JP6252096B2 - Observation method and microscope - Google Patents

Description

  The present invention relates to an observation method and a microscope.

An object to be observed such as a cell or tissue in culture may be observed with a microscope (see, for example, Patent Document 1).
[Patent Document 1] JP2013-138690A

  When observing at a high magnification, the objective lens is immersed in a culture solution in which the object to be observed is immersed. For this reason, there is a possibility that the observation sample is contaminated through the objective lens.

  In the first aspect of the present invention, there is provided an observation method for observing an object immersed in a culture solution with a microscope as an observation object, the method comprising an sterilization step of sterilizing an objective lens that has contacted the culture solution Is done.

  In the second aspect of the present invention, a microscope for observing an object immersed in a culture solution as an observation target by immersing the objective lens in the culture solution and sterilizing the objective lens in contact with the culture solution A microscope is provided.

  The above summary of the present invention does not enumerate all necessary features of the present invention. A sub-combination of these feature groups can also be an invention.

1 is a schematic diagram of a laser microscope 100. FIG. 1 is a schematic diagram illustrating the principle of a laser microscope 100. FIG. 3 is a schematic cross-sectional view of a culture vessel 200. FIG. It is a flowchart which shows an observation procedure. It is typical sectional drawing which shows a part of observation procedure. It is typical sectional drawing which shows a part of observation procedure. 3 is a schematic cross-sectional view of a revolver 170. FIG. It is typical sectional drawing which shows a part of observation procedure. It is typical sectional drawing which shows a part of observation procedure. It is typical sectional drawing which shows a part of observation procedure.

  Hereinafter, the present invention will be described through embodiments of the invention, but the following embodiments do not limit the invention according to the claims. Not all combinations of features described in the embodiments are essential for the solution of the invention.

  FIG. 1 is a schematic diagram showing the structure of a laser microscope 100 that can be used for cell observation. The laser microscope 100 includes a stage 110, a laser device 120, a scanning system 130, an optical system 140, a detection system 150, and a control system 160.

  The laser microscope 100 can observe the cultured cells, tissues, microorganisms, and the like by utilizing nonlinear optical effects such as CARS (coherent anti-Stokes Raman scattering) and SRS (stimulated Raman scattering). The laser microscope 100 can also be used as a phase contrast microscope.

  In the laser microscope 100, the stage 110 supports a sample 112 that is observed by the laser microscope 100. The sample 112 supported by the stage 110 is irradiated with laser light from one surface (the lower surface in the drawing in the drawing). Further, an observation image is formed by detecting CARS light emitted from the opposite side of the stage 110 to the side irradiated with the laser light.

The laser device 120 includes a plurality of laser light sources 122 and 124 and a combiner 126. The laser light sources 122 and 124 generate a pulse laser used for observation by CARS light detection. The pulse lasers generated by the laser light sources 122 and 124 have different wavelengths λ _P and λ _S.

As the laser light sources 122 and 124, for example, a mode-locked picosecond Nd: YVO ₄ laser, a mode-locked picosecond yttrium laser, or the like can be used. Note that one of the laser light sources 122 and 124 may be replaced with an optical parametric oscillator that converts the wavelength of the pulse laser generated by the other laser light sources 122 and 124.

Of the two-wavelength picosecond pulses generated by the laser light sources 122 and 124, the pulse laser having the shorter wavelength λ _P is used as pump light in CARS light observation. Of the picosecond pulses generated by the laser light sources 122 and 124, the pulse laser having the longer wavelength λ _S is used as Stokes light in CARS light observation.

  The combiner 126 integrates the laser beams generated by the plurality of laser light sources 122 and 124 and emits them as irradiation light on a single optical path. Thereby, the irradiation light which combined pump light and Stokes light can be irradiated to the sample 112, and CARS light can be generated.

  The laser device 120 may be provided with a delay optical path for the purpose of synchronizing the pump light generated by the laser light sources 122 and 124 and the Stokes light. The delay optical path can be formed by a plurality of reflecting mirrors whose intervals can be changed. In the laser device 120, the Stokes light may be broadened using a photonic crystal fiber.

  In the laser microscope 100, the scanning system 130 includes a galvano scanner 132 and a scan lens 134. The galvano scanner 132 includes a reflecting mirror that swings around two axes whose directions are different from each other, and two-dimensionally displaces the optical path of incident irradiation light in a direction intersecting the optical axis. The scan lens 134 focuses the irradiation light emitted from the galvano scanner 132 on a predetermined primary image plane 136. Thereby, the observation region of the sample 112 is scanned with the irradiation light emitted from the laser device 120, and the irradiation region can be irradiated with the irradiation region having a predetermined area.

  The optical system 140 includes an objective lens 142 disposed on the detection system 150 side with respect to the stage 110, and a lower objective lens 144 and a condenser lens 146 disposed on the laser device 120 side with respect to the stage 110. The lower objective lens 144 focuses the irradiation light irradiated toward the sample 112 within the observation region of the sample 112. The pair of objective lenses 142 and 144 have the same numerical aperture (NA).

  On the incident end side of the optical system 140, a reflecting mirror 148 is disposed on the optical path of the irradiation light between the laser device 120 and the objective lens 142. The reflecting mirror 148 bends the optical path of the irradiation light to prevent the structure of the laser microscope 100 from becoming excessively high. The reflecting mirror 148 may be a metal mirror that reflects incident light on the surface instead of a total reflection prism. A detection system 150 is disposed in the propagation optical path of the light emitted from the optical system 140.

  The detection system 150 includes a condenser lens 152, a relay lens 154, a photomultiplier tube 156, and a dichroic mirror 158, and is used to detect CARS light emitted from the sample 112. The dichroic mirror 158 branches the CARS light from the light emitted from the sample 112 and introduces it to the photomultiplier tube 156 through the condenser lens 152 and the relay lens 154. Therefore, the CARS light is efficiently received by the photomultiplier tube 156 without reducing the amount of light.

  The control system 160 includes a control unit 162, a keyboard 164, a mouse 166, and a display unit 168. The control unit 162 can be formed by mounting a program that causes a general-purpose personal computer to execute a control procedure described later.

  The keyboard 164 and the mouse 166 are connected to the control unit 162 and operated when a user instruction is input to the control unit 162. The display unit 168 returns feedback to the user's operation using the keyboard 164 and the mouse 166, and displays the image or character string generated by the control unit 162 toward the user.

  The control unit 162 is connected to each of the laser device 120, the detection system 150, and the scanning system 130, and controls each operation. Further, an image to be displayed on the display unit 168 is generated based on the detection result of the detection system 150. Further, the control unit 162 determines the state of the sample 112 based on the detection result of the detection system 150.

  Furthermore, the control system 160 controls the operation of the entire laser microscope 100 in accordance with instructions received from the user. Further, the control system 160 generates an observation image based on the detection result of the detection system 150. Further, the control system 160 may also output a character string or a code indicating a determination result reflecting the state of the sample 112.

FIG. 2 is a schematic diagram for explaining the CARS process in which the object to be observed generates CARS light in the laser microscope 100. CARS process, different optical frequencies omega _{1 together,} the excitation light including the two laser beams of the pump light and stokes light having a omega ₂ by irradiating the object under observation, light of the pump light of the optical frequency omega ₁ and the Stokes beam This occurs when the difference [ω ₁ −ω ₂ ] from the frequency ω ₂ matches the angular frequency ω ₀ of the natural vibration of the molecule contained in the observed object.

When the vibration mode of a specific molecular structure included in the object to be observed is excited by the CARS process, the molecular vibration interacts with the probe light, which is the third laser light having the optical frequency ω ₃ , so that the third order CARS light derived from nonlinear polarization is generated as Raman scattered light.

Furthermore, since the pump light can also be used as probe light, CARS light is generated under the condition [ω ₁ = ω ₃ ]. The CARS light generated in the object to be observed has an optical frequency satisfying [ω _CARS = ω ₁ −ω ₂ + ω ₃ ]. Therefore, by detecting the CARS light emitted from the object to be observed, it is possible to detect the presence of a specific molecular structure, such as a functional group, included in the object to be observed. Further, by repeatedly detecting the CARS light while changing the position where the irradiation object is irradiated with the irradiation light, the distribution of a specific molecular structure in the observation object can be imaged.

  Since CARS light has higher light intensity than spontaneous Raman scattered light or the like, it can be detected in a short time when a photoelectric conversion element is used. Therefore, the time required for detection is not only shortened, but observation at a video rate is also possible. Thereby, not only the distribution of the specific molecular structure but also the change of the distribution can be detected. In addition, by irradiating the object to be observed with an infrared light band that is less damaging to living cells, it is possible to observe while alive the living cells of the object to be observed.

  Further, the CARS light generated by the nonlinear effect is generated in a very narrow region where the irradiation light is narrowed down by the lower objective lens 144. For this reason, the area | region used as the object of CARS light detection becomes a narrow area | region regarding both the direction crossing the optical axis of irradiation light, and a direction parallel to an optical axis. Therefore, the observation of the object to be observed with the CARS light has a three-dimensionally high resolution.

  Therefore, the observation plane may be formed inside the object to be observed using the irradiation light in the infrared band or the near infrared band. Further, an image reflecting a three-dimensional distribution of a specific molecular structure can be generated by sequentially moving the observation plane in the depth direction of the object to be observed.

  Furthermore, a spectral image showing the frequency distribution of Raman scattered light emitted from the irradiation position is obtained by changing the optical frequency of the irradiation light irradiated to a specific position of the object to be observed. Further, by repeatedly irradiating the irradiation light while moving the observation object in a direction intersecting the optical path of the irradiation light, the distribution of specific molecules in the observation plane including the focal point of the upper objective lens 142 is imaged. A detection image can be generated.

  FIG. 3 is a schematic cross-sectional view of the sample 112 in the laser microscope 100. Here, the cell sheet 111 that becomes an object to be observed of the laser microscope 100 is placed on the stage 110 as the sample 112 while being accommodated in the culture vessel 200.

  Note that the cell sheet 111 means a sheet-like cell tissue prepared by culturing cells collected from a subject. The cultured cell sheet is used while being attached to the living tissue of the subject while the cells are alive. For this reason, even when inspecting the culture amount, cell viability, etc. in the course of culture, contamination of the cell sheet 111 by bacteria or the like is strictly prevented.

  In the laser microscope 100, the stage 110 is provided with an observation window 114 that exposes the bottom surface of the culture vessel 200 placed on the stage 110. Thereby, the culture vessel 200 placed on the stage 110 can be observed from both the bottom surface side and the top surface side. Moreover, it is possible to irradiate the culture vessel 200 placed on the stage 110 from the bottom surface side or the top surface side.

  The culture vessel 200 has a holding part 210 and a transmission part 220. The holding part 210 has a short cylindrical shape made of resin. The holding part 210 forms a cylindrical side wall and contacts the upper surface of the stage 110 at the lower end surface. An opening 212 is formed at the center of the lower surface of the holding unit 210.

  The permeation unit 220 is held by the holding unit 210, and the opening 212 of the holding unit 210 is liquid-tightly sealed to form the bottom surface of the culture vessel 200. Thereby, the culture container 200 can accommodate the culture solution 240 without leaking. In addition, the bottom surface of the culture vessel 200 formed by the transmission part 220 is optically highly transparent.

  The holding part 210 can be formed of a resin such as polycarbonate, polystyrene, polydimethylsiloxane, silicone polycarbonate, polyethylene terephthalate, or the like. The transmission part 220 can be formed of, for example, a transparent inorganic material such as a cover glass.

  In the culture container 200, the temperature-responsive polymer layer 230 is attached to the upper surface of the transmission part 220, that is, the bottom surface inside the culture container 200. The temperature-responsive polymer layer 230 can be formed of, for example, poly-N-isopropylacrylamide. Poly-N-isopropylacrylamide changes its hydration power at a temperature around 30 degrees. Therefore, the cell sheet 111 can be fixed or opened to the bottom surface of the culture vessel 200 without causing damage due to enzyme treatment or the like.

  The material of the temperature-responsive polymer layer 230 is not limited to poly-N-isopropylacrylamide, but N- (or N, N-di) alkyl-substituted (meta) such as (meth) acrylamide compounds and N-isopropylacrylamide. ) Any one of acrylamide derivatives and vinyl ether derivatives, or a combination of two or more thereof can be used. Further, copolymerization with monomers other than those described above, grafting or copolymerization of polymers, and a mixture of polymers and copolymers may be used. Furthermore, it can be used by crosslinking within a range not losing a given temperature responsiveness.

  In the culture container 200 as described above, the cell sheet 111 is immersed in the culture solution 240 and observed in a state of being fixed to the bottom surface by the temperature-responsive polymer layer 230. Since the transmission part 220 forming the bottom surface of the culture vessel 200 is transparent, the cell sheet 111 can be irradiated with excitation light while being accommodated and fixed in the culture vessel 200.

  FIG. 4 is a flowchart showing a procedure for observing the cell sheet 111 fixed to the culture vessel 200. When observing the cell sheet 111 with the laser microscope 100, first, the objective lens 142 of the laser microscope 100 to be used is sterilized (step S101).

Since living cells such as the cell sheet 111 itself deteriorate due to contamination and are finally brought into contact with the human body, contamination is severely warned. Therefore, the objective lens 142 that comes into contact with the culture solution 240 in which the cell sheet 111 is immersed is sterilized at a stage before the contact with the culture solution 240. Here, sterilization is performed, for example, to achieve a D value of 10 ⁻⁶ or more as a sterility assurance level.

  Sterilization of the objective lens 142 is performed by exposure to at least one of ozone gas, ethylene oxide gas, formaldehyde gas, hydrogen peroxide, sodium chlorite, hydrofluoric acid, ultraviolet rays, gamma rays, and electron beams. When the objective lens 142 is sterilized by exposure to gas, it is preferable to expose the objective lens 142 to gas in an airtightly closed environment to prevent the gas from leaking to the outside. In addition, after sterilization with a drug such as gas or liquid, it is preferable to execute a process for removing the component of the drug remaining on the objective lens 142.

  Other sterilization methods include sterilization using ultrasonic waves, pressurized steam, a surfactant, and an organic solvent.

  In the sterilization process, not all the microorganisms attached to the objective lens 142 are killed at the same time, and the survival number decreases logarithmically with the duration of the sterilization process. Therefore, it is preferable that the sterilization treatment conditions including the treatment time are determined in advance by microbial monitoring or the like executed in advance.

  In the case of sterilization treatment, it is preferable to remove a deposit on the objective lens 142 by performing a washing treatment with running water or the like prior to exposure to the gas, chemicals, radiation, or the like as described above. . Thereby, the objective lens 142 can be sterilized efficiently.

  Referring again to FIG. 4, in observing the cell sheet 111 with the laser microscope 100, the sample 112 is placed on the stage 110 with the sterilized objective lens 142 raised and sufficiently away from the upper surface of the stage 110. (Step S102), it is made to face the objective lens 142.

  FIG. 5 is a schematic cross-sectional view showing a state where the sample 112 is placed on the stage 110. A culture vessel 200 containing a cell sheet 111 as an object to be observed is placed on the stage 110 of the laser microscope 100. Here, the lower surface of the holding part 210 of the culture vessel 200 is in close contact with the upper surface of the stage 110, and the cell sheet 111 in the culture vessel 200 is fixed horizontally in parallel with the stage 110. The cell sheet 111 is immersed in the culture solution 240 inside the culture vessel 200.

  The water 143 filling the gap between the lower objective lens 144 and the transmission part 220 of the culture vessel 200 may be held in advance on the upper end of the objective lens 144 before placing the culture vessel 200 on the stage 110. Good. Alternatively, the water 143 may be poured into the gap between the objective lens 144 and the transmission part 220 after placing the culture vessel 200 on the stage 110.

  With respect to the sample 112 placed on the stage 110, the lower objective lens 144 of the laser microscope 100 approaches the lower surface of the transmission part 220 forming the bottom surface of the culture vessel 200 through the observation window 114. Thereby, the objective lens 144 can be made sufficiently close to the cell sheet 111 that is an object to be observed.

  In the laser microscope 100, the space between the objective lens 144 and the transmission part 220 can be filled with water 143 having a refractive index larger than that of air. Thereby, the numerical aperture (NA) of the objective lens 144 can be made larger than when it is used in the air.

  In the laser microscope 100, taking into account that the temperature-responsive polymer layer 230 is interposed between the lower objective lens 144 and the cell sheet 111, the laser beam irradiated when observing the cell sheet 111 is used. The wavelength may be selected on the condition that it easily transmits the temperature-responsive polymer layer 230.

  Alternatively, when the material of the temperature-responsive polymer layer 230 can be selected, a material that is transparent to the wavelength of the laser beam to be irradiated may be selected. Further, in the culture vessel 200, the temperature-responsive polymer layer 230 in a region sandwiched between the lower objective lens 144 and the cell sheet 111 may be removed in advance.

  Referring to FIG. 4 again, the objective lens 142 is then lowered and the lower end of the objective lens 142 is immersed in the culture solution 240 (step S103). FIG. 6 is a schematic cross-sectional view showing a state in which the sample 112 is observed with the laser microscope 100.

  As illustrated, the objective lens 142 can be sufficiently brought close to the cell sheet 111 by immersing the lower end of the objective lens 142 in the culture solution 240.

  Further, the upper objective lens 142 can be placed in an optically substantially symmetrical position with respect to the lower objective lens 144 with respect to the cell sheet 111 to be observed. Thus, a high-magnification image with few artifacts of the cell sheet 111 can be observed with the laser microscope 100 (step S104).

  In addition, when observing the cell sheet 111, it is preferable to avoid that the objective lens 142 contacts the cell sheet 111 directly. Therefore, in this embodiment, a contact preventing means for preventing the objective lens 142 from contacting the cell sheet 111 is provided.

  The contact prevention means includes a measurement unit that measures the distance between the cell sheet 111 and the objective lens 142. The measurement unit calculates the interval between the cell sheet 111 and the objective lens 142 based on the positional information on the upper surface of the cell sheet 111 and the positional information on the objective lens 142. The position of the objective lens 142 is measured by a position measuring instrument such as an encoder.

  When the position of the objective lens 142 is fixed and the stage 110 that holds the culture vessel 200 moves, the position of the cell sheet 111 is calculated from the position of the stage. The position of the cell sheet 111 is calculated from the thickness of the cell sheet 111. In this case, the thickness information of the cell sheet 111 is acquired from a manufacturing apparatus that manufactures the cell sheet 111, for example.

  When the distance between the cell sheet 111 and the objective lens 142 reaches a predetermined value, a limiting unit is provided that restricts the amount by which the objective lens 142 is lowered to a range that does not contact the cell sheet 111. In addition, a warning unit is provided that generates a warning by sound, light, video, or the like when the interval exceeds a predetermined value or when the objective lens 142 is lowered to a range in contact with the cell sheet 111.

  If the position of the surface of the cell sheet 111 to be observed has not been determined, such as the thickness of the cell sheet 111 varies depending on the sample 112, a sensor that detects the surface position of the cell sheet 111 in a non-contact manner, such as a laser rangefinder The laser microscope 100 may be provided. The laser microscope 100 may limit the descending amount of the objective lens 142 according to the detection result of the sensor.

  Alternatively, a plurality of objective lenses 142 having different optical specifications are provided, and a mechanism for exchanging the objective lenses 142 having a focal depth corresponding to the number of stacked layers is provided. In this case, a display unit that indicates to the user that replacement should be performed may be provided to recommend replacement.

  Furthermore, when the cell sheet 111 to be observed includes a plurality of stacked cell sheets 111, the lowering range of the objective lens 142 may be changed according to the number of stacked layers. The number of stacked cell sheets 111 may be displayed on the culture vessel 200 in a format that can be automatically detected by the laser microscope 100, such as a barcode.

  When the observation is completed, the objective lens 142 is raised (step S105), and as shown in FIG. 5, the space between the objective lens 142 and the stage 110 is expanded to collect the culture vessel 200 from the stage 110 (step S106). . Thus, when the observation with respect to one sample 112 is completed, the objective lens 142 immersed in the culture solution 240 in step S103 is sterilized (step S107). The sterilization in step S107 may be the same method as the sterilization in step S101.

  As the next observation procedure, it is checked whether there is another sample 112 to be observed (step S108). When it is determined that there is no other sample 112 to be observed (step S108: NO), the observation procedure is terminated. On the other hand, when it is found that the sample 112 to be observed next exists (step S108: YES), it is checked whether or not the time interval until the next observation of the sample 112 exceeds a predetermined threshold ( Step S109).

  If the time interval until the next observation of the sample 112 is short (step S109: NO), it is determined that the objective lens 142 is unlikely to be contaminated before the next observation, and the objective is Without sterilizing the lens 142 again, the sample 112 is placed on the stage 110 (step S102), and the next observation is started. If the time interval until the next observation is long (step S109: YES), it is determined that the objective lens 142 may be contaminated during that time, and the objective lens 142 is sterilized (step S101). ) Place the sample 112 on the stage 110 (step S102), and start the next observation.

  In this way, the sample 112 is prevented from being contaminated by another sample 112 through the culture solution 240 attached to the objective lens 142. Note that the threshold of the time interval referred to in step S109 is determined according to the environment in which the laser microscope 100 is placed, the sterility compensation level required for the sample 112, and the like.

  For example, when the laser microscope 100 is used in a state of being housed in a sterile box, there is a low possibility that the objective lens 142 is contaminated by the atmosphere. Therefore, the threshold value set for the time interval becomes long. On the other hand, when the level of sterility compensation required for the sample 112 is high, the time interval threshold is set short in order to eliminate the possibility of contamination as much as possible.

  FIG. 7 shows an example in which the laser microscope 100 includes a plurality of objective lenses 142. While one objective lens 142 is being used among the plurality of objective lenses 142, the other objective lens 142 is sterilized. It is an example. The plurality of objective lenses 142 are provided so as to be movable between an observation position where the object to be observed is observed and a sterilization position where the objective lens 142 is sterilized.

  In the example of FIG. 7, the laser microscope 100 includes a revolver 170 that supports a plurality of objective lenses 142. In the laser microscope 100, the objective lens 142 is held by the revolver 170. However, in FIG. 7, illustration of optical components in each of the objective lenses 142 is omitted.

  The revolver 170 is supported from the revolver base 174 via a rotation shaft 172 inclined with respect to the optical axis Q of the optical system 140 while holding a plurality of objective lenses 142. The rotation axis 172 has an inclination that forms an angle of 45 degrees with respect to the vertical optical axis Q. Thereby, one of the objective lenses 142 supported by the revolver 170 communicates with the optical path 176 provided through the revolver base 174 along the vertical optical axis Q, and the sample 112 in the laser microscope 100 is Used for observation.

  The other objective lens 142 supported by the revolver 170 is displaced laterally with respect to the optical axis Q and becomes substantially horizontal at a position away from the sample 112. Thereby, while using one objective lens 142 for observation by the laser microscope 100, other objective lenses 142 not used for observation can be sterilized while being attached to the revolver 170.

  In the drawing, a pair of objective lenses 142 supported by the revolver 170 is shown, but it goes without saying that the revolver 170 may hold more objective lenses 142. Further, the size of the revolver 170 and the inclination of the rotating shaft 172 are not limited to the illustrated example.

  FIG. 8 is a schematic cross-sectional view illustrating an example of a method for sterilizing one objective lens 142. After being used for observing the sample 112, the box-shaped sterilization chamber 180 is first approached from the side toward the optical axis Q with respect to the objective lens 142 that is moved away from the sample 112 by rotating the revolver 170. . Eventually, the tip of the objective lens 142 is inserted into the sterilization chamber 180 through the insertion hole 182 provided in the side wall of the sterilization chamber 180.

  A sterilizing gas such as ozone gas is circulated from the inlet 184 toward the outlet 186 in the sterilization chamber 180 into which the tip of the objective lens 142 is inserted. Thereby, when observing, the front-end | tip of the objective lens 142 immersed in the culture solution 240 can be sterilized. The objective lens 142 thus sterilized can be used again for observing the sample 112 by rotating the revolver 170 after the sterilization chamber 180 is retracted to the side.

  Note that the objective lens 142 may be sterilized by circulating a solution of hydrogen peroxide, sodium hypochlorite, hydrofluoric acid, or the like in the sterilization chamber 180. However, when a liquid is used for sterilization, it is preferable to subsequently dry the objective lens 142 by circulating sterilized dry air or the like in the sterilization chamber 180.

  Further, in the sterilization chamber 180, the objective lens 142 may be irradiated with ultraviolet rays, gamma rays, and electron beams. In this case, the wall surface of the sterilization chamber 180 is used as a shielding material for preventing gamma rays and the like from leaking to the outside.

  Furthermore, in the observation with the laser microscope 100, a step of washing the objective lens 142 in the sterilization chamber 180 may be provided prior to sterilization after observing the cell sheet 111. By this cleaning, the deposit on the objective lens 142 can be removed, and the efficiency and effect of sterilization can be improved.

  Cleaning can be selected from hand cleaning, immersion cleaning, ultrasonic cleaning, jet cleaning, etc., but since the objective lens 142 is an optical instrument, cleaning methods with large temperature changes such as hot water cleaning and steam cleaning are not available. It is not preferable. For cleaning the objective lens 142, pure water, distilled water, treated water (purified water) or the like can be used. Furthermore, it can also serve as a sterilization process by washing | cleaning using chemical | medical agents, such as pyrogen.

  In observation with the laser microscope 100, the objective lens 142 may be washed after the sterilization for the purpose of removing the residue of the medicine used for the sterilization. Thereby, it can prevent that the chemical | medical agent used for sterilization damages the cell sheet 111 observed next, and its culture solution 240. FIG.

  In this case, it is preferable to use pure water, distilled water, treated water (purified water) or the like that does not contain an electrolyte. Moreover, you may wash | clean using RO water in order to remove the remaining electric field quality. Furthermore, it is preferable to dry immediately after washing for the purpose of preventing the growth of bacteria attached from the atmosphere.

  The sterilization chamber 180 as described above may be provided as a part of the laser microscope 100. Further, the sterilization chamber 180 may be used in combination with the laser microscope 100 as an independent sterilization apparatus. Further, when the laser microscope 100 is used in an industrial application, a function of automatically executing an operation of sterilizing the objective lens 142 by rotating the revolver 170 every time the sample 112 to be observed is replaced. You may provide in microscope 100 itself.

  In this case, the laser microscope 100 has a detection unit that detects the end of observation or the completion of observation, a drive unit that drives the revolver 170 based on the detection result of the detection unit, and the sterilization chamber 180 facing the objective lens 142. And a moving unit for moving. The moving unit may move the sterilization chamber 180 in synchronization with the drive unit, or may move the sterilization chamber 180 after detecting that the objective lens 142 has moved to the sterilization position.

  Further, when the objective lens 142 is inserted into the sterilization chamber 180, the laser microscope 100 detects, for example, the position of the tip of the objective lens 142, the sealing degree of the insertion hole 182 of the sterilization chamber 180, and the like. An insertion detection unit that detects whether or not the lens 142 is properly cleaned and sterilized may be provided, and cleaning and sterilization may be started based on the detection result of the insertion detection unit.

  FIG. 9 is a schematic cross-sectional view of the revolver 170. In the illustrated example, the plurality of objective lenses 142 held by the revolver 170 can be individually attached to and detached from the revolver 170. Therefore, the objective lens 142 can be removed from the revolver 170 by rotating the revolver 170 after observing the cell sheet 111 and moving away from the sample 112. That is, the plurality of objective lenses 142 are provided so as to be movable between an observation position where the object to be observed is observed and an attachment / detachment position where the objective lens 142 is attached / detached.

  FIG. 10 is a schematic cross-sectional view of a sterilization case 190 used when the removed objective lens 142 is sterilized. The sterilization case 190 is disposed in the vicinity of the revolver 170, and the objective lens 142 removed from the revolver 170 is immediately accommodated in the sterilization case 190.

  The sterilization case 190 has an open / close lid 192 that can be opened and closed. Therefore, the objective lens 142 can be accommodated in the sterilization case by opening the opening / closing lid 192. Further, the objective lens 142 can be hermetically sealed in the sterilization case 190 by binding the open / close lid 192 in a state where the objective lens 142 is accommodated.

  As in the sterilization chamber 180, a sterilization gas such as ozone gas is circulated through the sterilization case 190 containing the objective lens 142 through the inflow port 194 and the outflow port 196. Thereby, when observing, the front-end | tip of the objective lens 142 immersed in the culture solution 240 can be sterilized. The sterilization case 190 can be hermetically sealed when the objective lens 142 is sterilized. Therefore, a sterilizing gas that has interpersonal toxicity but a high sterilizing effect, such as ethylene oxide gas, can be used.

  Further, the objective lens 142 may be sterilized by circulating a solution of hydrogen peroxide, sodium hypochlorite, hydrofluoric acid or the like in the sterilization case 190. Further, a cleaning agent such as pyrogen may be used. However, when a liquid is used for sterilization, it is preferable to dry the objective lens 142 by circulating sterilized dry air or the like through the sterilization case 190.

  Further, in the sterilization case 190, the objective lens 142 may be sterilized by irradiating ultraviolet rays, gamma rays, electron beams or the like. In this case, the wall surface of the sterilization case 190 is used as a shielding material for preventing gamma rays and the like from leaking to the outside.

  The sterilization case 190 as described above may be provided as a part of the laser microscope 100. Further, the sterilization case 190 may be prepared as an independent sterilization apparatus and used together with the laser microscope 100.

  Even when the objective lens 142 is removed from the revolver 170 and sterilized, the objective lens 142 may be washed in the sterilization case 190 prior to sterilization. By this cleaning, the deposit on the objective lens 142 can be removed, and the efficiency and effect of sterilization can be improved.

  Cleaning can be selected from hand cleaning, immersion cleaning, ultrasonic cleaning, jet cleaning, and the like. However, since the objective lens 142 is an optical instrument, cleaning methods with large temperature changes such as hot water cleaning and steam cleaning are not preferable. . For cleaning the objective lens 142, pure water, distilled water, treated water (purified water) or the like can be used. Furthermore, it can also serve as a part of sterilization by washing with a chemical such as pyrogen.

  Further, after the sterilization, the objective lens 142 may be washed for the purpose of removing the residue of the medicine used for the sterilization. Thereby, it can prevent that the chemical | medical agent used for sterilization damages the cell sheet 111 observed next, and its culture solution 240. FIG.

  In this case, it is preferable to use pure water, distilled water, treated water (purified water) or the like that does not contain an electrolyte. Moreover, you may wash | clean using RO water in order to remove the remaining electric field quality. Further, after the cleaning, it is preferable to dry the objective lens 142 after the cleaning immediately for the purpose of preventing the growth of bacteria attached from the atmosphere.

  The objective lens 142 thus sterilized may be stored in a sterile box for subsequent use. In addition, when observation of the cell sheet 111 with the laser microscope 100 is continued, it may be attached to the revolver 170 again.

  When the laser microscope 100 is used for industrial applications, the revolver 170 is rotated each time the sample 112 to be observed is replaced, and the objective lens 142 is removed from the revolver 170 and replaced with a sterilized objective lens 142. Equipment for automatically executing a series of operations may be provided.

  In this case, the laser microscope 100 includes an attachment / detachment mechanism that attaches / detaches the objective lens 142 to / from the revolver 170 and a conveyance unit that conveys the objective lens 142 removed by the attachment / detachment mechanism to the sterilization case 190. The attachment / detachment mechanism includes, for example, an attachment unit that holds the objective lens 142 at the attachment / detachment position and removes it from the revolver 170. After the sterilization operation, the transport unit transports the sterilized objective lens 142 to the revolver 170 and attaches it to the revolver 170 by an attaching / detaching mechanism. The position of the revolver 170 to which the objective lens 142 is attached may be the same as or different from the position where it was originally attached. The attaching / detaching step and the sterilizing step may be performed while observation is performed by the other objective lens 142.

  In addition, a housing case for housing a plurality of already sterilized objective lenses 142 is provided, and after the objective lens 142 is removed by the attaching / detaching mechanism, a new objective lens 142 is taken out from the housing case and transported to the revolver 170 by the transport unit. The objective lens 142 may be attached to the revolver 170 by an attaching / detaching mechanism. The objective lens 142 sterilized in the sterilization case 190 may be accommodated in the accommodation case after sterilization, or may be directly conveyed and attached to the revolver 170 after sterilization.

  In the above example, the case where the revolver 170 is provided in the laser microscope 100 and the objective lens 142 is sterilized in a state where the objective lens 142 is away from the stage 110 has been described. However, for example, in the laser microscope 100 including the objective lens 142 fixed without the revolver 170, for example, after observing the cell sheet 111 accommodated in the culture container 200, the container corresponding to the culture container 200 is used for sterilization. The medicine may be put on the stage 110 and sterilized by immersing the objective lens 142 in the medicine in the container.

  In this example, an example using a cell sheet as an object to be observed was shown, but instead of this, for observation of cells that do not form a sheet, such as tissues such as skin fragments, microorganisms such as bacteria, etc. The present invention can be used. As the cell type, an artificial multifunctional stem cell (iPS cell), an embryonic stem cell (ES cell) or the like may be used.

  As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

  The order of execution of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior to”. It should be noted that the output can be realized in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for convenience, it means that it is essential to carry out in this order. It is not a thing.

100 laser microscope, 110 stage, 111 cell sheet, 112 sample, 114 observation window, 120 laser device, 122, 124 laser light source, 126 combiner, 130 scanning system, 132 galvano scanner, 134 scan lens, 136 primary image plane, 140 optics System, 142, 144 objective lens, 143 water, 146 condenser lens, 148 reflector mirror, 150 detection system, 152 condenser lens, 154 relay lens, 156 photomultiplier tube, 158 dichroic mirror, 160 control system, 162 control unit, 164 keyboard, 166 mouse, 168 display unit, 170 revolver, 172 rotation axis, 174 revolver base, 176 optical path, 180 sterilization chamber, 182 insertion hole, 184, 194 inlet, 186, 196 outlet, 190 Sterilization case, 192 Open / close lid, 200 Culture vessel, 210 Holding part, 212 Opening part, 220 Permeation part, 230 Temperature-responsive polymer layer, 240 Culture solution

Claims (11)

A step of observing the first observation object with an objective lens supported by the revolver and arranged at a position to observe the observation object;
Rotating the revolver to dispose the objective lens in a sterilization unit for sterilizing the objective lens;
Sterilizing the objective lens supported by the revolver in the sterilization unit;
Rotating the revolver and disposing the sterilized objective lens at a position for observing the observation object;
Observing a second observation object with the objective lens arranged at a position for observing the observation object;
Including observation method. The sterilizing step includes exposing the objective lens to at least one of ozone gas, ethylene oxide gas, hydrogen peroxide, sodium hypochlorite, hydrofluoric acid, ultraviolet rays, gamma rays, and electron beams in the sterilization unit. The observation method of Claim 1 containing. The observation method according to claim 1 or 2, wherein the sterilization unit is located farther from the observation target than a position at which the observation target is observed by the objective lens . The observation method according to any one of claims 1 to 3 , further comprising a step of sterilizing the objective lens before the step of observing the first observation target . The observation method according to any one of claims 1 to 4 , further comprising a step of washing the objective lens after the sterilizing step . Step method of observation according to claim 5 including the step of exposing the objective lens, the flowing water to the washing. The revolver supports two or more objective lenses,
The other one of the objective lenses is disposed in the sterilization unit and is sterilized in the sterilization unit while one of the objective lenses is performing the step of observing the first observation target. 7. The observation method according to any one of 6. An objective lens;
A sterilization unit for sterilizing the objective lens;
A revolver that supports and rotates the objective lens;
When the revolver is rotated, the objective lens moves between a position where the observation target is observed and a position where the object is sterilized by the sterilization unit . The revolver supports two or more objective lenses,
The microscope according to claim 8, wherein when one of the objective lenses is arranged at a position for observing the observation target, another one of the objective lenses is arranged at the sterilized position. The sterilization unit, ozone, ethylene oxide gas, hydrogen peroxide, sodium hypochlorite, hydrofluoric acid, ultraviolet light, gamma ray, and a microscope according to claim 8 or 9 exposing said objective lens in at least one electron beam. The microscope according to any one of claims 8 to 10, wherein the sterilization unit is located farther from the observation target than a position at which the observation target is observed .

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