Microscope observation stage and microscope device

Abstract

The present invention addresses the problem of providing: a microscope observation stage that is equipped with a sealed container capable of reproducing in-vivo pressure (for example, pressure corresponding to in-vivo pulsation); and a microscope device. The present invention relates to a microscope observation stage and a microscope device, said microscope observation stage comprising: a sealed container that is capable of accommodating an observation target and has a port coupled to a pressure application unit; and a pressure sensor that is capable of measuring the pressure inside the sealed container in real time, wherein the observation target is a biological sample, the ceiling surface and the bottom surface of the sealed container are composed of optically transparent and smooth members, and the pressure sensor outputs a pressure sensor signal to outside.

Description

Microscope observation stage and microscope device

[0001] The present invention relates to a microscope observation stage and a microscope apparatus, and more particularly to a microscope observation stage and a microscope apparatus for observing biological samples such as cells in a sealed container that reproduces the pressure inside a living body.

[0002] Organs and cells in the body are exposed to various mechanical stresses (tension, shear stress, hydrostatic pressure, etc.). In particular, circulatory system organs such as the heart and blood vessels sense these mechanical stresses in response to cardiac pumping. Mechanical stress is known to be involved in cell differentiation, proliferation, and migration, but the cellular response to hydrostatic pressure, for example, remains largely unknown.

[0003] Cell culture instruments and microscopes equipped with cell culture functions have been developed. ２ Some incubators are also known that are provided with a means for introducing gas to increase the concentration, etc. For example, Patent Document 1 discloses an incubator for microscope observation that is provided with a container holder that detachably holds a sample container such as a dish, a water bath unit, a heater that heats the sample container and the water bath unit, and a gas supply means that supplies a predetermined gas to an incubation space defined by the water bath unit and a lid.

[0004] Patent Document 2 discloses a cell culture instrument that includes a main body having a culture vessel storage section that detachably stores a culture vessel, a lid, a first nozzle for supplying liquid from outside the main body to the culture vessel, a second nozzle for discharging liquid from the culture vessel to the outside of the main body, an air inlet for sending gas from outside the main body to the culture vessel storage section, and an exhaust port for discharging gas from the culture vessel storage section to the outside of the culture vessel storage section, and discloses that the cell culture instrument can be used in a microscope device.

[0005] JP 2004-141143 A JP 2009-65892 A

[0006] As described above, microscope devices having a cell culture space are known, but in conventional microscope devices, the in vivo environment (physiological condition) related to mechanical stress is not reproduced in the cell culture space, making it difficult to observe the state of cells or tissues under the in vivo environment in vitro. In particular, it has been difficult to observe over time cells, etc., that are subjected to cyclic mechanical stress in response to cardiac pumping.

[0007] Therefore, in order to solve these problems of the conventional technology, the inventors have conducted research to develop a microscope observation stage and microscope device for observing biological samples such as cells in a sealed container that reproduces periodically fluctuating pressure (e.g., pressure corresponding to the pulsation of a living body).

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that by providing a sealed container having a connection port to a pressure application unit on a microscope observation stage and a pressure sensor capable of measuring the pressure inside the sealed container in real time and outputting a pressure sensor signal to the outside, it is possible to observe biological samples such as cells in a state where a periodically fluctuating pressure is reproduced, and have completed the present invention. Specifically, the present invention has the following configuration.

[0009] [1] A microscope observation stage comprising: a sealed container capable of accommodating an observation object and having a connection port for connection to a pressure application unit; and a pressure sensor capable of measuring the pressure inside the sealed container in real time, wherein the observation object is a biological sample, the ceiling and bottom surfaces of the sealed container are made of optically transparent and smooth materials, and the pressure sensor outputs a pressure sensor signal to the outside. [2] The microscope observation stage according to [1], wherein the sealed container has at least one recessed portion in a region including an intersection of a diagonal line extending from the connection port and an inner wall of the sealed container. [3] The microscope observation stage according to [1] or [2], wherein the sealed container has two recessed portions in a region including an intersection of a diagonal line extending from the connection port and an inner wall of the sealed container, the two recessed portions facing each other. [4] The microscope observation stage according to any of [1] to [3], wherein the connection port is a connection port for an inlet / outlet pipe, and the inlet / outlet pipe is a pipe for supplying gas from the pressure application unit and recovering gas. [5] The microscope observation stage according to any one of [1] to [4], further comprising a temperature adjustment mechanism. [6] The microscope observation stage according to any one of [1] to [5], wherein the biological sample is a cell. [7] The microscope observation stage according to any one of [1] to [6], wherein one of the recesses further comprises a through-hole, and the through-hole is provided with a safety valve. [8] A microscope apparatus comprising the microscope observation stage according to any one of [1] to [7], and a pressure application unit connected to the sealed container, wherein the pressure application unit applies a physical stimulus to the biological sample by varying the pressure in the sealed container. [9] The microscope apparatus according to [8], wherein the pressure application unit receives a pressure sensor signal from the microscope observation stage and adjusts the pressure in the sealed container.

[10] The microscope apparatus according to [8] or [9], wherein the pressure application unit has a first pressure adjustment mechanism and a second pressure adjustment mechanism that adjust the pressure in the sealed container, and generates a pressure similar to in vivo pulsatile pressure in the sealed container by alternately operating the first pressure adjustment mechanism and the second pressure adjustment mechanism.

[0010] According to the present invention, it is possible to provide a microscope observation stage and microscope apparatus equipped with a sealed container that can reproduce in vivo pressure (for example, pressure corresponding to in vivo pulsation). Use of the microscope observation stage and microscope apparatus of the present invention is expected to dramatically advance research into cellular responses to in vivo pressure.

[0011] FIG. 1 is a schematic diagram illustrating the configuration of a microscope observation stage according to this embodiment. FIG. 2 is a plan view illustrating the configuration of a microscope observation stage according to this embodiment. FIG. 3 is a cross-sectional perspective view and a cross-sectional plan view illustrating the configuration of a sealed container in the microscope observation stage according to this embodiment. FIG. 4 is a schematic diagram illustrating the mechanism of a microscope apparatus according to this embodiment. FIG. 5 is a schematic diagram illustrating the mechanism of a microscope apparatus according to this embodiment. FIG. 6 is a diagram illustrating a pressure waveform when pressure fluctuations are generated in a sealed container using the microscope apparatus according to this embodiment. FIG. 7 is a graph of pressure fluctuations when cyclic pressurization is applied to human aortic smooth muscle cells and an observation image of the cells. FIG. 8 is a graph of pressure fluctuations when cyclic pressurization is applied to fluorescently labeled human aortic smooth muscle cells and an observation image of the cells.

[0012] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0013] (Microscope observation stage) The present invention relates to a microscope observation stage that includes a sealed container that can accommodate an observation object and has a connection port to a pressure application unit, and a pressure sensor that can measure the pressure inside the sealed container in real time. The observation object in the microscope observation stage of the present invention is a biological sample, and the ceiling and bottom surfaces of the sealed container are made of optically transparent and smooth materials. In addition, the pressure sensor outputs a pressure sensor signal to the outside.

[0014] The microscope observation stage of the present invention includes a sealed container having a connection port for connecting to a pressure application unit, and a pressure sensor that can measure the pressure inside the sealed container in real time and output a pressure sensor signal to the outside, so that it is possible to generate in vivo pressure (e.g., pressure corresponding to in vivo pulsation) inside the sealed container provided on the microscope observation stage. For example, if a liquid such as a culture medium is contained in the sealed container, it is possible to apply a pressure similar to in vivo pulsatile pressure to the liquid (static water).

[0015] FIG. 1 is a schematic diagram illustrating the configuration of a microscope observation stage according to this embodiment. The microscope observation stage 100 according to this embodiment includes a sealed container capable of housing a biological sample as an observation target. As shown in FIG. 1 , the sealed container has a sealed container body 10, which is provided with a transparent member 11 constituting the ceiling surface of the sealed container and a transparent member 12 constituting the bottom surface. Furthermore, O-rings 15 may be provided between the sealed container body 10 and each of the transparent members 11 and 12 to enhance the sealing performance within the sealed container. Examples of materials for the O-rings 15 include silicone, fluororesin, nitrile resin, and styrene resin. Furthermore, to enhance the engagement between the sealed container body 10 and each of the transparent members 11 and 12, a top lid 13 and an inner lid 14 may be engaged with the sealed container body 10, and these members are preferably fastened together with screws or other members. The top lid 13 and the inner lid 14 have hollowed-out central regions or are made of transparent materials. The sealed container may be a member composed of, for example, a sealed container body 10, a transparent member 11 constituting the ceiling surface of the sealed container, and a transparent member 12 constituting the bottom surface, and in this case, each of the transparent members 11 and 12 may be engaged with the sealed container body 10 so as to form a sealed storage section. Also, the sealed container may be a container in which the sealed container body 10, the ceiling surface, and the bottom surface of the sealed container are integrated.

[0016] The sealed container body 10, the top lid 13, and the inner lid 14 are preferably made of a non-metallic inorganic material such as a metal, an alloy, or a ceramic, or a resin. The size of the sealed container body 10 is not particularly limited as long as it fits within the base of the microscope observation stage 100. The transparent members 11 and 12 are optically transparent and smooth members. That is, the ceiling and bottom surfaces of the sealed container are made of optically transparent and smooth members. The transparent members 11 and 12 are engaged with the sealed container body 10 to form a sealed space. The transparent members 11 and 12 are arranged in parallel positions to form a light-transmitting region of the sealed container. Examples of materials for the transparent members 11 and 12 include glass, PMMA resin, PC resin, COP resin, COC resin, and PAR resin.

[0017] The sealed container (sealed container body 10) has a connection port 20 to the pressure application unit (pressure application unit connection port 20). By having the sealed container (sealed container body 10) have the connection port 20 to the pressure application unit (pressure application unit connection port 20), gas sent from the pressure application unit can be supplied into the sealed container, and pressure can be applied inside the sealed container.

[0018] The microscope observation stage 100 is equipped with a pressure sensor 30 that can measure the pressure inside the sealed container in real time. The pressure sensor 30 outputs a pressure sensor signal to the outside. The pressure sensor signal may be output to a pressure application unit (microscope device), or may be output to a display or controller provided in the pressure application unit or other device.

[0019] In this embodiment, the pressure sensor 30 is preferably a gauge pressure sensor. The pressure sensor 30 is provided inside or outside the sealed container and measures the pressure applied to the sealed container in real time. Then, in accordance with the pressure value detected by the pressure sensor, a pressure sensor signal is output to the outside so that the pressure inside the sealed container reaches a desired pressure value.

[0020] In this embodiment, for example, a periodic pressure can be generated within the sealed container, and a pressure similar to in vivo pulsatile pressure can also be applied. In this specification, "in vivo pulsatile pressure" refers to pressure that fluctuates in accordance with blood flow in a living body due to cardiac pulsation, and is also referred to as physiological pressure or physiological pulsatile pressure. "In vivo pulsatile pressure-like pressure" refers to a pressure that fluctuates to mimic the pressure that periodically fluctuates in accordance with blood flow in a living body due to cardiac pulsation. While in vivo pulsatile pressure values ​​measured at various locations within a living body vary, for example, the generation of a "in vivo pulsatile pressure-like pressure" refers to alternating low and high pressures within a range of 1 to 200 kPa (gauge pressure) (repeated so as to generate a minute pressure difference of several tens of kPa). In the present invention, for example, high pressures of approximately several tens of kPa and low pressures of several to several tens of kPa can be alternately generated (reducing the fluctuation range of pressure values), which is useful in that it more faithfully reproduces in vivo pulsation. When a "pressure similar to in vivo pulsatile pressure" occurs, the cycle of the pressure change is 10 to 300 times / minute, preferably 25 to 150 times / minute.

[0021] In this embodiment, a biological sample is preferably contained in the sealed container as an observation target. In this case, the biological sample may be placed directly in the sealed container, but it is preferable that a biological sample container be contained in the sealed container, and the biological sample is preferably cultured in the biological sample container. Examples of the biological sample container include a cell culture plate and a cell culture tank. The biological sample container is preferably filled with a medium for cell culture or the like. In this case, instead of applying pressure directly to the medium (liquid) containing the biological sample, pressure can be indirectly transmitted to the liquid by varying the pressure in the sealed container, thereby more faithfully reproducing physiological conditions.

[0022] Examples of biological samples include cells, tissues, blood, body fluids, DNA, RNA, proteins, etc. Among these, cells are preferred as biological samples. It can be said that tissues are aggregates of cells. Cells include all cells that make up a living organism, but also include unicellular organisms (amoeba, euglena, diatoms, etc.), multicellular organisms (daphnia, Spirogyra, etc.), and multicellular three-dimensional structures (organoids, spheroids, etc.).

[0023] Preferably, the sealed container contains a culture medium containing a biological sample. By applying in vivo pressure (e.g., pressure corresponding to in vivo pulsation) to the sealed container, the culture medium (static water) can be pressurized, thereby applying mechanical stress to the biological sample. This allows the biological sample to be cultured under conditions closer to the in vivo environment, making it possible to study cellular responses to mechanical stress.

[0024] The microscope observation stage 100 may further include a temperature and humidity control mechanism inside or outside the sealed container. Examples of the temperature and humidity control mechanism include a heater and a humidifier. For example, the heater is preferably provided below the sealed container. This allows the culture medium or the like inside the sealed container or the biological sample container to be heated and maintained at a temperature range suitable for cell culture (e.g., about 25 to 45°C).

[0025] It is preferable that at least one recess be provided in the sealed container in a region including the intersection of a diagonal line extending from the connecting port 20 and the inner wall of the sealed container. FIG. 3 is a line cross-sectional view of the sealed container 10 taken parallel to a horizontal plane at the midpoint of its height on the microscope observation stage 100. FIG. 3( a) is a perspective view of the cross-section, and FIG. 3( b) is a top view. In FIG. 3, the diagonal line extending from the connecting port 20 is indicated by a dotted line. This diagonal line is an extension of the axis of the discharge pipe 25 connected to the connecting port 20. In this embodiment, it is preferable that at least one recess be provided in a region including the intersection of the diagonal line extending from the connecting port 20 and the inner wall of the sealed container, and more preferable that two recesses be provided in a region including the intersection of the diagonal line extending from the connecting port and the inner wall of the sealed container. When only one recess is provided, it is preferable that the recess be provided at a position facing the connecting port 20 (on the opposite circumference). Furthermore, when two recesses 50 are provided, it is preferable that the two recesses 50 face each other. That is, it is preferable that one of the two recesses 50 is provided at a position facing the connecting port 20, and the other is provided at a position on the side where the connecting port 20 is present. It is preferable that the recesses 50 are provided so as to protrude outward from the inner wall of the sealed container body 10.

[0026] The shape of the recess 50 is preferably semi-elliptical. The volume of the recess is preferably 10% or less, more preferably 5% or less, of the volume surrounded by the inner wall of the sealed container (main body volume). When two recesses are provided, the total volume of the two recesses is preferably within the above range.

[0027] The recess 50 in the sealed container facilitates mitigating the impact pressure within the sealed container, enabling early pressure stabilization. For example, when a pressure of 30 kPa is applied to a sealed container without the recess 50, a pressure several kPa to several tens of Pa greater than 30 kPa tends to be applied (overshoot). In particular, when the volume of the sealed container is small, the gas flow rate supplied relative to the volume tends to be large, making the container more susceptible to overshoot. If an overshoot occurs, the pressure stabilizes through repeated expansion and recompression as a reaction, and it takes time for the pressure to stabilize. On the other hand, in the case of a sealed container with the recess 50, a pressure of approximately 30 kPa tends to be applied, reducing the discrepancy between the set pressure and the pressure within the sealed container. In other words, the presence of the recess 50 in the sealed container makes it possible to apply a pressure closer to the set pressure, facilitating pressure control. Furthermore, in the case of a sealed container with the recess 50, overshoot is suppressed, thereby shortening the time until the pressure stabilizes.

[0028] As shown in Figure 3, a flow path 55 is preferably provided between the connecting port 20 and the recess provided on the connecting port 20 side. In this case, the cross-sectional diameter (orifice diameter) of the flow path 55 is preferably 6 mm or less, and particularly preferably 1 mm or less. The cross-sectional diameter (orifice diameter) of the flow path 55 is preferably 0.1 mm or more, and more preferably 0.3 mm or more. In this way, by providing the flow path 55 between the connecting port 20 and the recess provided on the connecting port 20 side and setting the cross-sectional diameter (orifice diameter) of the flow path 55 within the above range, the time required for pressure stabilization in the sealed container can be more effectively shortened.

[0029] The recesses provided in the inner wall of the sealed container preferably have through holes, one of which is provided with a safety valve. In particular, it is preferable that the through hole be provided in the recess located opposite the connecting port 20. As shown in Figures 1 and 3, the sealed container body 10 has a recess 50 that protrudes outward from the inner peripheral edge of the sealed container body 10, and a through hole is formed at the tip of the recess 50, and this through hole is preferably provided with a safety valve 60. By opening the safety valve 60, the sealed container can be rapidly evacuated when the pressure inside the sealed container reaches an abnormal value, thereby reducing the pressure inside the sealed container.

[0030] The present invention also relates to a microscope apparatus including the above-described microscope observation stage and a pressure application unit connected to the sealed container. In the microscope apparatus of the present invention, the pressure application unit applies a physical stimulus (pressure stimulus) to the biological sample by varying the pressure in the sealed container.

[0031] In the microscope apparatus of this embodiment, it is preferable that the pressure application unit receives a pressure sensor signal output from a pressure sensor provided on the microscope observation stage and adjusts the pressure inside the sealed container. The pressure sensor provided on the microscope observation stage measures the pressure inside the sealed container in real time. The pressure application unit receives the signal detected by the pressure sensor, and controls the pressurization and / or depressurization operation.

[0032] The pressure application unit preferably has a first pressure adjustment mechanism that adjusts the pressure inside the sealed container and a second pressure adjustment mechanism that adjusts the pressure inside the sealed container. In this case, a pressure sensor signal detected by the pressure sensor is output to the pressure application unit, and the operation of the first pressure adjustment mechanism and the second pressure adjustment mechanism are respectively controlled. For example, the operation of the first pressure adjustment mechanism and the second pressure adjustment mechanism is respectively controlled so that, when pressurization by the first pressure adjustment mechanism is performed and the pressure sensor detects that a desired maximum pressure has been reached, depressurization by the second pressure adjustment mechanism is performed instead of pressurization by the first pressure adjustment mechanism. Next, depressurization by the second pressure adjustment mechanism is performed and, when the pressure sensor detects that a desired minimum pressure has been reached, pressurization by the first pressure adjustment mechanism is performed instead of depressurization by the second pressure adjustment mechanism is performed. Note that it is also possible to maintain the state in which the maximum pressure or the state in which the minimum pressure has been reached for a predetermined period of time.

[0033] In this embodiment, the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in series or in parallel. It is particularly preferable that the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel. FIG. 4 is a schematic diagram showing the mechanism of a microscope apparatus 200 in this embodiment when the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel. As shown in FIG. 4 , the microscope apparatus 200 of this embodiment includes a microscope observation stage 100, a first pressure adjustment mechanism 110, and a second pressure adjustment mechanism 120. When the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel, the microscope apparatus 200 has a first flow path 111 and a second flow path 121, with the first pressure adjustment mechanism 110 provided in the first flow path 111 and the second pressure adjustment mechanism 120 provided in the second flow path 121. In the microscope apparatus 200, the first pressure adjustment mechanism 110 and the second pressure adjustment mechanism 120 are alternately operated to generate a periodically fluctuating pressure (e.g., a pressure similar to in vivo pulsatile pressure) within the sealed container. The microscope apparatus 200 of this embodiment is equipped with multiple pressure adjustment mechanisms, such as the first pressure adjustment mechanism 110 and the second pressure adjustment mechanism 120, and by providing these pressure adjustment mechanisms on separate flow paths, it is possible to generate a periodically fluctuating pressure within the sealed container. When a liquid such as a culture medium is contained within the sealed container, it is possible to apply in vivo pressure to the liquid (hydrostatic pressure), and it is possible to observe biological samples such as cells within the sealed container in which in vivo pressure is reproduced.

[0034] In this specification, "pressure adjustment" refers to pressurizing and / or depressurizing the inside of the sealed container of the microscope observation stage 100. Note that, when the pressure inside the sealed container is at a desired pressure value, maintaining the pressure inside the sealed container without pressurizing or depressurizing is also included in "pressure adjustment." However, it is preferable that the first pressure adjustment mechanism 110 is a mechanism that pressurizes and / or depressurizes the inside of the sealed container, and it is preferable that the second pressure adjustment mechanism 120 is a mechanism that pressurizes and / or depressurizes the inside of the sealed container. In a particularly preferred embodiment of the present invention, it is preferable that the first pressure adjustment mechanism 110 is a mechanism that pressurizes the inside of the sealed container (high-pressure application mechanism), and it is preferable that the second pressure adjustment mechanism 120 is a mechanism that depressurizes the inside of the sealed container (low-pressure application mechanism).

[0035] The first pressure adjustment mechanism 110 is preferably a mechanism that adjusts (pressurizes) the pressure inside the sealed container of the microscope observation stage 100 so that a pressure (gauge pressure) of less than 200 kPa is applied inside the sealed container. The pressure applied by the first pressure adjustment mechanism 110 is more preferably 180 kPa or less, even more preferably 160 kPa or less, still more preferably 140 kPa or less, still more preferably 120 kPa or less, and particularly preferably 100 kPa or less. The pressure applied by the first pressure adjustment mechanism 110 is preferably 1 kPa or more, more preferably 5 kPa or more, and even more preferably 10 kPa or more.

[0036] Second pressure adjustment mechanism 120 is preferably a mechanism that adjusts (depressurizes) the pressure inside the sealed container so that the pressure is reduced by 1 kPa or more from the pressure applied by first pressure adjustment mechanism 110, and more preferably a mechanism that adjusts (depressurizes) the pressure inside the sealed container so that the pressure is reduced by 1 kPa or more and 50 kPa or less. Thus, the fluctuation range of the pressure applied inside the sealed container (maximum pressure - minimum pressure) is preferably 1 to 50 kPa.

[0037] If the maximum pressure (absolute pressure) inside the sealed container when first pressure adjustment mechanism 110 adjusts the pressure inside the sealed container is P and the minimum pressure (absolute pressure) inside the sealed container when second pressure adjustment mechanism 120 adjusts the pressure inside the sealed container is Q, the value of P / Q is preferably 1.000 or more, more preferably 1.001 or more, and even more preferably 1.010 or more. Furthermore, the value of P / Q is preferably 3 or less, more preferably 1.5 or less, even more preferably 1.35 or less, even more preferably 1.2 or less, particularly preferably 1.15 or less, and most preferably 1.1 or less.

[0038] The microscope device 200 is connected to a supply source 150. The supply source 150 is an airflow supply source and is a component that supplies airflow to the first pressure adjustment mechanism 110 and / or the second pressure adjustment mechanism 120. Preferably, the supply source 150 is a compressed gas supply source, and it is particularly preferable that the supply source 150 supplies compressed gas to the first pressure adjustment mechanism 110. Examples of the supply source 150 include a mechanism for supplying compressed gas, such as an air compressor or a gas cylinder. Compressed gas can be compressed from ordinary air, but any gas can also be used, such as carbon dioxide, inert gas, or a mixture of these. In this embodiment, compressed gas is introduced when applying pressure, thereby suppressing changes in pH, carbon dioxide concentration, nitrogen gas concentration, and the like within the culture medium containing the biological sample. The discharge pressure of the supply source 50 is preferably less than 1000 kPa (gauge pressure), at which pneumatic equipment operates. The discharge pressure of the supply source 50 must be greater than the maximum pressure applied within the sealed container.

[0039] In this embodiment, when the first pressure adjustment mechanism 110 and the second pressure adjustment mechanism 120 are connected in parallel, the first flow path 111 has the first pressure adjustment mechanism 110, the sealed container 10 is connected downstream of the first pressure adjustment mechanism 110, and the supply source 150 is connected upstream of the first pressure adjustment mechanism 110. In this way, the first flow path 111 is a flow path connecting the upstream supply source 150 to the downstream sealed container 10. Furthermore, the second flow path 121 has the second pressure adjustment mechanism 120, the sealed container 10 is connected downstream of the second pressure adjustment mechanism 120, and the supply source 150 or an exhaust mechanism (not shown) is connected upstream of the second pressure adjustment mechanism 120. In this way, the second flow path 121 is a flow path that has the second pressure adjustment mechanism 120 and connects to the downstream sealed container. The supply source 150 may be connected upstream of the second pressure adjustment mechanism 120, or an exhaust mechanism capable of exhausting may be provided.

[0040] The first flow path 111 is a flow path that mainly introduces compressed gas supplied from the supply source 150 into the sealed container, and the second flow path 121 is a flow path that mainly recovers gas from the sealed container. When the supply source 150 is connected to the second flow path 121 upstream of the second pressure adjustment mechanism 120, the recovered gas is preferably exhausted via the second pressure adjustment mechanism 120. Note that, when the increased pressure value in the sealed container is higher than the desired pressure value, the first flow path 111 can also serve as a flow path that recovers gas from the sealed container. In this case, the recovered gas is exhausted via the first pressure adjustment mechanism 110. On the other hand, when the decreased pressure value in the sealed container is lower than the desired pressure value, the second flow path 121 can also serve as a flow path that introduces compressed gas supplied from the supply source 150 into the sealed container.

[0041] The sealed container may be provided with a safety valve as described above. The pressure in the sealed container may be reduced by rapidly venting the air through the safety valve provided in the sealed container. Furthermore, both venting via the safety valve provided in the sealed container and venting via the second pressure adjustment mechanism 120 may be performed simultaneously.

[0042] The microscope device 200 in this embodiment may have a first common flow path 130. In this case, as shown in FIG. 4 , the first flow path 111 and the second flow path 121 join together upstream to form the first common flow path 130. The first common flow path 130 is preferably connected to a supply source 150. The microscope device 200 may also have a second common flow path 140. In this case, as shown in FIG. 2 , the first flow path 111 and the second flow path 121 join together downstream to form the second common flow path 140. The second common flow path 140 is preferably connected to a sealed container.

[0043] It is preferable that the first common flow path 130 and the second common flow path 140 each have a control valve at a point where they branch into the first flow path 111 and the second flow path 121. This control valve can, for example, block the airflow to one of the flow paths, thereby making it possible to supply compressed gas to only one of the flow paths (mainly the first flow path) or to discharge recovered gas to only one of the flow paths (mainly the second flow path).

[0044] The microscope device 200 may not have the second common flow path 140, and the first flow path 111 and the second flow path 121 may each be connected to a sealed container. The microscope device 200 may not have the first common flow path 130, and the first flow path 111 and the second flow path 121 may each be connected to a separate supply source, or one may be connected to a supply source and the other not connected to a supply source, and an exhaust mechanism may be provided to enable exhaust.

[0045] 5 is a schematic diagram illustrating another aspect of the microscope device 200 of this embodiment. As shown in FIG. 5 , in this embodiment, the first pressure adjustment mechanism 110 may include a first regulator 112 and a first control valve 114, and the second pressure adjustment mechanism 120 may include a second regulator 122 and a second control valve 124.

[0046] The control valve used in this embodiment is not particularly limited as long as it has the function of passing or blocking gas through the flow path. Examples of the control valve include a solenoid valve, a proportional control valve, an air-operated valve, a manual valve, and a mechanically operated valve. Among these, the control valve is preferably a solenoid valve, and the use of a solenoid valve makes it easier to achieve more regular cyclic pressurization.

[0047] In this embodiment, the first control valve 114 and the second control valve 124 are preferably two-port valves. In this embodiment, the first regulator 112 and the second regulator 122 are connected in parallel, and the first regulator 112 is disposed upstream of the first control valve 114 in the first flow path. In addition, the second regulator 122 is disposed upstream of the second control valve 124 in the second flow path.

[0048] The first regulator 112 and the second regulator 122 may each include a valve therein. The pressure at which the valve opens (operates) (the pressure at which compressed gas on the primary side of the regulator begins to be supplied to the secondary side) is adjusted as desired to determine the set pressure of the compressed gas introduced from the upstream (primary side) of the regulator to the downstream (secondary side). If the secondary side pressure of the regulator is lower than the set pressure, the regulator increases the secondary side pressure until it reaches the set pressure. If the secondary side pressure of the regulator is higher than the set pressure, the regulator releases excess pressure to the atmosphere via the regulator's relief mechanism, thereby reducing the secondary side pressure. In this way, the regulator increases or decreases the secondary side pressure based on the set pressure. The pressure at which the regulator's valve opens, i.e., the set pressure, may be adjusted manually or automatically.

[0049] The first control valve 114 and the second control valve 124 are two-port valves (on-off valves) that open and close the respective flow paths. The first control valve is provided downstream of the first regulator 112, and the second control valve 124 is provided downstream of the second regulator 122. The first control valve 114 and the second control valve 124 are downstream switching control valves that switch between the operation of the first regulator 112 and the operation of the second regulator 122. In other words, the first control valve 114 and the second control valve 124 serve to switch between the connection between the sealed container and the first regulator 112 and the connection between the sealed container and the second regulator 122.

[0050] For example, in this embodiment, when the pressure application unit of the microscope device 200 is operating, the first regulator 112 and the second regulator 122 are in an operating state and can apply and / or reduce pressure to a predetermined pressure. When applying pressure to the sealed container, the first control valve 114 opens for a predetermined time. Here, the predetermined time refers to the pressure increase time shown in FIG. 6 , e.g., 0.5 seconds. The set pressure of the first regulator 112 is manually or automatically adjusted so that the pressure inside the sealed container reaches a predetermined pressure value (maximum pressure value) within this predetermined time. When the pressure increase time is set to a short time, such as 0.5 seconds, if the set pressure value and the maximum pressure value are the same (set pressure value = maximum pressure value), the maximum pressure value may not be reached within the predetermined time. Therefore, it is preferable to set the set pressure value of the first regulator 112 higher than the maximum pressure value of the sealed container (maximum pressure value < set pressure value). Note that if the "maximum pressure value < set pressure value" is set, the maximum pressure value inside the sealed container may be reached earlier than the predetermined time. In this case, the amount of compressed gas supplied to the sealed container in a predetermined time may be reduced by lowering the set pressure of the first regulator 112. In this way, it is preferable that the first regulator 112 is adjusted so that the pressure inside the sealed container reaches the maximum value in a predetermined time.

[0051] When the first control valve 114 is opened and the first regulator 112 is operated, compressed gas is discharged from the secondary side of the first regulator 112, and the inside of the sealed container is pressurized. If the predetermined pressure inside the sealed container becomes higher than the set pressure, the excess pressure may be released from the relief mechanism of the first regulator 112.

[0052] Next, simultaneously with the closing of the first control valve 114, the second control valve 124 opens for a predetermined time, and the second regulator 122 operates, thereby reaching a predetermined pressure value (minimum pressure value). The predetermined time is the pressure drop time shown in FIG. 6 , e.g., 0.5 seconds. The set pressure of the second regulator 122 is adjusted manually or automatically so that the pressure inside the sealed container reaches the minimum pressure value within this predetermined time. When the pressure drop time is set to a short time, such as 0.5 seconds, if the set pressure value and the minimum pressure value are the same (set pressure value = minimum pressure value), the minimum pressure value may not be reached within the predetermined time. Therefore, the set pressure value of the second regulator 122 is set lower than the minimum pressure value of the sealed container (minimum pressure value > set pressure value). Furthermore, if the "minimum pressure value > set pressure value" is set, the minimum pressure value inside the sealed container may be reached earlier than the predetermined time. In this case, the set pressure of the second regulator 122 is increased to reduce the amount of compressed gas discharged from the sealed container within the predetermined time. In this way, the second regulator 122 is adjusted so that the pressure inside the sealed container reaches the minimum pressure value within a predetermined time.

[0053] When depressurizing the sealed container, the second regulator 122 typically recovers gas from the sealed container and releases it through its relief mechanism. When the second control valve 124 opens, the internal volume expands from the sealed container to the second regulator 122, causing the gas stored in the sealed container to expand. This causes the pressure inside the sealed container to drop rapidly (t in FIG. 6 ). Immediately thereafter, the compressed gas is gradually released through the relief mechanism of the second regulator 122 (m in FIG. 6 ). The waveform during pressure drop may be two-stage, similar to pulsatile pressure, as shown in FIG. 6 . To ensure sufficient internal volume, a tank may be appropriately installed between the second control valve 124 and the second regulator 122, or the flow path between the second control valve 124 and the second regulator 122 may be lengthened. The gas released through the relief mechanism of the second regulator 122 may be released into the pressure application unit; however, because the gas contains components different from those of the atmosphere, it may affect the human body. In this case, an exhaust port may be provided in the second flow path of the pressure application unit, and piping may be provided between the second regulator 22 and the exhaust port, so that the gas can be exhausted to the outside of the pressure application unit.

[0054] As described above, if one cycle is defined as the process of adjusting pressure by first pressure adjustment mechanism 110 (first regulator 112 and first control valve 114) followed by pressure adjustment by second pressure adjustment mechanism 120 (second regulator 122 and second control valve 124), first pressure adjustment mechanism 110 and second pressure adjustment mechanism 120 operate so as to be repeated 10 to 300 cycles per minute. That is, if one cycle of the pressure application method is defined as the process of performing step (a) followed by step (b), 10 to 300 cycles are repeated per minute. The number of cycles is preferably 20 or more per minute, more preferably 25 or more per minute, and even more preferably 30 or more per minute. Furthermore, the number of cycles is preferably 250 or less per minute, more preferably 200 or less per minute, even more preferably 180 or less per minute, and particularly preferably 150 or less per minute. In this manner, in this embodiment, periodic pressure fluctuations can be generated within the sealed container. Furthermore, in this embodiment, a minute pressure difference of approximately several tens of kPa can be generated in the sealed space at a period equivalent to the pulsation, simulating the pulsation in a living body, and therefore a periodically fluctuating pressure can be applied to the sealed space.

[0055] The microscope device 200 may further include a tank 116 downstream of the first regulator 112. The tank 116 is preferably provided between the first regulator 112 and the first control valve 114. Depending on the insufficient supply pressure from the supply source 150 or the volume of the sealed container, the first regulator 112 may have difficulty increasing the pressure inside the sealed container to a predetermined level within a predetermined time. In such cases, the tank 116 can be appropriately installed to compensate for the insufficient supply of compressed gas.

[0056] The microscope device 200 preferably further includes a controller 160. The controller 160 can set the pressure application period, the maximum pressure value, the minimum pressure value, and the time and pressure value for two-stage pressure reduction. Time-dependent pressure value information detected by the pressure sensor 30 is provided to the controller 160, which feedback-controls the first pressure adjustment mechanism 110 (the first regulator 112 and the first control valve 114) and the second pressure adjustment mechanism 120 (the second regulator 122 and the second control valve 124) according to the pressure value detected by the pressure sensor 30 and the pressure and period set by the controller 160. The controller 160 performs feedback control by performing calculations in a calculation unit and outputting adjusted signals to each mechanism so that the pressures coincide with target values ​​(set values).

[0057] For example, when pressurization is performed by first pressure adjustment mechanism 110 (first regulator 112 and / or first control valve 114), if the output value from pressure sensor 30 does not reach a predetermined pressure value within the pressure increase time (pressure application period) set in controller 160, the output value from controller 160 to first pressure adjustment mechanism 110 is controlled, and first pressure adjustment mechanism 110 is adjusted so that it matches the predetermined pressure value. Alternatively, if the pressure increase time (pressure application period) when the output value from pressure sensor 30 reaches the maximum pressure value does not match the time set in controller 160, the output value from controller 160 to first pressure adjustment mechanism 110 is controlled, and first pressure adjustment mechanism 110 is adjusted so that it matches the set time.

[0058] When the pressure is reduced by the second pressure adjustment mechanism 120 (second regulator 122 and / or second control valve 124) and the output value from the pressure sensor 30 does not reach a predetermined pressure value within the pressure reduction time (pressure application period) set in the controller 160, the output value from the controller 160 to the second pressure adjustment mechanism 120 is controlled, and the second pressure adjustment mechanism 120 is adjusted so that it matches the predetermined pressure value. Alternatively, if the pressure reduction time (pressure application period) when the output value from the pressure sensor 30 reaches the minimum pressure value does not match the time set in the controller 160, the output value to the controller 160 is controlled, and the second pressure adjustment mechanism 120 is adjusted so that it matches the set time. For example, the first control valve 114 and the second control valve 124 are controlled by output from the controller to each control valve according to the pressure application period set in the controller 160.

[0059] The microscope device 200 preferably further includes an input / output terminal 40. Input / output signals to each mechanism within the microscope device 200 are indicated by dotted arrows in FIG. 5 . In addition to the pressure sensor 30 described above, the observation stage 100 can also include a temperature sensor, a humidity sensor, a gas sensor, and the like. The observation stage 100 can also include devices for maintaining the culture environment, such as a heater and a humidifier. Wiring for outputting signals acquired in real time by various sensors and wiring for inputting operation commands for the heater, humidifier, and the like are preferably integrated into the input / output terminal 40. Output signals are output primarily as analog signals to the controller 160 or an external display. When output signals from the observation stage, such as pressure, cycle, temperature, humidity, and gas composition, are input to the controller 160, the controller 160 outputs signals adjusted so that these values ​​reach pre-input target values, and the observation stage receives (input of) operation commands for the heater, humidifier, and the like. The controller 160 can also store data on these input / output signals over time.

[0060] The microscope device 200 is preferably an inverted microscope device equipped with a lens and an imaging device and capable of time-lapse observation.

[0061] (Applications) The microscope observation stage and microscope device of the present invention can reproduce in vivo pressure (e.g., pressure corresponding to in vivo pulsation) on the microscope observation stage, making it possible to observe the state of cells and tissues under conditions close to the conditions of the in vivo environment. This makes it possible to observe cells exposed to mechanical stress in real time, for example, and is expected to be applied to research on cellular responses to mechanical stress.

[0062] A typical blood pressure waveform is observed in which the pressure rises from low to high and then drops in two stages. The microscope apparatus of the present invention can also reproduce such a two-stage pressure waveform. Specifically, by using the microscope apparatus of the present invention, the pressure in a sealed container can be raised from low to high in one go and then lowered in two stages. For example, FIG. 6 is a graph showing an example of a pressure waveform generated when a pressure fluctuation is generated in a sealed container using a microscope apparatus having a pressure application unit of this embodiment. In the example shown in FIG. 6, the minimum pressure (low-pressure side pressure) in the pressure fluctuation is 17 kPa, the maximum pressure (high-pressure side pressure) is 27 kPa, the pressure rise time is 0.5 seconds, the pressure drop time is 0.5 seconds, and the time required for one cycle (one period) is 1 second. Furthermore, the pressure waveform shown in FIG. 6 rises from low to high and then drops in two stages. In this way, this embodiment can generate a pressure similar to in vivo pulsatile pressure in a sealed container of a microscope observation stage.

[0063] Furthermore, by using the microscope observation stage and microscope device of the present invention, it is possible to artificially create an environment of hypotensive or hypertensive state, and to observe in real time the state of cells or tissues placed in a hypotensive or hypertensive environment. By observing in real time the state of cells or tissues placed in a hypotensive or hypertensive environment, it is possible to conduct research on cellular responses brought about by hypotensive or hypertensive conditions, and to evaluate the efficacy of drugs such as antihypertensive drugs.

[0064] Furthermore, the microscope observation stage and microscope device of the present invention can be used to study tissues that are periodically subjected to pressure (such as masticatory muscles and joints). For example, applying high pressure to cells or tissues that are periodically subjected to pressure can be used to study the mechanisms of adaptation to abnormal environments.

[0065] Furthermore, by using the microscope observation stage and microscope device of the present invention, it is possible to study the responses of tissues and cells to pressure in tissues other than the circulatory system that are periodically subjected to pressure (such as masticatory muscles and joints), and it can also be used to study the mechanisms of adaptation to abnormal environments such as high pressure.

[0066] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0067] (Example 1) (Preparation of Biological Sample) Commercially available human aortic smooth muscle cells were suspended in Dulbecco's Modified Eagle Medium (hereinafter referred to as DMEM) containing 10% bovine serum, then seeded on a 35 mm diameter dish and allowed to stand at room temperature for 10 minutes.

[0068] (Microscopic Observation) A 35 mm diameter dish seeded with cells was placed in a sealed container of a microscope observation stage having the configuration shown in Figure 1, heated to 37°C, and set on the stage of an inverted microscope (IX83, Olympus). A heating device attachment unit was provided on the rear side wall of the microscope observation stage shown in Figure 1, allowing for temperature control of the observation stage. Simultaneously with time-lapse imaging (1 image / 5 seconds) of phase-contrast images of the cells using image acquisition software (cellSens, Olympus), signals from a pressure sensor attached to the microscope observation stage were captured (0.5 Hz) by an external device (PowerLab, BRC).

[0069] The set pressures of the first and second pressure adjustment mechanisms in the pressure application unit of the microscope apparatus of this embodiment ( FIG. 4 ) were respectively set so that the pressure inside the sealed container was between 17 kPa and 100 kPa, and cyclic pressure was applied at 0.002 Hz for 1 hour and 15 minutes, with one cycle being 17 kPa / 100 kPa. FIG. 7 shows a graph of pressure fluctuations over time and observation images of cells at each time point.

[0070] (Example 2) (Preparation of Biological Sample) Commercially available human aortic smooth muscle cells were suspended in a DMEM medium containing 10% bovine serum, and then 3 × 10 cells were placed in a 35 mm diameter dish. ４ The cells were seeded at 100 cells / dish and cultured for 24 hours (37°C, 5% CO ２ Then, calcein-AM (5 μM), which stains the cytoplasm uniformly, was added to the cells (10 min, 37°C), and the cells were incubated in an extracellular solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl ２ , 2 mM MgCl ２ , 26mM NaHCO ３ , 1.25 NaH ２ P.O. ４ , 11 mM glucose) and washed.

[0071] (Microscopic Observation) A 35 mm diameter dish seeded with cells was placed in a sealed container on a microscope observation stage with the configuration shown in Figure 1, which was heated to 37°C, and set on the stage of an inverted microscope (IX83, Olympus). Time-lapse imaging (exposure 400 ms, 1 image / 5 sec) of fluorescent images of the cells (excitation wavelength 470-495 nm, fluorescence wavelength 510-550 nm) was performed using image acquisition software (cellSens, Olympus). Simultaneously, signals from a pressure sensor installed on the microscope observation stage were captured (0.5 Hz) by an external device (PowerLab, BRC).

[0072] The pressure settings of the first and second pressure adjustment mechanisms in the pressure application unit of the microscope apparatus of this embodiment (FIG. 4) were adjusted so that the pressure inside the sealed container varied from 14 kPa to 73 kPa. Cyclic pressure application was performed at 0.002 Hz for 33 minutes, with one cycle consisting of 14 kPa / 73 kPa. Calcein-AM generates calcein through the action of esterase within the cells, emitting green fluorescence (excitation 490 nm, emission 510 nm). FIG. 8 shows an observation image of a cell emitting green fluorescence.

[0073] As described above, using the microscope observation stage and microscope device of this embodiment, cells could be observed in real time while periodically pressurizing the inside of a sealed container containing cells. In this embodiment, pressure fluctuations inside the sealed container and cell behavior could be recorded simultaneously. Furthermore, although an inverted microscope was used in this example, other microscopes capable of optically observing biological samples may also be used.

[0074] REFERENCE SIGNS LIST 10 Sealed container (sealed container body) 11 Transparent member (ceiling surface) 12 Transparent member (bottom surface) 13 Top lid 14 Inner lid 15 O-ring 20 Pressure application unit connection port 25 Conduit 30 Pressure sensor 40 Input / output terminal 50 Recess 55 Flow path 60 Safety valve 100 Microscope observation stage 110 First pressure adjustment mechanism 111 First flow path 112 First regulator 114 First control valve 116 Tank 120 Second pressure adjustment mechanism 121 Second flow path 122 Second regulator 124 Second control valve 130 First common flow path 140 Second common flow path 150 Supply source 160 Controller 200 Microscope device

Claims

1. A microscope observation stage comprising: a sealed container capable of accommodating an observation object and having a connection port for connecting to a pressure application unit; and a pressure sensor capable of measuring the pressure inside the sealed container in real time, wherein the observation object is a biological sample; the ceiling and bottom surfaces of the sealed container are made of optically transparent and smooth materials; and the pressure sensor outputs a pressure sensor signal to the outside.

2. A microscope observation stage according to claim 1, wherein at least one recess is provided in the sealed container in an area including the intersection of a diagonal line extending from the connecting port and the inner wall of the sealed container.

3. A microscope observation stage as described in claim 2, wherein two recesses are provided within the sealed container in an area including the intersection of a diagonal line extending from the connecting port and the inner wall of the sealed container, and the two recesses are opposite each other.

4. A microscope observation stage according to claim 1, wherein the connecting port is a conduit connecting portion, and the conduit is a conduit for supplying gas from the pressure application portion and recovering the gas.

5. The microscope observation stage according to claim 1, further comprising a temperature adjustment mechanism.

6. The microscope observation stage of claim 1, wherein the biological sample is a cell.

7. The microscope observation stage according to claim 2, wherein one of the recesses further comprises a through-hole, and the through-hole is provided with a safety valve.

8. A microscope apparatus comprising a microscope observation stage according to any one of claims 1 to 7 and a pressure application unit connected to the sealed container, wherein the pressure application unit applies a physical stimulus to the biological sample by varying the pressure in the sealed container.

9. The microscope apparatus according to claim 8, wherein the pressure applying unit receives the pressure sensor signal from the microscope observation stage and adjusts the pressure inside the sealed container.

10. The microscope device according to claim 8, wherein the pressure application unit has a first pressure adjustment mechanism and a second pressure adjustment mechanism for adjusting the pressure inside the sealed container, and generates a pressure inside the sealed container similar to a pulsatile pressure in a living body by alternately operating the first pressure adjustment mechanism and the second pressure adjustment mechanism.

Images