, pressurized gas is supplied to the insulated container 565 through a pressurized gas inlet 561. A pressure gauge 562 measures the pressure of the pressurized gas. If the pressure detected by the pressure gauge 562 reaches or exceeds a specified pressure, a safety valve 563 releases the pressurized gas.

[0142] A refrigerant channel 571 is connected to the lower side of the insulated container 565. Using pressurized gas, the refrigerant flows downwards through the refrigerant channel 571. The refrigerant channel 571 passes through a pre-cooling chamber 574. The pre-cooling chamber 574 is pre-cooled by liquid nitrogen 572. A solenoid valve 573 is located midway through the refrigerant channel 571. The bottom surface of the pre-cooling chamber 574 serves as a refrigerant supply port. Refrigerant is supplied from directly above the sample 200. By opening the solenoid valve 573, refrigerant is supplied to the sample 200, freezing the sample 200.

[0143] Function generator 552 generates a control signal for controlling the solenoid valve. Solenoid valve 573 opens and closes based on the control signal from function generator 552. Function generator 552 outputs a trigger signal to stimulation electrode 553. The trigger signal is a signal for electrically stimulating the isolated heart, which serves as sample 200. Sample 200 is stimulated using the trigger signal applied to stimulation electrode 553. Function generator 552 independently controls both the control signal and the trigger signal. Function generator 552 can control the intensity and phase of the applied voltage.

[0144] The solenoid valve 573 opens at a predetermined time offset from the timing of applying electrical stimulation to the sample 200. When the solenoid valve opens based on a control signal from the function generator 552, refrigerant is supplied to the sample 200. Thus, the timing of refrigerant application can be controlled according to the trigger signal used to stimulate the sample 200. Furthermore, since the timing of the control signal is variable due to the function generator 552, the timing of refrigerant application can be adjusted. The sample 200 can be frozen after a desired time has elapsed since the start of electrical stimulation. The sample 200 can be frozen according to the timing of the heartbeat.

[0145] Furthermore, an electrocardiogram (ECG) measuring device 530 is connected to the sample 200. The ECG measuring device 530 measures the electrocardiogram corresponding to the heartbeat of the sample 200.

[0146] Figure 18 It is a diagram showing the waveforms of the electrocardiogram and the trigger signal. Figure 18 The upper side shows the waveform of an electrocardiogram, and the lower side shows the waveform of a trigger signal applied to the stimulation electrode 553. Here, after the trigger signal is generated, a control signal reaches the solenoid valve 573 after approximately 40 msec. Subsequently, after approximately 40 msec, the refrigerant comes into contact with the sample 200.

[0147] Furthermore, in the above description, although the timing of refrigerant supply is controlled based on the trigger signal from the function generator 552, it is also possible to control the timing of refrigerant supply based on the electrocardiogram measured by the electrocardiogram measuring device 530. That is, the timing of opening the solenoid valve 573 can be adjusted according to the electrocardiogram waveform. Since the timing of the heart's diastolic and systolic phases can be determined based on the electrocardiogram waveform, the sample 200 can be frozen at the desired time. Therefore, the timing of refrigerant supply can be controlled based on the signal from the sample 200.

[0148] Implementation method 5.

[0149] In Embodiment 5, after freezing the sample 200 with a refrigerant, the sample 200 is melted by irradiating it with a laser. Figure 19 The microscope of Embodiment 5 will be described. Figure 19 This is a schematic diagram showing the optical system of microscope 700. Here, microscope 700 is described as a fluorescence microscope for detecting fluorescence from sample 200.

[0150] The microscope 700 includes an observation light source 701, a stage 710, an observation optical system 720, a two-dimensional light detector 723, a thawing light source 730, a thawing optical system 740, a refrigerant dispensing device 750, and a container 751. The observation optical system 720 includes a lens 702, a dichroic mirror 703, a dichroic mirror 704, an objective lens 705, a filter 721, and a tube lens 722. The thawing optical system 740 includes a shutter 741, a scanner 742, a dichroic mirror 703, a dichroic mirror 704, and an objective lens 705.

[0151] The stage 710 holds the container 751 that holds the sample 200. Thus, the sample 200 is placed above the stage 710. A refrigerant supply device 750 is provided above the sample 200. The refrigerant supply device 750 is the aforementioned sample freezing unit, supplying refrigerant to the sample 200. The sample 200 is frozen using the refrigerant supplied from the refrigerant supply device 750.

[0152] Next, the observation optical system 720 for guiding light from the observation light source 701 will be described. The observation light source 701 is, for example, a laser light source, which generates excitation light for exciting the sample 200. The laser (excitation light) from the observation light source 701 is focused by the lens 702 and enters the dichroic mirror 703. The dichroic mirror 703 has wavelength characteristics that allow light of the excitation wavelength to pass through while reflecting light used for thawing.

[0153] The laser light passing through dichroic mirror 703 enters dichroic mirror 704. Dichroic mirror 704 has the wavelength characteristic of reflecting the excitation light wavelength while allowing fluorescence to pass through. The excitation light reflected by dichroic mirror 704 enters objective lens 705. Objective lens 705 focuses the excitation light onto sample 200.

[0154] The sample 200 is placed on a substrate (not shown) through which the wavelengths of excitation light and thawing light are transmitted. The sample 200 and the substrate are placed on a stage 710. The stage 710 has an opening at the lower part of the sample 200 portion. Therefore, the excitation light passes through the opening of the stage 710 and enters the sample 200.

[0155] When the excitation light excites the sample 200, fluorescence is generated from the sample 200. The fluorescence from the sample 200 is refracted by the objective lens 705 and enters the dichroic mirror 704. Regarding the fluorescence, the fluorescence passing through the dichroic mirror 704 enters the filter 721.

[0156] The filter 721 has wavelength characteristics that block the excitation light from the observation light source 701 and the light from the thawing light source 730. Furthermore, the filter 721 allows the fluorescence generated by the sample 200 to pass through. The fluorescence passing through the filter 721 enters the lens 722. The lens 722 focuses the fluorescence onto the two-dimensional photodetector 723. The two-dimensional photodetector 723 is capable of capturing an image of the fluorescence of the sample 200.

[0157] The thawing light source 730 generates, for example, infrared light for heating. Although the thawing light source 730 is described as a laser source that generates laser light, it can also be a lamp source or the like. The laser light from the thawing light source 730 is transmitted to the scanner 742 via the shutter 741. The scanner 742 is an optical scanner such as a galvanometer. The scanner 742 is a dual-axis scanner that performs a two-dimensional scan of the position of the laser irradiation on the sample 200. Thus, laser light can be irradiated at any position on the sample 200.

[0158] The light reflected from the scanner 742 enters the dichroic mirror 703. The dichroic mirror 703 has the wavelength characteristic of reflecting infrared light. Therefore, the dichroic mirror 703 reflects the laser light from the scanner 742. Thus, the laser light from the thawing light source 730 and the excitation light from the observation light source 701 become coaxial and propagate. The laser light reflected from the dichroic mirror 703 enters the dichroic mirror 704.

[0159] The dichroic mirror 704 has the wavelength characteristic of reflecting infrared light towards the objective lens 705. Therefore, the laser light reflected by the dichroic mirror 704 enters the objective lens 705. The objective lens 705 refracts the laser light in a manner that focuses it onto the sample 200. By irradiating the sample 200 with laser light, the sample 200, which is frozen using a refrigerant, can be locally heated. As a result, at least a portion of the sample 200 can be melted.

[0160] Furthermore, the laser from the thawing light source 730 and the excitation light from the observation light source 701 become coaxial. Therefore, the laser and excitation light are incident on the same position of the sample 200. This allows for the capture of a fluorescence image of the sample 200 at the laser-heated location. Thus, it is possible to observe the thawing process of the frozen sample 200. Moreover, since a scanner 742 for scanning the laser is provided, the laser irradiation position can be controlled. This allows for proper thawing of the sample 200. Furthermore, the thawing position and time can be controlled during observation.

[0161] Furthermore, a shutter 741 is configured in the optical path of the laser from the defrosting light source. The shutter 741 is configured to open and close. By opening the shutter 741, the laser can enter the sample 200 and heat the sample 200. By closing the shutter 741, the laser can be prevented from irradiating the sample 200. By controlling the opening and closing of the shutter 741, the sample 200 can be melted at an appropriate time.

[0162] Implementation method 6.

[0163] use Figure 20 The microscope 700 of Embodiment 6 will be described. Figure 20 This diagram illustrates the structure of the microscope 700. In Embodiment 6, the sample 200 contains a cage-like compound and a calcium indicator. The microscope 700 is used to freeze and observe the sample 200 containing the cage-like compound and the calcium indicator. Therefore, the light sources 761 and 762 differ from those in Embodiment 5. Structures other than the light sources, such as the optical system, are the same as in Embodiment 5 and therefore descriptions are omitted.

[0164] In Embodiment 6, the light source 761 is a light source for observing the calcium indicator. The light source 761 generates excitation light to excite the calcium indicator contained in the sample 200. When the excitation light from the light source 761 irradiates the sample 200, fluorescence is generated from the calcium indicator. By detecting the fluorescence using a two-dimensional photodetector 723, the calcium concentration can be determined.

[0165] Light source 762 is a light source that generates light to irradiate the cage-like compound. Light source 762 produces ultraviolet and visible light. Sample 200 has a photodegradable protecting group. The protecting group temporarily inactivates the physiologically active molecules. The protecting group is dissociated by light from light source 762, thereby activating the physiologically active molecules.

[0166] By controlling the timing of the opening and closing of the shutter 741, the timing of activation can be controlled. Furthermore, by controlling the opening and closing of the valve located midway through the refrigerant flow path, the timing of freezing can be controlled.

[0167] The invention made by the inventors has been specifically described above based on the embodiments. However, it is self-evident that the invention is not limited to the above embodiments, and various modifications can be made without departing from its spirit.

[0168] This application claims priority based on Japanese Application Special Purpose 2021-080906 filed on May 12, 2021, the entire contents of which are incorporated herein by reference.

[0169] Explanation of reference numerals in the attached figures

[0170] 1: Microscope;

[0171] 4: Microscope;

[0172] 5: Microscope;

[0173] 10: Stage;

[0174] 11: Objective lens;

[0175] 100: Sample holder;

[0176] 110: First board;

[0177] 111: Gas supply port;

[0178] 113: Opening;

[0179] 117: Sample setting section;

[0180] 120: Second board;

[0181] 125: Hinge;

[0182] 127: Refrigerant vent;

[0183] 130: Refrigerant Supply Department;

[0184] 133: Refrigerant Storage Section;

[0185] 134: Refrigerant inlet;

[0186] 160: Refrigerant;

[0187] 401: Sample holder;

[0188] 402: Base;

[0189] 403: Flow channel;

[0190] 404: Opening;

[0191] 405: Sample clamping component;

[0192] 450: Dewar flask;

[0193] 451: Refrigerant;

[0194] 460: Refrigerant Supply Department;

[0195] 461: Supply Management;

[0196] 462: Valve;

[0197] 464: Nozzle;

[0198] 465: Control Department;

[0199] 470: Refrigerant recovery tank;

[0200] 471: Refrigerant;

[0201] 473: Discharge pipe;

[0202] 600: Sample holder;

[0203] 610: First board;

[0204] 613: Opening;

[0205] 617: Sample setting section;

[0206] 620: Second board;

[0207] 625: Hinge;

[0208] 627: Refrigerant vent;

[0209] 630: Refrigerant Storage Section;

[0210] 633: Supply Management;

[0211] 634: Refrigerant inlet;

[0212] 636: Lens configuration section;

[0213] 637: Bevel;

[0214] 800: Sample holder;

[0215] 810: Base;

[0216] 813: Discharge outlet;

[0217] 820: Side wall;

[0218] 830: Refrigerant supply port;

[0219] 833: Refrigerant storage tank.

Claims

1. A microscope having: Objective lens, which is illuminated by light from the sample; and A sample holder for holding the sample; The sample holder includes a sample freezing unit that freezes the sample by bringing a cryoprotectant into contact with the sample within the field of view of the objective lens. The sample holder has a first plate with an opening. The opening and its surrounding area form a sample setting area for setting the sample. The objective lens is configured such that the sample setting section becomes the focal plane. The sample freezing unit has a refrigerant storage section for storing liquid refrigerant, and the refrigerant storage section is positioned directly above the opening. By allowing the refrigerant to flow in from the refrigerant inlet located on the upper side of the refrigerant storage section, the refrigerant is supplied to the sample from above.

2. The microscope according to claim 1, wherein, The sample holder also has a second plate disposed on the first plate and a refrigerant supply section. The refrigerant supply unit is connected to the second plate. The refrigerant supply section is opened and closed. By closing the refrigerant supply section, the refrigerant supply section is positioned above the opening; by opening the refrigerant supply section, the upper side of the opening is opened.

3. The microscope according to claim 1, wherein, The sample can be observed while the refrigerant is being supplied.

4. The microscope according to claim 1, wherein, The microscope is equipped with an introduction mechanism that introduces the cryoprotectant into the sample based on a signal from the sample or a signal used to stimulate the sample.

5. The microscope according to claim 4, wherein, The sample freezing unit has a supply pipe through which the refrigerant flows. The valve installed on the refrigerant supply pipe serves as the inlet mechanism.

6. The microscope according to claim 1, wherein, The microscope also has a pressurization unit that pressurizes the sample while freezing it.

7. The microscope according to claim 1, wherein, The sample is pressurized using a pressurization unit, and the sample is observed while being pressurized.

8. The microscope according to claim 7, wherein, The microscope is equipped with a control unit that controls the timing of pressurizing the sample and the timing of introducing the cryoprotectant.

9. The microscope according to claim 1, wherein, The sample freezing unit is provided with a refrigerant outlet that communicates with the opening.

10. The microscope according to claim 2, wherein, The refrigerant storage section is connected to the second plate via a hinge. The refrigerant storage section is opened and closed by rotating about the axis of rotation of the hinge.

11. The microscope according to any one of claims 1 to 10, wherein, The microscope has the function of melting at least a portion of the frozen sample by irradiating it with light.

12. An observation method, having: The step of positioning the specimen held in the specimen holder within the field of view of the objective lens; The sample freezing unit freezes the sample by supplying or moving a cryoprotectant into or within the field of view of the objective lens; and The procedure for observing the frozen sample. The sample holder has a first plate with an opening for holding the sample. A sample freezing unit is disposed above the first plate. The sample freezing unit has a refrigerant storage section for storing refrigerant, and the refrigerant storage section is positioned directly above the opening. By allowing the refrigerant to flow in from the refrigerant inlet located on the upper side of the refrigerant storage section, the refrigerant is supplied to the sample from above.

13. The observation method according to claim 12, wherein, The sample holder also has a second plate disposed on the first plate and a refrigerant supply section. The refrigerant supply unit is connected to the second plate. The refrigerant supply section is opened and closed. By closing the refrigerant supply section, the refrigerant supply section is positioned above the opening; by opening the refrigerant supply section, the upper side of the opening is opened.

14. The observation method according to claim 13, wherein, The sample is observed while the refrigerant is supplied.

15. The observation method according to claim 13, wherein, The cryoprotectant is introduced into the sample based on a signal from the sample or a signal used to stimulate the sample.

16. The observation method according to claim 15, wherein, A supply pipe through which the refrigerant flows is provided. The timing of introducing the refrigerant is adjusted by a valve installed in the refrigerant supply pipe.

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