JP2015152370A - Detection device, microscope and processing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem in which excitation light may ablate an observation object.SOLUTION: A detection device comprises a detection unit that detects occurrence of ablation when irradiating excitation light causing a nonlinear optical effect to be generated in an object. In the detection device, the detection unit may detect the ablation occurring in any of the object and a support body when the object supported by the support body is irradiated with the excitation light, and in the detection device, the object may include a cell sheet, a cellular tissue piece and a microorganism.

Description

  The present invention relates to a detection device, a microscope, and a processing device.

There is an observation apparatus using a nonlinear optical effect (see Patent Document 1).
Japanese Patent Application Laid-Open No. 2011-257691

  The excitation light that excites the non-linear optical effect is irradiated on the observation target and the container that supports the observation target, so that thermal excitation occurs in the observation target and the container and the quality thereof may change.

  In a first aspect of the present invention, a detection device is provided that includes a detection unit that detects that ablation has occurred when an excitation light that causes a nonlinear optical effect is irradiated on an object.

  In a second aspect of the present invention, there is provided a microscope that includes the above-described detection device and detects scattered light emitted from an object irradiated with excitation light and observes the object.

  In a third aspect of the present invention, there is provided an observation object processing apparatus that includes the detection device and includes a removal unit that removes an object damaged by ablation detected by the detection device.

  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.

2 is a schematic diagram showing a structure of an evaluation apparatus 101. FIG. 2 is a sectional view of a sample 200. FIG. 4 is a schematic diagram showing a state of a sample 200 in the observation unit 100. FIG. FIG. 6 is a diagram for explaining the operation of an observation unit 100. It is a schematic diagram explaining the state of the focus 145 vicinity. 3 is a block diagram of a detection unit 300. FIG. 5 is a diagram illustrating an example of writing to a sample 200. FIG. It is a flowchart which shows the operation | movement procedure of the evaluation apparatus.

  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 an evaluation apparatus 101 that evaluates the quality of a cell sheet. Evaluation apparatus 101 includes an observation unit 100 and a detection unit 300.

  The observation unit 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. A part of the control system 160 is shared by the detection unit 300.

  In the observation unit 100, the stage 110 has an observation region that is supported when observing the loaded sample 200, and a processing region that supports the sample 200 when the writing device 370 writes the observation result in the sample 200. . A writing device 370 and a transport device 380 are provided in the processing area.

  The writing device 370 writes information to the sample 200 by a method that does not affect the quality of the sample 200, such as an inkjet method or an ink pen method. The transport device 380 is used when the sample 200 is moved on the stage 110 and the sample 200 is unloaded from the stage 110. However, the sample 200 on the stage 110 may be moved by the user's hand.

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 excitation light on a single optical path. Accordingly, the sample 200 can be irradiated with excitation light that is a combination of pump light and Stokes light, and CARS light can be generated in the sample 200.

  The laser device 120 may be provided with a delay optical path for the purpose of synchronizing the pump light pulse generated by the laser light sources 122 and 124 and the Stokes light pulse. 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.

  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 the incident excitation light in a direction intersecting the optical axis.

  The scan lens 134 focuses the excitation light emitted from the galvano scanner 132 on a predetermined primary image plane 136. As a result, the excitation light emitted from the laser device 120 is focused on the observation region of the sample 200. Further, when the galvano scanner 132 is operated, the focus of the excitation light is scanned in parallel with the plane indicated by the arrow xy in the drawing, and the observation light having a predetermined area is irradiated with the excitation light. , CARS light can be generated.

  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 excitation light irradiated toward the sample 200 within the observation region of the sample 200. 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 excitation light between the laser device 120 and the objective lens 142. The reflecting mirror 148 bends the optical path of the excitation light to prevent the structure of the observation unit 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 200. The dichroic mirror 158 branches the CARS light from the light emitted from the sample 200 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, the control unit 162 generates an observation image of the sample 200 based on the detection result of the detection system 150 and displays it on the display unit 168. Furthermore, the control unit 162 controls the operation of the detection unit 300 and controls the observation unit 100 according to the detection result by the detection unit 300.

  The observation unit 100 can observe the cultured cells, tissues, microorganisms, and the like using a nonlinear optical effect such as CARS (coherent anti-Stokes Raman scattering) and SRS (stimulated Raman scattering). The nonlinear optical effect is generated by irradiating the observation target with excitation light. The detection system 150 detects the CARS light emitted from the side opposite to the side irradiated with the excitation light and forms an observation image.

  Furthermore, the evaluation apparatus 101 includes a detection unit 300. The detection unit 300 includes an imaging unit 310 and a recording medium 360. The imaging unit 310 captures the sample 200 placed on the stage 110 from the front and back, and generates an image indicating the state of the sample 200 observed by the observation unit 100. The recording medium 360 records the detection result by the detection unit 300. The detection unit 300 includes a part of the control unit 162 in addition to the imaging unit 310 and the recording medium 360, which will be described later with reference to other drawings.

  FIG. 2 is a cross-sectional view of a sample 200 that is an inspection target of the evaluation apparatus 101 and is an observation target of the observation unit 100. The sample 200 includes a cell sheet 201 to be inspected and a culture container 210 that contains the cell sheet 201.

  The cell sheet 201 is a living cell tissue in the form of a thin sheet created by culturing cells collected from a subject, and is handled in a state immersed in a culture solution. The cultured cell sheet is finally attached to the living tissue of the subject while alive.

  The culture vessel 210 has a holding frame 212 and a transmission part 214. The holding frame 212 is formed by easy molding of resin or the like, and has a short cylindrical shape. In the culture vessel 210, the holding frame 212 forms a cylindrical side wall, and contacts the upper surface of the stage 110 or the like at the lower end surface of the side wall. The holding frame 212 has an opening 211 on the lower surface.

  The culture container 210 is only an example of a support that supports the cell sheet 201. The cell sheet 201 can be taken out of the culture solution and supported by a flat support such as a slide glass and transported for a short time required for observation. Further, when the stage 110 is in an aseptic operation environment, the stage 110 can be directly supported.

  The transmission part 214 is held by the holding frame 212, and the opening 211 of the holding frame 212 is sealed in a liquid-tight manner to form the bottom surface of the culture vessel 210. Thereby, the culture container 210 can accommodate the culture solution 220 without leaking. In addition, the bottom surface of the culture vessel 210 formed by the transmission part 214 is optically transparent.

  The holding frame 212 can be formed of a polymer material such as polycarbonate, polystyrene, polydimethylsiloxane, silicone polycarbonate, or polyethylene terephthalate. The transmission part 214 can be formed of, for example, a transparent inorganic material such as a cover glass.

  In the culture vessel 210, a temperature-responsive polymer layer 216 is disposed on the upper surface of the transmission part 214, that is, on the bottom surface inside the culture vessel 210. The temperature-responsive polymer layer 216 is grafted to the permeation part 214 and fixed to the culture vessel 210. The temperature-responsive polymer layer 216 can be formed of a polymer material such as poly-N-isopropylacrylamide.

  A temperature-responsive polymer such as poly-N-isopropylacrylamide changes in hydration power at a preset critical temperature. The critical temperature can be set to around 30 degrees Celsius that does not damage the cell sheet 201. On the other hand, the cell sheet 201 adheres to the temperature-responsive polymer layer 216 that exhibits hydrophobicity and can be easily peeled off from the temperature-responsive polymer layer 216 that exhibits hydrophilicity. Utilizing such properties, the cell sheet 201 is placed on the bottom surface of the culture vessel 210 without being damaged by enzyme treatment or the like by heating or cooling the temperature-responsive polymer layer 216 disposed in the culture vessel 210. Can be fixed or open.

  Note that the material of the temperature-responsive polymer layer 216 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.

  FIG. 3 is a schematic diagram illustrating a state of the sample 200 in the observation unit 100. In the sample 200 placed on the stage 110, the cell sheet 201 is adhered to the surface of the temperature-responsive polymer layer 216.

  The stage 110 is provided with an observation window 114 that exposes the bottom surface of the culture vessel 210 placed on the stage 110. Thereby, the culture vessel 210 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 210 placed on the stage 110 from the bottom surface side or the top surface side.

  A lower objective lens 144 faces the transmission part 214 of the culture vessel 210 placed on the stage 110 through the observation window 114. Further, the upper objective lens 142 faces the cell sheet 201 accommodated in the culture vessel 210 on the stage 110 from above. The pair of objective lenses 142 and 144 have a common focal point.

  Note that the gap between the lower objective lens 144 and the transmission part 214 of the culture vessel 210 is filled with water 143. Thereby, the effective numerical aperture of the lower objective lens 144 is increased, and the effective magnification of the objective lens can be increased.

FIG. 4 is a schematic diagram for explaining the CARS process in which CARS light is generated at the focal point of the excitation light irradiated on the cell sheet 201 in the observation unit 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 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. In addition, by repeatedly detecting CARS light while changing the position where the observation object is irradiated with the excitation 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 making the excitation light band irradiated on the object to be observed an infrared band that causes little damage to the living cells, the living cells of the object to be observed can be observed.

  Furthermore, the CARS light generated by the nonlinear effect is generated in a very narrow region where the excitation 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. 5 is a schematic diagram illustrating a state in which the cell sheet 201 is being observed in the observation unit 100. 5 corresponds to a region surrounded by a dotted line A in FIG.

  When observing the cell sheet 201, the focal point of the excitation light connected by the objective lens 144 is located inside the cell sheet 201. Thereby, the detection system 150 of the observation unit 100 detects the CARS light generated in the cell sheet 201 and generates an observation image of the cell sheet 201.

  Here, the optical linear effect that generates CARS light occurs in the immediate vicinity of the focal point of the excitation light. For this reason, when observing the vicinity of the lower surface of the cell sheet 201, at least one of the stage 110 and the optical system 140 is moved to move the focal point to the vicinity of the lower surface of the cell sheet 201.

  On the other hand, the excitation light excites the irradiation object thermally as well as optically. Thermal excitation by the excitation light extends around the focal point 145. In the figure, a thermal excitation zone 147 in which thermal excitation can occur is indicated by a dotted line.

  Since the thermal excitation zone 147 is wider than the range where optical excitation occurs, when the focal point is located near the lower surface of the cell sheet 201, the thermal excitation zone 147 extends to the outside of the cell sheet 201. For this reason, the temperature-responsive polymer layer 216 fixing the cell sheet 201 in the sample 200 may be thermally excited by excitation light.

  Since the temperature-responsive polymer layer 216 is formed of a polymer material, the temperature-responsive polymer layer 216 is easily affected by heat and, since it is a solid, diffusion of heat is relatively slow. Therefore, when excitation light is irradiated on the temperature-responsive polymer layer 216 or when the temperature-responsive polymer layer 216 enters the thermal excitation zone 147, a part of the light energy absorbed by the molecules of the temperature-responsive polymer layer 216 is absorbed. By so-called thermal excitation that is converted into thermal energy, the temperature of the temperature-responsive polymer layer 216 rises above a predetermined temperature, or the surface layer that has become locally hot evaporates, causing atoms and molecules The so-called ablation emitted by the plasma and their clusters can occur in the temperature-responsive polymer layer 216.

  For example, when the temperature of the temperature-responsive polymer layer 216 increases, heat is transferred from the temperature-responsive polymer layer 216 to the cell sheet 201, and the cells of the cell sheet 201 may die or denature. The temperature at which the cell may die or denature is, for example, 37 ° C. or higher. Further, if ablation occurs in the temperature-responsive polymer layer 216, the cell sheet 201 may be damaged.

  When the temperature-responsive polymer layer 216 rises above a predetermined temperature or causes ablation, the temperature-responsive polymer rapidly vaporizes or becomes plasma. For this reason, the temperature-responsive polymer layer 216 is not only damaged, but the cell sheet 201 may be damaged.

  In addition to the ablation that occurs with the observation as described above, when the focal point 145 moves to the holding frame 212 of the culture vessel 210 for some reason, a temperature increase or ablation may occur in the holding frame 212. Furthermore, when the output of the laser device 120 is excessively high, temperature rise and ablation may occur in the cell sheet 201 itself.

  FIG. 6 is a block diagram of the detection unit 300. In addition to the imaging unit 310, the detection unit 300 includes a determination unit 320, a specification unit 330, a counting unit 340, and a recording unit 350. The determination unit 320, the identification unit 330, the counting unit 340, and the recording unit 350 are formed as a part of the control unit 162.

  The imaging unit 310 images the entire sample placed on the stage 110. The imaging unit 310 has a lower resolution than the observation unit 100, but has high sensitivity from the visible light band to the infrared band, and images light and heat generated from the sample 200. Such an imaging unit 310 can be formed by a camera having a solid-state imaging device such as a CCD or CMOS sensor.

  The determination unit 320 extracts a bright spot from the image generated by the imaging unit 310 and determines whether the bright spot is due to ablation. For example, when the brightness of a bright spot exceeds a predetermined threshold, it can be determined that the bright spot is due to the occurrence of ablation. Alternatively, it may be determined that ablation has occurred when the spectrum of the bright spot has a broad shape having no peak. The determination result is transferred to the specifying unit 330, the counting unit 340, and the recording unit 350 of the detection unit 300, and is notified to the determination unit 161 of the control unit 162.

  The identifying unit 330 identifies in which part of the sample 200 the bright spot that the determining unit 320 has determined to have occurred due to the occurrence of ablation has occurred. The specifying unit 330 may specify a member that has caused ablation based on, for example, a material that has been determined by the brightness, spectrum, or the like of a bright spot that has been generated by ablation. The ablated material can be specified by matching with the spectral pattern of each member recorded in advance.

  Thereby, the specifying unit 330 specifies whether the part where the ablation occurred in the sample 200 is the cell sheet 201, the holding frame 212, the permeation unit 214, or the temperature-responsive polymer layer 216 of the culture vessel 210. The specifying unit 330 may further specify the position where ablation has occurred in each element. The specifying unit 330 may further specify the position where ablation has occurred in each element. The occurrence position of ablation can be identified based on the image of the imaging unit 310 from the image such as the breakage of the vessel and the scattering of the fragments of the container. The identification result by the identification unit 330 is passed to the counting unit 340 and the recording unit 350 of the detection unit 300 and is notified to the determination unit 161 of the control unit 162.

  The specification by the specifying unit 330 can be specified by, for example, the brightness of the bright spot in the image generated by the imaging unit 310. For example, in the case of a material that is prone to ablation such as a temperature-responsive polymer, the brightness of the bright spot is remarkably increased, so that the ablation can be identified.

  The specification by the specifying unit 330 can also be specified by, for example, the spectrum of bright spots in the image generated by the imaging unit 310. That is, by storing the spectrum of the constituent elements of the sample 200 in advance, it is possible to specify the ablation caused from the wavelength of the bright spot.

  The counting unit 340 counts the number of times that the determination unit 320 determines that it is ablation for each sample 200. The counting unit 340 may count the number of occurrences of ablation for each target specified by the specifying unit 330. Thereby, it is possible to estimate the damage that the sample 200 receives when minor ablation that does not satisfy the stop condition occurs many times. The counting result is transferred to the recording unit 350 and notified to the determination unit 161 of the control unit 162.

  The recording unit 350 accumulates the determination result of the determination unit 320, the specification result of the specification unit 330, and the count result of the counting unit 340 for each sample 200 as a record. The accumulated record is referred to by the determination unit 161.

  The determination unit 161 acquires ablation information detected by the detection unit 300 and controls the overall operation of the evaluation apparatus 101. That is, when the generated ablation satisfies a stop condition that is considered to have a serious effect on the observation unit 100, the sample 200, etc., the determination unit 161 suppresses the irradiation of the excitation light in the observation unit 100 through the issuing unit 163 or Generates a command to stop.

  As a case where the stop condition is satisfied, for example, a case where ablation in the culture vessel 210 is specified can be exemplified. In such a case, there is a high possibility that the cell sheet 201 accommodated in the culture vessel 210 is also damaged.

  The suppression of the excitation light can take various measures such as reducing the output of the laser device 120, changing the irradiation position by the scanning system 130, and changing the focal position by the optical system 140. When it is determined that the influence of the generated ablation is large, the observation of the sample 200 may be terminated by inserting a light shielding plate in the optical path or stopping the laser device 120.

  On the other hand, if it is determined that the generated ablation is a minor one that does not satisfy the stop condition, the determination unit 161 determines that the observation by the observation unit 100 continues as it is, although the record about the generated ablation is retained. Also good. However, even if the influence of individual ablation is slight, when the number of occurrences of ablation increases, the determination unit 161 performs observation by the observation unit 100 when the stop condition that the influence of ablation is considered to be serious is satisfied. finish.

  Further, when the location where the ablation occurs is outside the holding frame 212, the determination unit 161 determines that the observation unit 100 itself is damaged, although the cell sheet 201 is not damaged. It may be determined that the observation by the unit 100 is finished.

  Furthermore, the determination unit 161 may make a determination with reference to the observation result in the detection system 150. Acquisition of the observation result from the detection system 150 may be automatic processing such as image processing or the like, or may be an observation result input to the control unit 162 by a user who observed the sample 200 by the detection system 150.

  As described above, in the evaluation apparatus 101, the occurrence of ablation is detected by the detection unit 300 in parallel with the observation of the sample 200 irradiated with the excitation light. Thereby, while being able to suppress that the sample 200 receives damage by the observation part 100, quality of test object, such as the cell sheet 201, can be evaluated and productivity and quality can be balanced.

  Further, the determination unit 161 may send a command for recording the detection result, the identification result, and the count result in the detection unit 300 to the sample 200 to the writing device 370 through the issuing unit 163. Thereby, since the quality evaluation result is displayed for every sample 200, a detection result can be easily utilized in a subsequent process. As a subsequent process, for example, the repair of the cell sheet 201 in the repair unit 384 described below can be exemplified.

  Furthermore, the determination unit 161 may transport the sample 200 to a transport destination corresponding to the evaluation result by supporting the transport device 380 through the issuing unit 163. For example, a sample evaluated as high quality as a result of the evaluation is transported to the collection unit 382 and stored in a culture environment until used for treatment or the like.

  In addition, as a result of the evaluation, the sample 200 that is only partially damaged is transported to the repair unit 384, and after removing the damaged part specified by the specifying unit 330, by continuing the culture, Can be shipped as an intact cell sheet. Furthermore, as a result of the evaluation, the sample 200 evaluated as unrepairable is transported to the discarding unit 386 and discarded. However, even in the discarded sample 200, the culture vessel 210 that is not damaged may be collected for reuse.

  In the above example, the determination unit 161 automatically executes the end of observation, suppression of excitation light, and the like. However, a message indicating that the observation should be terminated or the excitation light should be suppressed may be output to the display unit 168, and operations such as stopping may be left to the user.

  FIG. 7 is a diagram illustrating an example of writing to the sample 200 by the writing device 370. In the example shown in the drawing, a label 250 on which the detection result is written in a barcode is attached to the side surface of the culture vessel 210.

  The label 250 is formed of, for example, thermal paper, and the thermal head of the writing device 370 writes the detection result on what was initially blank. Thereby, the damage by ablation can be known in a post process by reading a barcode with a reader in a post process. Therefore, for example, the cell sheet 201 can be shipped after being selected according to the application, or a low-quality one can be discarded before shipment.

  Needless to say, the recording description method is not limited to the above-described method, and the label 250 printed earlier may be affixed to the culture vessel 210, or an ink jet printer, a stamp may be used without using the label 250. For example, it may be directly written in the culture vessel 210. Further, the description format is not limited to the barcode, and may be described with characters, icons, and the like.

  FIG. 8 is a flowchart showing an operation procedure of the evaluation apparatus 101 including the detection unit 300 as described above. In the evaluation apparatus 101, first, the sample 200 including the cell sheet 201 to be observed is carried into the stage 110 (step S101).

  Next, the control unit 162 starts the irradiation of excitation light with the focus of the objective lenses 142 and 144 set to the initial position in the cell sheet 201, for example, the surface of the cell sheet 201. Further, the focal point 145 is scanned by the galvano scanner 132 (step S102), and a two-dimensional observation image is generated based on the CARS light generated from a certain region on the surface of the cell sheet 201.

  When the irradiation of the excitation light is started on the sample 200 including the cell sheet 201, the control unit 162 monitors whether the detection unit 300 detects ablation in parallel with the observation of the cell sheet 201 (step S103). ).

  When the generation of ablation is notified in step S103, the determination unit 161 in the control unit 162 checks whether the generated ablation satisfies the stop condition (step S104). If the ablation stop condition is not satisfied, the determination unit 161 The observation started in S102 is continued (step S104: NO). On the other hand, when the generated ablation satisfies the stop condition, the determination unit 161 suppresses the excitation light and ends the observation by the observation unit 100 (step S104: YES).

  When the generation of ablation is not notified in step S103 (step S103: NO), or when it is found that the ablation generated in step S104 does not satisfy the stop condition (step S104: NO), the control unit 162 It is checked whether or not the scanning in step S102 is completed (step S105). Here, when the scanning is not completed, the control is returned to step S102 and the scanning is continued (step S105: NO).

  When it is found in step S105 that the scanning is completed (step S105: YES), and the observation image on the first plane is generated by the scanning in step S102, the control unit 162 focuses the objective lenses 142 and 144 on the focus of FIG. Is moved in the thickness direction of the cell sheet 201 indicated by the arrow z (step S105). Here, the control unit 162 checks whether or not the focal points of the objective lenses 142 and 144 are outside the cell sheet 201 (step S106).

  When it is determined in step S104 that the focal point has moved within the thickness of the cell sheet 201 (step S106: NO), the control unit 162 performs CARS light on the next observation plane that is different in the thickness direction of the cell sheet 201. The observation image by is generated. Hereinafter, as long as the position of the focal point is within the thickness of the cell sheet 201, the control unit 162 repeats the movement in the z direction and the generation of the observation image to generate a three-dimensional observation image of the cell sheet 201.

  On the other hand, when it is determined in step S106 that the focal point has moved beyond the thickness of the cell sheet 201 (step S106: YES), the control unit 162 resets the focal point in the z direction, and again returns to the cell sheet. It returns to the surface of 201 (step S107). Next, the control unit 162 checks whether or not an observation image of the entire area has been generated in the surface direction of the cell sheet 201 (step S108).

  Note that “entire area” does not necessarily mean the entire area of the cell sheet 201. Since the resolution of observation with CARS light is high, if the cell sheet 201 is observed without any difficulty, it takes an enormous amount of time for observation. Therefore, the term “entire area” means all of the partial areas selected in advance to be observed on the cell sheet 201.

  In step S108, when the whole area of the cell sheet 201 has not been observed yet (step S108: NO), the control unit 162 moves the focal position in the surface direction of the cell sheet 201 indicated by the arrow xy in FIG. (Step S109). Next, the control part 162 returns control to step S102 again, and repeats the production | generation of the observation image of the cell sheet 201 by CARS light. When it is determined in step S106 that the entire region of the cell sheet 201 has been observed (step S108: YES), the control unit 162 ends the observation by the observation unit 100.

  In the above-described example, the example in which the ablation is detected in the cell sheet 201, the culture vessel 210, or the temperature-responsive polymer layer 216 has been shown, but whether or not these temperatures have risen above the predetermined temperature described above. May be detected by the same method as in the above example to detect whether or not the cell sheet 201 is damaged. Moreover, in order to detect temperature, without disturbing irradiation of the excitation light to the cell sheet 201, you may use thermography, for example.

  In the above example, when a general-purpose upper processing device is used as the control unit 162, the determination unit 320, the specifying unit 330, the counting unit 340, and the recording unit 350 that form a part of the detection unit 300 are connected to the recording medium or the communication unit. It can also be formed by a program implemented through a line. In addition, a part of the determination unit 320, the identification unit 330, the counting unit 340, and the recording unit 350 can be shared with other parts of the evaluation apparatus 101 such as the observation unit 100.

  In this embodiment, an example in which a cell sheet that is an object to be observed is used as an excitation target is shown. However, the laser microscope using the photolinear effect can use the present invention for observing cells that do not form a sheet, for example, tissue such as a skin fragment, microorganisms such as bacteria, etc., instead of a cell sheet. it can. 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.

DESCRIPTION OF SYMBOLS 100 Observation part, 101 Evaluation apparatus, 110 stage, 114 Observation window, 120 Laser apparatus, 122, 124 Laser light source, 126 Combiner, 130 Scan system, 132 Galvano scanner, 134 Scan lens, 136 Primary image plane, 140 Optical system, 142 144 objective lens, 143 water, 146 condenser lens, 145 focal point, 147 thermal excitation zone, 148 reflector, 150 detection system, 152 condenser lens, 154 relay lens, 156 photomultiplier tube, 158 dichroic mirror, 160 control system 161 judgment unit 162 control unit 163 instruction unit 164 keyboard 166 mouse 168 display unit 200 samples 201 cell sheet 210 culture vessel 211 opening 212 holding frame 214 permeation unit 216 temperature Responsive polymer layer, 220 culture solution, 250 label, 300 detection unit, 310 imaging unit, 320 determination unit, 330 identification unit, 340 counting unit, 350 recording unit, 360 recording medium, 370 writing device, 380 conveying device, 382 Recovery unit, 384 repair unit, 386 discard unit

Claims (25)

  A detection apparatus comprising: a detection unit that detects whether or not the object may be damaged when irradiated with excitation light that causes a nonlinear optical effect on the object.   The detection device according to claim 1, wherein the detection unit detects a possibility of damage to the object by detecting ablation that occurs in at least one of the object and a support that supports the object.   The detection apparatus according to claim 1, wherein the object includes any one of a cell, a tissue, and a microorganism.   The detection apparatus according to claim 3, wherein the object includes a cell sheet.   The detection apparatus according to any one of claims 1 to 4, wherein the object is irradiated with the excitation light in a state of being supported by a support including a portion formed of a polymer material.   The detection device according to any one of claims 1 to 5, wherein the detection unit includes an imaging unit that captures an area including a focus of the excitation light.   The detection device according to claim 6, wherein the imaging unit has sensitivity in a visible light band to a near infrared band.   The detection device according to claim 6, wherein the detection unit includes a determination unit that determines that ablation has occurred when a luminance of a bright spot in a captured image of the imaging unit exceeds a predetermined threshold.   The detection device according to claim 6, wherein the detection unit includes a determination unit that determines that ablation has occurred when a peak width of a spectrum pattern of a bright spot in a captured image of the imaging unit is widened.   The detection device according to any one of claims 6 to 9, further comprising a specifying unit that specifies a position where the ablation is generated when the detection unit detects the occurrence of the ablation.   The detection device according to claim 10, wherein the specifying unit specifies an ablation occurrence position based on a position of a bright spot in a captured image of the imaging unit.   The detection according to claim 10, wherein the specifying unit includes an image pickup unit that picks up an area including a focal point of the excitation light, and specifies an ablation occurrence position based on brightness of a bright spot in a captured image of the image pickup unit. apparatus.   The said specific | specification part specifies the generation | occurrence | production position of the ablation corresponding to the said luminescent spot based on the matching with the pattern stored in advance with the spectrum pattern of the luminescent spot in the captured image of the said imaging part. Detection device.   The detection device according to any one of claims 1 to 13, further comprising: an estimation unit configured to estimate a degree of damage to the object caused by ablation when the detection unit detects occurrence of ablation. .   15. The detection according to claim 14, wherein the estimation unit counts the number of occurrences of ablation for one of the objects, and estimates that the object has been damaged when the number of occurrences exceeds a predetermined threshold. apparatus.   The detection device according to claim 14 or 15, wherein the estimation unit estimates that the object supported by the support is damaged when ablation occurs in the support that supports the object. .   The detection device according to any one of claims 14 to 16, wherein the detection of the target object is continued until a degree of damage of the target object estimated by the estimation unit exceeds a predetermined threshold value. A detection device according to any one of claims 1 to 17, comprising:
A microscope for observing the target object by detecting scattered light emitted from the target object irradiated with the excitation light.   The microscope according to claim 18, further comprising a suppressing unit that suppresses the intensity of the excitation light irradiated to the object when the detection device detects ablation.   A processing apparatus comprising the detection device according to any one of claims 1 to 17, further comprising a removal unit that removes the object damaged by ablation detected by the detection device. When the detection unit detects the occurrence of ablation, the detection unit further includes a specifying unit for specifying the generation position of the ablation,
21. The processing apparatus according to claim 20, wherein the removing unit partially removes the occurrence location of ablation specified by the specifying unit from the object. When the detection unit detects the occurrence of ablation, the detection unit further includes an estimation unit that estimates the degree of damage to the object caused by ablation,
The processing device according to claim 20 or 21, wherein the removing unit removes the object estimated by the estimating unit to be damaged.   The processing apparatus according to claim 22, further comprising a recording unit that records a degree of damage to the object estimated by the estimation unit. The processing apparatus according to claim 23, wherein the recording unit records on a support that supports the object.
  The detection unit detects the possibility of damage to the object by detecting whether or not the temperature of at least one of the object and the support that supports the object is equal to or higher than a predetermined temperature. The detection device according to claim 1.

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