Microscope and observation method

Abstract

A microscope (1) according to the present embodiment comprises: an objective lens (11) for receiving light from a sample (200); and a sample freezing means for freezing the sample (200) by supplying a cryogen (160) to or bringing the cryogen (160) into contact with the sample (200) in the field of view of the objective lens (11). A sample in the field of view of the objective lens (11) can be frozen and observed by using the microscope (1) having such a configuration.

Description

[Technical field]

[0001] The present invention relates to a microscope and an observation method, and more particularly to a technique for freezing and observing a sample. [Background technology]

[0002] In optical microscopy of biological samples, samples are sometimes fixed in advance to prevent changes to the sample during observation or due to pretreatment. In this case, chemical fixation using aldehydes that crosslink proteins or dehydration fixation using organic solvents are used. These fixation methods are sufficient to maintain morphological information at the level of optical microscopy. However, some molecules may be oxidized by aldehydes or dissolved in organic solvents. In addition, the fixation reaction may take minutes, and thick samples may require even more time to fix deep inside. Another problem is that not all molecules are fixed. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Fuest, et al., J. Microsc. 272, 87 (2018). Summary of the Invention

[0004] In light of the above, freeze fixation has recently begun to attract attention as a new method for fixing biological samples. Freezing fixation is a method that physically stops the movement of molecules and ions in a sample by freezing water. If freezing is performed in a short period of time, the biological sample can be fixed in its original state.

[0005] Non-Patent Document 1 discloses a method using a heat sink and a heater. In this method, a thin NiCr heater is placed on a heat sink cooled by liquid nitrogen. A sample placed on the heater is frozen at any timing by controlling the on / off of the heater. The sample volume is reduced by placing the sample in a microchannel. This method has succeeded in freezing nematodes in a few tens of milliseconds. However, in this conventional technology, the freezing speed is limited by the thickness of the heater.

[0006] The microscope of this embodiment comprises an objective lens that receives light from a sample, and a sample freezing means that freezes the sample by supplying or contacting a cryogen to the sample in the field of view of the objective lens.

[0007] In the above microscope, the sample may be observed while the cooling agent is being supplied.

[0008] The microscope may be provided with an introduction mechanism for introducing the cryogen toward the sample based on a signal from the sample or a signal for stimulating the sample.

[0009] In the above microscope, the sample freezing means may include a supply pipe through which the cryogen flows, and a valve provided in the cryogen supply pipe may serve as the introduction mechanism.

[0010] The above microscope may further comprise a pressurizing means for pressurizing the sample, so that the sample is frozen while being pressurized.

[0011] In the above microscope, a pressurizing means for pressurizing the sample may be used so that the sample is observed while being pressurized.

[0012] The microscope may be provided with a control unit that controls the timing of pressurizing the sample and the timing of introducing the cryogen.

[0013] In the above microscope, the liquid cryogen may be supplied to the sample from above the sample.

[0014] The microscope described above may further comprise a sample holder for holding the sample, and the sample holder may be provided with a cryogen reservoir for storing a cryogen or a flow path through which a cryogen flows.

[0015] In the above microscope, the objective lens may be disposed above the sample, the sample freezing means may include a nozzle for spraying the cryogen from above toward the sample, and a cover for protecting the objective lens may be provided below the objective lens.

[0016] The microscope may have a function of irradiating light toward the frozen sample to melt at least a portion of the sample.

[0017] The observation method according to the present embodiment includes the steps of: placing a sample in a field of view of an objective lens; The method includes the steps of freezing the sample by supplying or moving a cryogen to the sample in the field of view of the objective lens, and observing the frozen sample.

[0018] In the above observation method, the sample may be observed while the cooling agent is being supplied.

[0019] In the above observation method, the cryogen may be introduced toward the sample based on a signal from the sample or a signal for stimulating the sample.

[0020] In the above observation method, a supply pipe through which the cryogen flows may be provided, and a timing for introducing the cryogen may be adjusted by a valve provided in the supply pipe for the cryogen.

[0021] In the above observation method, the frozen sample may be irradiated with light to melt at least a portion of the sample.

[0022] According to the present invention, it is possible to provide a microscope and an observation method capable of freezing and observing a sample. [Brief description of the drawings]

[0023] [Figure 1] FIG. 2 is a perspective view showing a configuration of a sample holder. [Diagram 2] FIG. 2 is a perspective view showing a configuration of a sample holder. [Diagram 3] FIG. 1 is a diagram showing a configuration of an optical microscope according to a first embodiment. [Figure 4] FIG. 2 is a cross-sectional view illustrating a schematic configuration of a sample. [Diagram 5] FIG. 2 is a schematic diagram showing an example of a pressurizing means for pressurizing a sample. [Figure 6] 13 shows images of the sample before and after freezing. [Figure 7] FIG. 11 is a diagram showing the configuration of an inverted microscope according to a second embodiment. [Figure 8] FIG. 4 is a cross-sectional view showing a detailed configuration of a cryogen supply unit. [Figure 9] FIG. 4 is a perspective view showing a detailed configuration of a cryogen supply unit. [Figure 10] FIG. 11 is a diagram showing the configuration of an upright microscope according to a third embodiment. [Figure 11] FIG. 4 is a side view showing a detailed configuration of a cryogen supply unit. [Figure 12] 1 is a captured image showing how a sample is frozen. [Figure 13] FIG. 13 is a perspective view showing a sample holder according to Modification 1. [Figure 14] FIG. 13 is a perspective view showing a sample holder according to Modification 1. [Figure 15] FIG. 11 is a perspective view showing a sample holder according to Modification 2. [Figure 16] FIG. 11 is a perspective view showing a sample holder according to Modification 2. [Figure 17] FIG. 13 is a schematic diagram showing the configuration of a microscope according to a fourth embodiment. [Figure 18] FIG. 1 is a waveform diagram showing timing of cryogen injection relative to an electrocardiogram and electrical stimulation. [Figure 19] FIG. 13 is a schematic diagram showing the configuration of a microscope according to a fifth embodiment. [Figure 20] FIG. 13 is a schematic diagram showing the configuration of a microscope according to a sixth embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The following describes embodiments to which the present invention can be applied. The following description describes the embodiments of the present invention, and the present invention is not limited to the following embodiments. For clarity of explanation, the following description has been omitted and simplified as appropriate. Furthermore, a person skilled in the art would be able to easily modify, add, or convert each element of the following embodiments within the scope of the present invention. In addition, elements with the same reference numerals in each drawing indicate similar elements, and descriptions thereof will be omitted as appropriate.

[0025] First embodiment The optical microscope (hereinafter also simply referred to as the microscope) according to this embodiment freezes a sample using a cryogen. The microscope has a sample freezing means that supplies a cryogen. The sample freezing means supplies a cryogen to a sample in the field of view of the objective lens. The cryogen supplied from the sample freezing means freezes the sample. The sample in the field of view of the objective lens can be frozen. This allows the frozen sample to be easily observed.

[0026] The structure of the sample holder 100 having a sample freezing means will be described. In the following figures, an XYZ three-dimensional orthogonal coordinate system is appropriately shown. The Z direction is parallel to the optical axis. The XY plane is a plane perpendicular to the Z direction, and is the focal plane on which the sample is placed.

[0027] 1 and 2 are perspective views showing the configuration of a sample holder 100. The sample holder 100 includes a first plate 110, a second plate 120, and a cryogen supply unit 130. The cryogen supply unit 130 supplies a cryogen toward a sample, thereby functioning as a freezing means for freezing the sample.

[0028] The sample holder 100 is used in an inverted microscope in which an objective lens is placed directly under a sample. The cryogen can be liquid or solid. Figure 1 shows the cryogen supply unit 130 in a closed state, and Figure 2 shows the cryogen supply unit 130 in an open state. The sample is observed in the state shown in Figure 1, and the sample is placed in the sample holder 100 in the state shown in Figure 2.

[0029] The first plate 110 and the second plate 120 are disk-shaped members. 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 of metal plates such as titanium. Alternatively, the first plate 110 and the second plate 120 can be formed of a polymer plate that has low thermal conductivity and is difficult to break even at low temperatures. The polymer plate can be made of a material such as Teflon (registered trademark), polycarbonate, or polylactic acid. The first plate 110 and the second plate 120 may also be a ceramic plate used as a heat insulating material.

[0030] The first plate 110 serves as a base that is placed on the stage of a microscope. A disk-shaped second plate 120 is fixed onto the first plate 110. In the XY plane, the outer shapes of the first plate 110 and the second plate 120 have approximately the same size.

[0031] An opening 113 is provided in the center of the first plate 110 in the XY plane. The opening 113 penetrates in the Z direction. The opening 113 and its periphery form a sample mounting portion 117. A sample is mounted on the sample mounting portion 117. Therefore, the sample mounting portion 117 becomes the focal plane of the objective lens.

[0032] A cryogen supply unit 130 is connected to the second plate 120 via a hinge 125. The cryogen supply unit 130 serves as a cover that covers the opening 113. The cryogen supply unit 130 opens and closes by rotating around the axis of the hinge 125. By closing the cryogen supply unit 130, the cryogen supply unit 130 is disposed above the opening 113. Furthermore, by opening the cryogen supply unit 130, the upper side of the opening 113 is opened. This allows the sample in the sample installation unit 117 to be replaced.

[0033] A cylindrical cryogen reservoir 133 is provided on the upper part of the cryogen supply unit 130. When the cryogen supply unit 130 is closed, the cryogen reservoir 133 is located directly above the opening 113. A cryogen inlet 134 is provided on the upper side of the cryogen reservoir 133. By flowing liquid cryogen through the cryogen inlet 134, the cryogen is stored in the cryogen reservoir 133. The cryogen comes into contact with the sample in the sample placement unit 117, and the cryogen cools the sample. This causes the sample to freeze. For example, to supply a cryogen, the user can simply transfer the cryogen in a container to the cryogen reservoir 133.

[0034] A gas supply port 111 is disposed on a side surface of the first plate 110. The gas supply port 111 communicates with an opening 113. The gas supply port 111 is supplied with anti-condensation gas to be sprayed onto the objective lens.

[0035] A cryogen exhaust port 127 is provided on the upper surface of the second plate 120. The cryogen exhaust port 127 communicates with the opening 113. X Cryogen outlets 127 are arranged on both sides of the cryogen supply unit 130 in the direction. The cryogen supplied from the cryogen supply unit 130 is discharged from the cryogen outlet 127. In other words, the cryogen used to cool the sample is discharged from the cryogen outlet 127. This makes it possible to continue to supply new cryogen, thereby increasing the cooling rate. Of course, the cryogen outlet 127 can be omitted.

[0036] FIG. 3 is a side cross-sectional view showing a state in which the sample holder 100 having the cryogen supply unit 130 is installed on the stage 10 of the microscope 1. The sample holder 100 shown in FIG. 3 does not strictly match the sample holder 100 shown in FIG. 1 and FIG. 2. For example, the axis of the hinge 125 is parallel to the X direction in FIG. 1 and FIG. 2, but the axis of the hinge 125 is parallel to the Y direction in FIG. 3. In addition, O-rings O1 to O4 are arranged to prevent the liquid cryogen 160 from leaking. By preventing the cryogen 160 from leaking from the sample holder 100, handling can be made easier.

[0037] A sample holder 100 is fixed on a stage 10. An objective lens 11 that receives light from a sample 200 is disposed in an opening 113 of the sample holder 100. The objective lens 11 is inserted into the opening 113 from below. A sample 200 is disposed directly above the objective lens 11. The sample 200 is disposed in the field of view of the objective lens 11. Thus, freezing while observing the sample 200 is possible. An O-ring O1 seals the space between the sample 200 and the first plate 110. A microscope main body 12 is disposed below the objective lens 11. The microscope main body 12 includes an optical system including a light source, a camera, a mirror, and an imaging lens. Light from the objective lens 11 propagates through the microscope main body 12. The camera captures an image of the sample 200 formed by the imaging lens. Alternatively, the microscope main body 12 may have an eyepiece for a user to directly observe.

[0038] A cryogen supply unit 130 is disposed on the first plate 110. The space between the first plate 110 and the cryogen supply unit 130 is sealed with an O-ring O2. The cryogen supply unit 130 has a cryogen reservoir 133 for storing a cryogen at its upper portion. The cryogen reservoir 133 is disposed directly above the sample 200. The cryogen reservoir 133 is connected to the sample 200 in the sample installation section (not shown in FIG. 3). A cryogen 160 is stored in the cryogen reservoir 133 of the cryogen supply unit 130. The cryogen 160 flows into the cryogen reservoir 133 from a cryogen inlet 134. When the cryogen 160 comes into contact with the sample 200, the sample 200 is frozen.

[0039] A specimen holder 135 is provided in the cryogen reservoir 133. The specimen holder 135 holds the specimen 200 from above, thereby fixing the specimen 200 in place. The specimen holder 135 is provided detachably with respect to the cryogen supply unit 130. The specimen holder 135 can be replaced depending on the thickness, size, etc. of the specimen 200. An O-ring is provided between the specimen holder 135 and the specimen 200. O4 The sample holder 135 is sealed with an O-ring between the sample holder 135 and the inner wall of the cryogen storage section 133. O3 It is sealed with.

[0040] A gas supply port 111 reaching the opening 113 is provided on the side wall of the first plate 110. Dry nitrogen gas is supplied to the gas supply port 111. The nitrogen gas from the gas supply port 111 flows through the space between the objective lens 11 and the first plate 110. The nitrogen gas from the gas supply port 111 is sprayed toward the objective lens 11. By spraying the nitrogen gas for preventing condensation onto the objective lens 11, it is possible to prevent condensation on the objective lens 11 and the sample 200. In addition, a thermocouple 17 for measuring temperature is provided on the wall surface of the first plate 110 on the opening 113 side or on the underside of the sample 200. This allows the cooling temperature by the cryogen 160 to be evaluated.

[0041] The gas supply port 111 may be used as a gas suction port for sucking in gas around the objective lens 11. That is, a vacuum pump or the like sucks in gas through the gas supply port 111. This evacuates the space between the objective lens 11 and the first plate 110. The area around the objective lens 11 can be placed in a reduced pressure or vacuum state. This can prevent condensation on the objective lens 11. Furthermore, the reduced pressure or vacuum state can reduce heat transferred from the objective lens 11 to the sample 200. This can improve the thermal insulation.

[0042] 4 is a detailed cross-sectional view showing an example of a frozen sample 200. The sample 200 includes a substrate 201, a spacer 202, a cover glass 203, and an object 204 to be observed.

[0043] 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 may 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 is 0.02 mm thick.

[0044] The substrate 201 and the cover glass 203 are disposed opposite to each other via a spacer 202. In other words, a gap is formed between the substrate 201 and the cover glass 203 according to the thickness of the spacer 202. An observation target 204 is disposed in the gap between the substrate 201 and the cover glass 203.

[0045] Excitation light is incident on the observation target 204 from the cover glass 203 side. Then, fluorescence emitted on the cover glass 203 side is incident on the objective lens. Liquid cryogen is supplied from the substrate 201 side. The liquid cryogen is a mixture of liquid propane / isopentane at a ratio of 2:1 or 3:1.

[0046] In this manner, the cryogen supply unit 130 is provided to supply a cryogen toward the sample 200. Therefore, the observation target 204 in the field of view of the objective lens can be quickly frozen, and the frozen sample 200 can be observed.

[0047] In this manner, in this embodiment, the cryogen is supplied directly to the sample 200. In other words, since the cryogen comes into contact with the substrate 201 of the sample 200, the sample 200 can be frozen in a short time. The biological sample can be fixed as it is. Cells and small pieces of biological tissue can be frozen in the order of milliseconds. By supplying liquid cryogen directly to organs in a living body, it is possible to fix the organs in their original state in the living body.

[0048] Since the cryogen is supplied directly, even a large sample 200 can be frozen in a short time. There are fewer restrictions on the thickness and size of the sample 200. Therefore, various samples 200 can be frozen and observed. In particular, since only a thin substrate 201 is placed between the observation target 204 and the cryogen, rapid cooling and freezing are possible. Even if the sample 200 is at room temperature before the cryogen is added, rapid freezing is possible.

[0049] Furthermore, the substrate 201 disposed between the objective lens 11 and the observation target 204 may be a transparent substrate. In this way, the observation target can be irradiated with illumination light or excitation light from the substrate 201 side. By making the substrate 201 a transparent substrate, observation can be made from the substrate 201 side.

[0050] The cryogen is not limited to a liquid, and may be a solid such as a powder. For example, a cooled metal can be used as the cryogen. The metal can be moved to contact the sample 200, thereby freezing the sample 200. The cryogen supply unit 130 can move a cryogen located away from the sample 200 toward the sample 200, thereby freezing the sample 200. The sample freezing means for freezing the sample with a cryogen is not limited to the above example. The sample freezing means may be any means that freezes the sample by supplying or moving a cryogen toward the sample in the field of view of the objective lens 11. The cryogen may be cooled to a temperature lower than the freezing temperature of the sample.

[0051] The sample freezing means is not limited to a configuration that supplies a cryogen so as to bring the cryogen into contact with the sample 200. In other words, the sample freezing means may be configured to supply a cryogen so as not to bring the cryogen into contact with the sample 200. For example, the sample freezing means may be configured to cool the sample via a substrate, film, sheet, or the like. In other words, the sample freezing means that supplies a cryogen to the sample brings the cryogen into contact with a substance other than the sample. The sample is frozen by cooling the sample via the other substance. In this way, the sample freezing means may supply a cryogen with the other substance interposed between the sample 200 and the cryogen.

[0052] Two or more types of cryogens may be used. For example, a first cryogen that freezes the sample 200 and a second cryogen that keeps the frozen sample 200 frozen may be used separately. First, the first cryogen is supplied toward the sample 200 at room temperature. After the sample 200 freezes, the supply of the first cryogen is stopped and a second cryogen is supplied. The second cryogen keeps the sample 200 frozen. Thus, the frozen state of the sample 200 can be observed. The first and second cryogens may be supplied from the same cryogen supply unit or from different cryogen supply units. Furthermore, cooling means other than cryogens may be used. For example, electronic cooling such as a Peltier element may be used in combination.

[0053] It is also possible to observe the sample 200 while supplying the cryogen. In other words, the cryogen may be continuously supplied from the cryogen supply unit 130 while observing the sample 200. For example, by providing a cryogen outlet 127 or the like, it is possible to continuously supply new cryogen. This allows the sample 200 to be continuously cooled so that it does not melt.

[0054] Also, a cryogen may be supplied to the sample 200 while applying pressure to the sample. This allows the sample to be frozen while pressure is being applied to the sample. By applying pressure to the sample, the generation of ice crystals can be suppressed. For example, the pressurizing means applies a pressure of 210 MPa or less to the sample 200. Even if the sample 200 is thick, the sample 200 can be frozen without ice crystals.

[0055] A pressure jet can be used as a pressurizing means for pressurizing the sample. For example, nozzles for ejecting gas are provided obliquely above and below the sample 200. Then, pressure is applied to the sample by ejecting high-pressure gas from above and below. In this way, it is possible to freeze the sample while applying pressure.

[0056] Alternatively, a pressurizing mechanism such as a piston may be used as the pressure applying means. Fig. 5 is a schematic diagram showing a configuration in which a piston 300 is used as the pressure applying means. In Fig. 5, the piston 300 is disposed above the sample 200, and the objective lens 11 is disposed below. A cryogen supply unit 301 is provided inside the piston 300. Specifically, a through hole provided from the side to the bottom of the piston 300 serves as the cryogen supply unit 301. In this way, the pressure applying mechanism and the freezing mechanism are provided independently.

[0057] 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 through hole that passes from one opening 302 to the other opening 302 via the opening 303 serves as a cryogen supply section 301. One opening 302 serves as a cryogen supply port, and the other opening 302 serves as a cryogen discharge port. The cryogen supplied from one opening 302 flows to the opening 303. As a result, the sample 200 facing the opening 303 is frozen by the cryogen. Then, the cryogen used for freezing is discharged from the other opening 303. In this way, it is possible to freeze the sample 200 while applying pressure to it. It is also possible to observe while applying pressure.

[0058] Furthermore, the timing of applying pressure may be synchronized with the timing of introducing a cryogen and the timing of applying a stimulus to the sample 200. Control of the timing of introducing a cryogen, the timing of pressurization, and the timing of applying a stimulus to the sample 200 will be described later.

[0059] Figure 6 shows microscopic images of a sample at room temperature and a sample after freezing. The sample is a HeLa cell labeled with Fluo-4. The objective lens is 60x, the numerical aperture is 0.95, and the excitation light source is a mercury lamp. The intensity of the excitation light is The power consumption is 1.04mW.

[0060] The room temperature sample is imaged with a microscope camera exposure time of 30 msec. The frozen sample has a microscope camera exposure time of 5 sec. In this way, by supplying a cryogen toward the sample 200, the sample can be frozen instantly. The sample can be frozen while maintaining the shape of the cells. Since the camera exposure time can be extended, the sample can be imaged with a high signal-to-noise ratio.

[0061] By simply introducing a cryogen at any timing, the sample 200 under microscope observation can be fixed in a state close to its original state. In addition, since the type of optical microscope and the optical response observed are not important, the present invention can be applied to any optical microscope. The microscope according to this embodiment can capture a snapshot of the dynamics of a biological sample, which is difficult to integrate signals over a long period of time. The above microscope 1 can be applied to research observing biological samples. The above microscope 1 can also be applied to clinical practice.

[0062] Embodiment 2 In the second embodiment, the cryogen supply unit includes a supply pipe and a valve. A microscope 4 according to the second embodiment will be described with reference to Figs. 7 to 9. Fig. 7 is a schematic diagram of the overall configuration of the microscope 4. Fig. 8 is a cross-sectional view showing the configuration of the sample holder 401. Fig. 9 is a perspective view of the sample holder 401. Here, the microscope 4 is an inverted microscope. Liquid nitrogen is used as the cryogen.

[0063] As shown in Fig. 7, a sample holder 401 is disposed on the microscope main body 412. A cryogen supply unit 460 includes a dewar 450, a supply pipe 461, and a valve 462. The supply pipe 461 of the cryogen supply unit 460 is connected to the sample holder 401. One end of the supply pipe 461 is connected to the sample holder 401, and the other end is installed in the dewar 450. The dewar 450 and the sample holder 401 are connected via the supply pipe 461. A valve 462 is provided midway along the supply pipe 461. The supply pipe 461 is preferably covered with a heat insulating material.

[0064] A cryogen 451 is stored in the dewar 450. A pressurized gas supply pipe 453 is connected to the dewar 450 via a valve 454. The pressurized gas supply pipe 453 supplies nitrogen gas into the dewar 450. The nitrogen gas from the pressurized gas supply pipe 453 makes the pressure in the dewar 450 higher than 1 atmosphere. The pressure in the sample holder 401 is also 1 atmosphere. A cryogen is filled from the other end of the supply pipe 461 to the valve 462. When the valve 462 opens, the cryogen 451 in the supply pipe 461 is discharged to the sample holder 401. The cryogen 451 passes through the supply pipe 461 and reaches the sample.

[0065] A discharge pipe 473 for discharging the cryogen is connected to the sample holder 401. The discharge pipe 473 and the supply pipe 461 are connected via a flow path 403. The cryogen supplied from the supply pipe 461 to the sample holder 401 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 installed in the cryogen recovery tank 470. The inside of the cryogen recovery tank 470 is at 1 atmosphere. The cryogen used to cool the sample is stored in the cryogen recovery tank 470. In this way, the used cryogen 471 can be collected. It is preferable that the discharge pipe 473 is covered with a heat insulating material.

[0066] It is preferable to cool the supply pipe 461, the valve 462, and the discharge pipe 473 in advance. For example, a cryogen is flowed in advance before a sample is placed on the sample holder 401. This allows the sample holder 401, the supply pipe 461, the valve 462, the discharge pipe 473, etc. to be cooled in advance. Then, after the sample holder 401, etc. are heated, a sample is placed on the sample holder 401. Thereafter, the valve 462 is opened to flow a cryogen from the supply pipe 461, thereby freezing the sample 200.

[0067] As shown in Fig. 8 and Fig. 9, the sample holder 401 includes a base 402 and a sample holder 405. 8 As shown in FIG. 98, the cover 407 is omitted. A flow path 403 through which a cryogen flows is formed in the base 402 or the sample holder 405. Furthermore, the cover 407 covers the sample holder 405. Furthermore, an opening 404 is formed in the base 402. The opening 404 is a through hole that penetrates the base 402 in the Z direction. As shown in FIG. 8, the objective lens 11 is disposed in the opening 404.

[0068] As shown in Fig. 8, a flow path 403 is provided in a sample holder 401. The flow path 403 is provided along the X direction. A supply pipe 461 and a discharge pipe 473 are connected via the flow path 403. A sample 200 is placed directly below the flow path 403. A cryogen flows directly above the sample 200. The cryogen flowing through the flow path 403 comes into contact with the sample 200. This allows the sample 200 to be cooled.

[0069] The sample holder 405 is detachably attached to the base 402. With the sample holder 405 detached from the base 402, the sample 200 is placed in the opening 404 of the base 402. The sample 200 is placed so as to cover the opening 404. Then, by attaching the sample holder 405 to the base 402 from above the sample 200, the sample 200 can be fixed to the sample holder 401.

[0070] In this way, by providing valve 462 on supply pipe 461 of cryogen supply unit 460, sample 200 can be frozen at an appropriate timing. The measurement results obtained by measuring the change in temperature over time using a thermocouple installed in flow path 403 will be described. Immediately after valve 462 is opened, the temperature becomes -189.5°C. Therefore, sample 200 can be frozen by contacting the cryogen with sample 200 immediately after valve 462 is opened. This makes it possible to freeze sample 200 in a short time.

[0071] The valve 462 may be opened and closed by a user operating a switch or the like. Alternatively, a control unit 465 may be provided to control the opening and closing of the valve 462. The control unit 465 can control the opening and closing timing of the valve 462. The valve 462 opens and closes based on a control signal output from the control unit 465.

[0072] Various signals can be used as the signal that triggers the opening and closing of the valve 462. For example, the control unit 465 may control the valve based on a signal from the sample 200. Specifically, the control unit 465 outputs a control signal based on an optical signal or an electrical signal from the sample 200. As the optical signal from the sample 200, the intensity of scattered light or the intensity of fluorescence can be used. Furthermore, the sample 200 may be imaged and used as a control signal according to its form, such as its shape and size. For example, when the sample 200 is a pulsating organ, the control unit 465 outputs a control signal according to the change in form of the sample 200. This allows freezing to be performed in accordance with the pulsation timing.

[0073] The control unit 465 controls the timing of opening and closing the valve 462 based on an image of the sample 200. Furthermore, when performing fluorescent observation, the sample can be frozen in conjunction with the timing of fluctuations in the amount of fluorescent light. When the sample 200 is a heart, the control unit 465 may control the valve 462 based on an electrical signal from an electrocardiogram. In this way, the control unit 465 may control the timing of freezing based on a signal from the sample 200. This allows the sample 200 to be frozen at a desired timing.

[0074] Alternatively, when, for example, an optical stimulus, an electrical stimulus, an electromagnetic stimulus, a magnetic stimulus, or the like is applied to the sample 200 based on a signal for stimulating the sample 200, the control unit 465 opens and closes the valve 462 in response to the signal for applying the stimulus. This allows the sample 200 to be frozen in conjunction with the timing of applying the stimulus. In addition, the stimulus for the sample 200 is not limited to an optical stimulus, an electrical stimulus, an electromagnetic stimulus, or a magnetic stimulus. The stimulus for the sample 200 can be applied by a drug, temperature, a mechanical stimulus, an electromagnetic wave, an electron beam, radiation, optogenetics, photodissociation of a caged compound, or the like. By using a signal for stimulating the sample 200, it is possible to freeze the sample at an appropriate timing.

[0075] In this way, the control unit 465 controls the opening and closing of the valve 462, thereby adjusting the timing of introducing the cryogen. Of course, the control unit 465 may control the timing of introducing the cryogen using two or more signals. Also, the introduction mechanism for introducing the cryogen into the cryogen supply unit and the flow path 403 is not limited to the valve 462. For example, in the configurations of Figs. 1 to 3, a mechanism for driving a cryogen container so as to pour the cryogen into the cryogen inlet 134 may be used. A container storing the cryogen is disposed above the cryogen inlet 134. Then, the container may be tilted according to a control signal. The cryogen can be introduced into the cryogen inlet 134 by tilting the container. Alternatively, the cryogen may be introduced into the flow path 403 by controlling the valve 454 for pressurizing the dewar 450. For example, the valve 462 is opened at the timing of introducing the cryogen. As a result, the space in the dewar 450 is pressurized, and the cryogen 451 flows through the flow path 403. Alternatively, a method of supplying cryogen to the sample may be used by switching the path of the cryogen flowing through a flow path near the sample. For example, two flow paths, one in contact with the sample and one not in contact with it, are prepared, and a flow path switching electromagnetic valve is placed near the sample. First, cryogen flows through the non-contact flow path, and then the path is switched by controlling the electromagnetic valve with an external signal to introduce the cryogen to the sample. This allows a lower temperature cryogen to be introduced to the sample with less time lag.

[0076] Furthermore, the timing of introducing the cryogen may be synchronized with the pressurization timing of the first embodiment. For example, the control unit 465 controls so that the timing of opening and closing the valve is synchronized with the pressurization timing. Similarly, the timing of stimulating the sample may also be synchronized with the pressurization timing of the first embodiment. 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 a stimulus to the sample 200.

[0077] Third embodiment The microscope according to the third embodiment will be described with reference to FIGS. 10 and 11. FIG. 10 shows a microscope. 5 11 is a schematic diagram showing the overall configuration of the microscope 5. FIG. 11 is an enlarged view of the objective lens 11 and the sample 200 and its surroundings. In the third embodiment, the microscope 5 is an upright microscope. Also, similar to the second embodiment, the timing of freezing is controlled by opening and closing the valve 462.

[0078] A sample 200 is disposed directly below the objective lens 11. The sample 200 is placed on a stage 10. The sample 200 is disposed in the field of view of the objective lens 11. The sample 200 includes a substrate 201 and an observation target 204 disposed on the substrate 201. A microscope main body 412 is disposed above the objective lens 11. The microscope main 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 through the microscope main body 412. The camera captures an image of the sample 200 formed by the imaging lens. Alternatively, the microscope main body 412 may have an eyepiece for direct observation by a user.

[0079] The cryogen supply unit 460 includes a dewar 450, a supply pipe 461, and a valve 462. One end of the supply pipe 461 is disposed near the sample 200, and the other end is installed inside the dewar 450. A valve 462 is provided midway along the supply pipe 461. The valve 462 is preferably disposed near the objective lens 11.

[0080] As in the second embodiment, a cryogen 451 is stored in a dewar 450. A pressurized gas supply pipe 453 is connected to the dewar 450 via a valve 454. The pressurized gas supply pipe 453 supplies nitrogen gas into the dewar 450. The pressure in the dewar 450 is higher than 1 atmosphere due to the nitrogen gas from the pressurized gas supply pipe 453. A cryogen is filled from the other end of a supply pipe 461 to a valve 462. When the valve 462 opens, the cryogen 451 in the supply pipe 461 is discharged to the sample holder 401. The cryogen 451 passes through the supply pipe 461 and reaches the sample.

[0081] As shown in FIG. 11, the tip of the supply pipe 461 serves as a nozzle 464 that sprays a cryogen. The inner diameter of the nozzle 464 is 3 mm. The objective lens 11 is a long working objective lens. The tip of the nozzle 464 is disposed between the objective lens 11 and the sample 200. The nozzle 464 sprays the cryogen toward the sample 200. The tip of the nozzle 464 may be disposed directly above the sample 200.

[0082] A valve 462 is disposed to the side of the objective lens 11. Before observation, the sample 200 and the objective lens 11 are warmed up. The valve 462 is preferably disposed near the sample 200. When the valve 462 is opened, a cryogen is ejected from a nozzle 464 in the direction of the arrow. The cryogen is sprayed onto the observation target 204 from diagonally above. The cryogen ejected onto the sample 200 freezes the observation target 204 of the sample 200. Therefore, the sample 200 can be frozen in a short time.

[0083] A protector 19 for protecting the objective lens 11 is provided around the tip of the objective lens 11. Furthermore, the protector 19 holds a transparent protective glass 18. The protective glass 18 is interposed between the objective lens 11 and the sample 200. This makes it possible to prevent the objective lens 11 from being sprayed with a cryogen.

[0084] Also, a dry gas may be flowed into the space between the protector 19 and the objective lens 11. The space between the protector 19 and the objective lens 11 may be evacuated to create a vacuum or reduced pressure state. In this way, condensation and cooling of the objective lens 11 can be prevented. Similarly, a dry gas may be flowed into the space between the protective glass 18 and the objective lens 11. Also, the space between the protector 19 and the objective lens 11 may be evacuated. In this way, the lens function can be maintained without condensation on the surface of the objective lens 11.

[0085] Figure 12 is a micrograph taken by observing the freezing of a sample. Figure 12 shows a micrograph of a rat perfused heart taken from above with a stereomicroscope. The tip of the nozzle is located to the left of the sample. The timing when the cryogen is ejected from the nozzle is set as 00'00". The micrographs shown were taken at intervals of 0.03 sec or 0.04 sec. In this way, the sample is frozen immediately after the cryogen is ejected from the nozzle. This makes it possible to distinguish between the diastole and systole of the heart.

[0086] Variation 1 A modified example of the sample holder serving as the sample freezing means will be described with reference to Figs. 13 and 14. Figs. 13 and 14 are perspective views showing a sample holder 600 according to the modified example 1. The sample holder 600 is used in an upright microscope. Therefore, an objective lens is disposed above the sample holder 600.

[0087] The sample holder 600 includes a first plate 610, a second plate 620, and a cryogen reservoir 630. The cryogen reservoir 630 is connected to the second plate 620 via a hinge 625. The angle of the cryogen reservoir 630 around the hinge 625 is different between FIG. 13 and FIG. 14. The sample is observed in the state shown in FIG. 13. Also, the sample can be removed from the sample holder 600 in the state shown in FIG. 14.

[0088] The first plate 610 and the second plate 620 are disk-shaped members. The first plate 610 and the second plate 620 are, for example, metal plates such as stainless steel. They can be made of metal plates such as titanium, not limited to stainless steel. Alternatively, the first plate 610 and the second plate 620 can be made of polymer plates that have low thermal conductivity and are difficult to break even at low temperatures. The polymer plates can be made of materials such as Teflon, polycarbonate, and polylactic acid. The first plate 610 and the second plate 620 can also be ceramic plates used as heat insulating materials.

[0089] The first plate 610 serves as a base to be placed on the stage of a microscope. A disk-shaped second plate 620 is fixed onto the first plate 610. In the XY plane, the outer shapes of the first plate 610 and the second plate 620 are approximately the same size.

[0090] 14, an opening 613 is provided in the center of the first plate 610 in the XY plane. The opening 613 penetrates in the Z direction. The opening 613 and its periphery serve as a sample mounting portion 617. A sample is mounted on the sample mounting portion 617. Therefore, the sample mounting portion 617 serves as the focal plane of the objective lens.

[0091] The cryogen reservoir 630 serves as a cover for holding the sample. For example, the sample 200 is placed between the cryogen reservoir 630 and the first plate 610. The cryogen reservoir 630 rotates around the axis of the hinge 625. By rotating the cryogen reservoir 630 so as to approach the first plate 610, the sample 200 is sandwiched between the cryogen reservoir 630 and the first plate 610. As a result, the sample holder 600 holds the sample, and the sample becomes observable. Also, by rotating the cryogen reservoir 630 so as to move away from the first plate 610, the upper side of the sample mounting portion 617 is opened. As a result, the sample can be mounted in the sample mounting portion 617. Alternatively, the sample in the sample mounting portion 617 can be removed.

[0092] The cryogen reservoir 630 is provided with a lens arrangement section 636 in which an objective lens is arranged. The lens arrangement section 636 includes an opening penetrating in the Z direction. The lens arrangement section 636 is arranged directly above the opening 613. Here, the lens arrangement section 636 is formed with a tapered inclined surface 637 in accordance with the shape of the objective lens.

[0093] A supply pipe 633 that functions as a cryogen supply section is obliquely provided in the cryogen storage section 630. With the sample 200 sandwiched between the cryogen storage section 630 and the first plate 610, the supply pipe 633 is located obliquely above the lens arrangement section 636. The shape of the sample 200 is not limited to the configuration in FIG. 4. For example, the sample 200 may be an organ as it is. A cryogen inlet 634 is provided on the upper side of the supply pipe 633. A tube through which the cryogen flows may be connected to the cryogen inlet 634. By flowing liquid cryogen from the cryogen inlet 634, the cryogen flows down from the supply pipe 633 to the inclined surface 637. The cryogen comes into contact with the sample in the sample installation section 617, and the cryogen cools the sample. This causes the sample to freeze. For example, in order to supply the cryogen, the user may transfer the cryogen in a container to the supply pipe. Alternatively, the timing of the cryogen supply may be controlled by opening and closing a valve as in the second embodiment. This configuration allows the sample in the field of view of the objective lens to be frozen using a simple configuration and method.

[0094] A cryogen exhaust port 627 is provided on the upper surface of the second plate 620. The cryogen exhaust port 627 communicates with the opening 613. X Cryogen outlets 627 are arranged on both sides of the cryogen reservoir 630 in the direction of the axis of rotation. The cryogen supplied from the supply pipe 633 is discharged from the cryogen outlet 627. In other words, the cryogen used to cool the sample is discharged from the cryogen outlet 627. This makes it possible to continue to supply new cryogen, thereby increasing the cooling rate. Of course, the cryogen outlet 627 can be omitted.

[0095] Variation 2 A second modified example of the sample holder serving as the sample freezing means will be described with reference to Figs. 15 and 16. Figs. 15 and 16 are perspective views showing the configuration of a sample holder 800. The sample holder 800 according to the second modified example can be used in a stereomicroscope. More specifically, the sample holder 800 can be applied to a stereomicroscope for perfusion in which the heart is observed as an object of observation. The sample holder 800 can be applied to an upright microscope. The sample holder 800 may also be applied to an inverted microscope.

[0096] The sample holder 800 includes a base 810 and a side wall 820. The base 810 is a flat plate-like member along the XY plane. The base 810 is made of a transparent member, which allows illumination or observation from below. A cryogen reservoir 833 is provided in the center of the base 810 when viewed from the XY plane. The cryogen reservoir 833 is a circular recess. A step provided around the cryogen reservoir 833 serves as a sample mounting section 817. A sample (not shown) is mounted on the sample mounting section 817. An objective lens (not shown) is disposed above or below the sample mounting section 817. Thus, the sample is disposed in the field of view of the objective lens.

[0097] An outlet 813 is provided on the -Y side of the base 810. The outlet 813 is connected to the sample mounting section 817 or the cryogen reservoir 833. The perfusion fluid is discharged from the outlet 813. Alternatively, the cryogen may be discharged from the outlet 813. The outlet 813 is provided along the Y direction. The outlet 813 penetrates the inside of the base 810 until it is connected to the sample mounting section 817 or the cryogen reservoir 833. A groove 814 is formed on the upper surface of the base 810 for arranging an electrode or a perfusion core tube. The groove 814 is provided along the Y direction. The groove 814 is connected to the cryogen reservoir 833. The grooves 814 extend from both sides of the cryogen reservoir 833.

[0098] A side wall 820 is provided at the end of the +X side of the base 810. The side wall 820 is erected on the +Z side from the upper surface of the base 810. A cryogen supply port 830 is formed in the side wall 820. The cryogen supply port 830 penetrates the side wall 820 at an angle. Specifically, the cryogen supply port 830 descends from the +X side toward the -X side. Therefore, when a cryogen is put into the cryogen supply port 830 from the +X side, the cryogen flows down from the -X side of the cryogen supply port 830 toward the cryogen storage tank 833. Thus, the cryogen comes into contact with the sample, and the sample can be frozen. With this configuration, a sample in the field of view of the objective lens can be frozen with a simple configuration and method.

[0099] This embodiment is applicable not only to inverted microscopes, upright microscopes, and stereo microscopes, but also to other types of microscopes. For example, it is also applicable to a microscope that performs side illumination in which illumination light is incident on a sample from the side of the objective lens. The microscope is suitable for observing biological samples such as cultured cells and cardiomyocytes.

[0100] The light observed by the microscope may be any of the following responses: scattering, absorption, emission, and reflection. The microscope can also be used for fluorescence observation and Raman scattering observation. The sample observed by the microscope can be frozen with a cryogen, and the frozen sample can be observed at low temperatures.

[0101] While observing the calcium ions of an isolated perfused rat heart placed in the sample holder 600 with a fluorescent microscope using an upright microscope or a stereo microscope, a liquid propane-isopentane mixture cryogen is introduced from the inflow channel into the reservoir at any desired timing to freeze and fix the heart. By freezing and fixing the heart during both the diastole and systole, it is possible to extend the signal integration time, which was previously limited by the speed of the phenomenon, and it becomes possible to observe both the diastole and systole.

[0102] Freezing and observing the sample 200 enables various applications. For example, the quantum yield can be estimated by measuring the fluorescence lifetime. In addition, the cell membrane potential can be measured by membrane potential imaging. The cell membrane potential and the surrounding ion distribution can be measured simultaneously.

[0103] The sample 200 can be observed in association with the redox potential or redox state of the sample 200. When the sample 200 is a biological sample, the sample 200 can be observed in association with the measurement results of blood flow or oxygen concentration.

[0104] The sample 200 may be melted by irradiating the sample 200 with light after freezing the sample 200. For example, the sample 200 can be melted by irradiating it with infrared laser light. The laser light may be irradiated to the sample 200 via the objective lens 11. 、 The sample 200 may be irradiated from the opposite side to the objective lens 11. Furthermore, by adjusting the irradiation area of ​​the sample 200, it is possible to adjust the sample 200 to be partially melted. For example, the laser light may be irradiated only to the part of the sample 200 where it is desired to melt the sample 200.

[0105] Moreover, after the sample 200 has melted by the laser irradiation, the sample 200 can be frozen again by stopping the irradiation of the laser light. Thus, the sample 200 can be observed while repeatedly freezing and melting the sample. In this way, by melting the sample by the laser light irradiation, the melted portion can be observed. Alternatively, the sample can be observed in a state where the sample has been melted and then frozen again.

[0106] Furthermore, the sample observed under the microscope is not limited to a sample placed on a sample substrate. For example, a sample flowing through a channel such as a microchannel may be observed. In other words, the sample is contained in the fluid flowing through the channel. A cryogen is introduced toward the channel in the field of view of the objective lens. For example, the cryogen is brought into contact with a substrate on which the channel is provided. This allows the sample in the channel to be instantly frozen.

[0107] Fourth embodiment In the fourth embodiment, a cryogen is supplied based on a signal from a sample or a signal for stimulating the sample. A configuration for supplying a cryogen based on a signal from a sample or a signal for stimulating the sample will be described with reference to Fig. 17. Fig. 17 is a diagram showing the configuration of a microscope according to the fourth embodiment.

[0108] The microscope 500 includes a camera 501, an imaging lens 502, a housing 503, an objective lens 510, a mirror 511, a support 521, a lifting stand 522, a stage 523, a jack 524, and a sample freezing means 550. The microscope 500 further includes a stimulation electrode 553 for applying a stimulus to the sample 200, a function generator 552, an electrocardiogram measuring device 530, and the like.

[0109] The specimen 200 is held in a specimen chamber 540. The specimen 200 is in the form of an excision core. The specimen chamber 540 is fixed on a height-adjustable jack 524. A specimen freezing means 550 is disposed directly above the specimen 200. The specimen 200 is illuminated by room lighting.

[0110] A mirror 511 is disposed to the side of the sample 200. The mirror 511 is supported by a support 521, a raised platform 522, and a stage 523. An objective lens 510, a housing 503, an imaging lens 502, and a camera 501 are provided above the mirror 511. Light incident on the mirror 511 from the sample 200 is reflected by the mirror 511 and enters the objective lens 510. The light incident on the objective lens 510 is refracted by the objective lens 510 and enters the housing 503.

[0111] Light from the objective lens 510 enters the imaging lens 502 through the housing 503. The imaging lens 502 forms an image of the sample 200 on the light receiving surface of the camera 501. This allows the camera 501 to capture an image of the sample 200. As the optical system for guiding the light from the light source to the sample 200 and the optical system for guiding the light from the sample 200 to the camera 501 can be a known one, a description thereof will be omitted.

[0112] The sample freezing means 550 includes a function generator 552. The sample freezing means 550 includes a pressurized gas inlet 561, a pressure gauge 562, a safety valve 563, a sealing lid 564, a heat-insulating container 565, a cryogen flow path 571, an electromagnetic valve 573, and a pre-cooling box 574.

[0113] Liquid cryogen 570 is stored in the insulated container 565. The upper side of the insulated container 565 is sealed with a sealing lid 564. Pressurized gas is supplied to the insulated container 565 through a pressurized gas inlet 561. A pressure gauge 562 measures the gas pressure of the pressurized gas. When the pressure detected by the pressure gauge 562 reaches or exceeds a predetermined pressure, a safety valve 563 releases the pressurized gas.

[0114] A cryogen flow path 571 is connected to the bottom of the insulated container 565. The cryogen flows downward through the cryogen flow path 571 by pressurized gas. The cryogen flow path 571 passes through a pre-cooling box 574. The pre-cooling box 574 is pre-cooled by liquid nitrogen 572. An electromagnetic valve 573 is provided midway along the cryogen flow path 571. The bottom of the pre-cooling box 574 serves as a cryogen supply port. The cryogen is supplied from directly above the sample 200. When the electromagnetic valve 573 is opened, the cryogen is supplied to the sample 200, and the sample 200 is frozen.

[0115] The function generator 552 generates a control signal for controlling the electromagnetic valve. The electromagnetic valve 573 opens and closes according to the control signal of the function generator 552. The function generator 552 outputs a trigger signal to the stimulation electrode 553. The trigger signal is a signal that electrically stimulates the isolated heart, which is the sample 200. The sample 200 is stimulated by the trigger signal applied to the stimulation electrode 553. The function generator 552 controls the control signal and the trigger signal independently. The function generator 552 can control the intensity and phase of the applied voltage.

[0116] The electromagnetic valve 573 opens at a predetermined time delay from the timing when the electrical stimulus is applied to the sample 200. When the electromagnetic valve opens in response to a control signal from the function generator 552, a cryogen is supplied to the sample 200. In this manner, the timing of applying the cryogen can be controlled in response to a trigger signal for stimulating the sample 200. Furthermore, the function generator 552 can vary the timing of the control signal, thereby adjusting the timing of applying the cryogen. The sample 200 can be frozen after a desired time has elapsed since the electrical stimulus. The sample 200 can be frozen in response to the timing of the heartbeat.

[0117] Furthermore, an electrocardiogram measuring device 530 is connected to the sample 200 . The electrocardiogram measuring device 530 measures an electrocardiogram according to the heart rate of the sample 200 .

[0118] Fig. 18 shows the waveforms of an electrocardiogram and a trigger signal. The upper part of Fig. 18 shows the waveform of an electrocardiogram, and the lower part shows the waveform of a trigger signal (stimulation) given to the stimulation electrode 553. Here, after generation of the trigger signal, a control signal reaches the electromagnetic valve 573 about 40 msec. After another 40 msec, the cryogen comes into contact with the sample 200.

[0119] In the above explanation, the timing of supplying the cryogen is controlled based on the trigger signal from the function generator 552, but it is also possible to control the timing of supplying the cryogen based on the electrocardiogram measured by the electrocardiogram measuring device 530. In other words, the timing of opening the solenoid valve 573 can be adjusted according to the electrocardiogram waveform. Since the timing of the heart's diastole and systole can be measured from the electrocardiogram waveform, the sample 200 can be frozen at the desired timing. Therefore, the timing of supplying the cryogen can be controlled according to the signal from the sample 200.

[0120] Embodiment 5. In the fifth embodiment, the sample 200 is frozen with a cryogen, and then thawed by irradiating the sample 200 with laser light. The microscope according to the fifth embodiment will be described with reference to Fig. 19. Fig. 19 is a schematic diagram showing the optical system of a microscope 700. Here, the microscope 700 will be described as a fluorescent microscope that detects fluorescence from the sample 200.

[0121] The microscope 700 includes an observation light source 701, a stage 710, an observation optical system 720, a two-dimensional photodetector 723, a thawing light source 730, a thawing optical system 740, a cryogen injection 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, the dichroic mirror 703, the dichroic mirror 704, and the objective lens 705.

[0122] The stage 710 holds a container 751 that contains the sample 200. Thus, the sample 200 is placed on the stage 710. A cryogen supply device 750 is provided above the sample 200. The cryogen supply device 750 is the above-mentioned sample freezing means, and supplies a cryogen to the sample 200. The cryogen supplied from the cryogen supply device 750 freezes the sample 200.

[0123] Next, an 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, and generates excitation light for exciting the sample 200. The laser light (excitation light) from the observation light source 701 is collected by a lens 702 and enters a dichroic mirror 703. The dichroic mirror 703 has wavelength characteristics that transmit light of the excitation light wavelength and reflect the light for thawing.

[0124] The laser light transmitted through the dichroic mirror 703 is incident on the dichroic mirror 704. The dichroic mirror 704 has wavelength characteristics of reflecting light of the excitation light wavelength and transmitting the fluorescence. The excitation light reflected by the dichroic mirror 704 is incident on the objective lens 705. The objective lens 705 focuses the excitation light on the sample 200.

[0125] The sample 200 is placed on a substrate (not shown) that transmits the wavelengths of the excitation light and the thawing light. The sample 200 and the substrate are placed on a stage 710. The stage 710 has an opening below the sample 200. Therefore, the excitation light passes through the opening of the stage 710 and enters the sample 200.

[0126] 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. The fluorescence that has passed through the dichroic mirror 704 enters the filter 721.

[0127] 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. The filter 721 transmits the fluorescence generated in the sample 200. The fluorescence that has transmitted through the filter 721 is incident on a tube lens 722. The tube lens 722 focuses the fluorescence on a two-dimensional photodetector 723. The two-dimensional photodetector 723 can capture a fluorescent image of the sample 200.

[0128] The thawing light source 730 generates, for example, infrared light for heating. The thawing light source 730 will be described as a laser light source that generates laser light, but the thawing light source 730 may be a lamp light source or the like. The laser light from the thawing light source 730 enters the scanner 742 via a shutter 741. The scanner 742 is an optical scanner such as a galvanometer mirror. The scanner 742 is a two-axis scanner, and two-dimensionally scans the irradiation position of the laser light on the sample 200. This allows the laser light to be irradiated to any position on the sample 200.

[0129] The light reflected by the scanner 742 is incident on the dichroic mirror 703. The dichroic mirror 703 has a wavelength characteristic of reflecting infrared light. Therefore, the dichroic mirror 703 reflects the laser light from the scanner 742. As a result, the laser light from the thawing light source 730 and the excitation light from the observation light source 701 propagate coaxially. The laser light reflected by the dichroic mirror 703 is incident on the dichroic mirror 704.

[0130] The dichroic mirror 704 has wavelength characteristics that reflect infrared light toward 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 so as to focus it on the sample 200. By irradiating the sample 200 with the laser light, the sample 200 frozen with a cryogen is partially heated. This allows at least a portion of the sample 200 to melt.

[0131] Furthermore, the laser light from the thawing light source 730 and the excitation light from the observation light source 701 are coaxial. Therefore, the laser light and the excitation light are incident on the same position of the sample 200. This makes it possible to capture a fluorescent image of the sample 200 at the position heated by the laser light. This makes it possible to observe the behavior of the frozen sample 200 while it is thawing. In addition, since a scanner 742 for scanning the laser light is provided, the irradiation position of the laser light can be controlled. The sample 200 can be appropriately thawed. Furthermore, the thawing position and time can also be controlled during observation.

[0132] Further, a shutter 741 is disposed in the optical path of the laser light from the thawing light source. The shutter 741 is provided so as to be able to open and close. When the shutter 741 is opened, the laser light is incident on the sample 200 and heats the sample 200. When the shutter 741 is closed, the laser light is no longer irradiated onto the sample 200. By controlling the opening and closing of the shutter 741, the sample 200 can be melted at an appropriate timing.

[0133] Embodiment 6 A microscope 700 according to the sixth embodiment will be described with reference to FIG. 20. FIG. 20 is a diagram showing the configuration of the microscope 700. In the sixth embodiment, the sample 200 contains a caged compound and a calcium indicator. The microscope 700 is for freezing and observing the sample 200 containing the caged compound and the calcium indicator. Therefore, the light sources 761 and 762 are different from those in the fifth embodiment. The configuration other than the light sources, for example, the configuration of the optical system, is the same as that in the fifth embodiment, and therefore will not be described.

[0134] In the sixth embodiment, light source 761 serves as a light source for observing the calcium indicator. Light source 761 generates excitation light that excites the calcium indicator contained in sample 200. When the excitation light from light source 761 is irradiated onto sample 200, fluorescence is generated from the calcium indicator. The calcium concentration can be measured by detecting the fluorescence with two-dimensional photodetector 723.

[0135] A light source 762 is a light source that generates light to be irradiated to the caged compound. The light source 762 generates ultraviolet light or visible light. The sample 200 has a photodegradable protecting group. The protecting group temporarily inactivates the biologically active molecule. The protecting group is dissociated by the light from the light source 762. This activates the biologically active molecule.

[0136] The timing of activation can be controlled by controlling the opening and closing timing of the shutter 741. Furthermore, the timing of freezing can be controlled by controlling the opening and closing of a valve provided midway through the cryogen flow path.

[0137] The invention made by the inventor has been specifically described above based on an embodiment, but it goes without saying that the present invention is not limited to the above embodiment and can be modified in various ways without departing from the gist of the invention.

[0138] This application claims priority based on Japanese Patent Application No. 2021-080906, filed on May 12, 2021, the disclosure of which is incorporated herein in its entirety. [Explanation of symbols]

[0139] 1. Microscope 4. Microscope 5. Microscope 10 Stages 11 Objective lens 100 Sample holder 110 First Plate 111 Gas supply port 113 Opening 117 Sample placement section 120 Second Plate 125 Hinge 127 Cryogen outlet 130 Cryogen supply section 133 Cryogen storage section 134 Cryogen inlet 160 Cooling agent 401 Sample holder 402 Base 403 Flow Path 404 Opening 405 Sample holder 450 Dewar 451 Cooling agent 460 Cryogen supply section 461 Supply pipe 462 Valve 464 Nozzle 465 Control Unit 470 Cryogen recovery tank 471 Cooling agent 473 Discharge pipe 600 Sample Holder 610 First Plate 613 Opening 617 Sample placement section 620 Second Plate 625 Hinge 627 Cryogen outlet 630 Cryogen storage section 633 Supply pipe 634 Cryogen inlet 636 Lens placement section 637 Slope 800 Sample Holder 810 Base 813 Outlet 820 side wall 830 Refrigerant supply port 833 Cryogen storage tank

Claims

1. an objective lens that receives light from the sample; a sample holder for holding the sample; the sample holder includes a sample freezing means for contacting a cryogen with the sample in the field of view of the objective lens to freeze the sample; the sample holder comprising a first plate; The first plate has an opening therein; The opening and its periphery serve as a sample placement area for placing the sample, the objective lens is disposed so that the sample mounting portion is a focal plane; the sample freezing means has a cryogen reservoir disposed directly above the opening for storing a liquid cryogen; A microscope in which the cryogen is supplied to the sample from above the sample by flowing the cryogen through a cryogen inlet provided on the upper side of the cryogen reservoir.

2. The sample holder further comprises a second plate disposed on the first plate and a cryogen supply unit; the cryogen supply is connected to the second plate via a hinge; The cryogen supply unit opens and closes by rotating about an axis of the hinge; By closing the cryogen supply unit, the cryogen supply unit is disposed above the opening, 2. The microscope according to claim 1, wherein an upper side of the opening is opened by opening the cryogen supply unit.

3. 2. The microscope of claim 1, wherein the sample can be observed while the cryogen is being supplied.

4. 2. The microscope according to claim 1, further comprising an introduction mechanism for introducing the cryogen toward the sample based on a signal from the sample or a signal for stimulating the sample.

5. The sample freezing means a supply pipe through which the cryogen flows; 5. The microscope according to claim 4, wherein the introduction mechanism is a valve provided in a supply pipe of the cryogen.

6. 2. The microscope according to claim 1, further comprising a pressurizing means for pressurizing the sample, the sample being frozen while being pressurized.

7. 2. The microscope according to claim 1, wherein a pressure means for pressurizing the sample is used, and the sample is observed while being pressurized.

8. 8. The microscope according to claim 7, further comprising a control unit for controlling the timing of pressurizing the specimen and the timing of introducing the cryogen.

9. 2. The microscope according to claim 1, wherein said sample freezing means is provided with a cryogen outlet communicating with said opening.

10. 10. The microscope according to claim 1, further comprising a function of irradiating light toward the frozen sample to melt at least a portion of the sample.

11. placing a sample held in a sample holder in a field of view of an objective lens; a sample freezing means contacting a cryogen with the sample in the field of view of the objective lens, thereby freezing the sample; and observing the frozen sample, the sample holder comprising a first plate; The first plate has an opening therein; The opening and its periphery serve as a sample placement area for placing the sample, the objective lens is disposed so that the sample mounting portion is a focal plane; the sample freezing means has a cryogen reservoir disposed directly above the opening for storing a liquid cryogen; An observation method in which the cryogen is supplied to the sample from above the sample by flowing the cryogen through a cryogen inlet provided on the upper side of the cryogen reservoir.

12. The sample holder further comprises a second plate disposed on the first plate and a cryogen supply unit; the cryogen supply is connected to the second plate via a hinge; The cryogen supply unit opens and closes by rotating about an axis of the hinge; By closing the cryogen supply unit, the cryogen supply unit is disposed above the opening, The observation method according to claim 11, wherein an upper side of the opening is opened by opening the cryogen supply unit.

13. 13. The observation method according to claim 11, wherein the sample is observed while the cooling agent is being supplied.

14. 13. The observation method according to claim 11, further comprising introducing the cryogen toward the sample based on a signal from the sample or a signal for stimulating the sample.

15. a supply pipe through which the cryogen flows; 15. The observation method according to claim 14, wherein a timing for introducing the cryogen is adjusted by a valve provided in a supply pipe for the cryogen.

Images