JP6211389B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus capable of performing fluorescence observation.

  A fluorescence microscope capable of performing fluorescence observation without using a dark room has been proposed. For example, an inverted fluorescence microscope described in Patent Document 1 includes a main body case, a specimen cover, and a lower cover. The specimen cover and the lower cover are provided on the front surface of the main body case. A stage, a bright field transmission illumination system, an epi-illumination system, an imaging system, a power supply unit, a power supply board, a controller, and the like are mounted inside the main body case.

  In the inverted fluorescence microscope, the stage and the imaging system are in a dark room state during fluorescence observation. Thereby, weak fluorescence emitted from the object is guided to the imaging system.

  In addition, a microscope imaging apparatus that generates an image that can be used to create a three-dimensional image by optical sectioning (cutting) has been proposed (see Patent Document 2).

  In the microscope imaging apparatus described in Patent Literature 2, a mask is provided in a light source so that an object is illuminated with a spatially periodic pattern. Thereby, a mask pattern is projected on the sample. In order to adjust the spatial phase of the mask pattern, the mask is moved to at least three positions by the carriage. By analyzing three or more images of the object illuminated at at least three positions of the mask, a three-dimensional image of the object is generated.

JP 2006-162664 A Special table 2000-506634

  Also in the inverted fluorescence microscope of Patent Document 1, if the object can be irradiated with the light of the mask pattern of Patent Document 2, a three-dimensional image of the object can be obtained. In this case, it is necessary to provide a mask and a mask moving mechanism for adjusting the spatial phase of the mask pattern in the main body case.

  Therefore, when switching between observation using a mask pattern and observation without using a mask pattern, the specimen cover and lower cover of the main body case are opened and the mask is attached to the main body case or the mask in the main body case is removed. Necessary. When changing the mask pattern, it is necessary to open the specimen cover and the lower cover of the main body case and replace the mask provided in the main body case. Such a mask replacement operation is complicated and takes time.

  Further, if dust is generated in the main body case due to frequent mask attachment / detachment and replacement operations or movement of the mask during adjustment of the spatial phase of the mask pattern, the reliability of fluorescence observation decreases. .

  An object of the present invention is to provide a microscope apparatus capable of easily and quickly performing a plurality of types of fluorescence observation without using a darkroom.

  (1) A microscope apparatus according to the present invention includes a stage on which an object is placed, a first observation mode for observing the object using first measurement light having a pattern, and a second that does not have a pattern. An operation device operated by a user to switch between a second observation mode for observing an object using measurement light, a light source device for generating light, and a first measurement from light generated by the light source device A light modulation element that generates light is generated, the first measurement light is generated by the light modulation element from the light generated by the light source device in the first observation mode, and the target is irradiated with the generated first measurement light And a measurement light irradiation device that irradiates the object with the light generated by the light source device in the second observation mode as the second measurement light, and a light reception signal that receives the fluorescence emitted from the object and indicates the amount of light received Light receiving unit and light receiving unit An image data generation unit that generates image data based on a light reception signal output from the signal, and pattern generation that generates a pattern of the first measurement light to be irradiated onto the object while the spatial phase is sequentially moved by a predetermined amount And in the first observation mode, the light modulation element is controlled based on the pattern generated by the pattern generation unit, and sectioning image data is generated based on a plurality of image data generated in a plurality of phases of the pattern. A control unit that controls the image data generation unit to control the image data generation unit so as to generate image data indicating a normal observation image of the object in the second observation mode, a stage, and a measurement light irradiation device And a first housing that houses the light receiving unit, and a second housing that houses the light modulation element in the first housing, the first housing includes an object on the stage and The measurement space including the light unit is configured to be in a dark room state, the operating device is provided outside the first housing, and the second housing is the first measurement light generated by the light modulation element. The other light is configured so as not to reach the measurement space from the light modulation element.

  In the microscope apparatus, a pattern of the first measurement light to be irradiated on the object is generated by the pattern generation unit while the spatial phase is sequentially moved by a predetermined amount. In the first observation mode, the light modulation element is controlled based on the pattern generated by the pattern generation unit. Accordingly, first measurement light having a pattern is generated from the light generated by the light source device, and the generated first measurement light is irradiated onto the object by the measurement light irradiation device. At this time, the fluorescence emitted from the object is received by the light receiving unit, and a light reception signal is output. A plurality of image data is generated at a plurality of phases of the pattern based on the received light signal. Sectioning image data is generated based on the plurality of image data.

  In the second observation mode, light generated by the light source device is irradiated to the object by the measurement light irradiation device as second measurement light having no pattern. At this time, the fluorescence emitted from the object is received by the light receiving unit, and a light reception signal is output. Based on the received light signal, image data indicating a normal observation image of the object is generated.

  The stage, the measuring light irradiation device, and the light receiving unit are accommodated in the first casing. In the first housing, the measurement space including the object on the stage and the light receiving unit is in a dark room state. In this case, light from the outside of the first housing does not enter the object and the light receiving unit in the first and second observation modes. Therefore, it is not necessary to install the microscope apparatus in the darkroom.

  Further, the light modulation element is accommodated in the second casing in the first casing. In the first observation mode, unnecessary light other than the first measurement light is generated when the first measurement light is generated by the light modulation element. Even in such a case, light other than the first measurement light generated by the light modulation element does not reach the measurement space from the light modulation element. Therefore, highly reliable fluorescence observation can be performed.

  Furthermore, according to the above configuration, the user can switch between the first observation mode and the second observation mode from the outside of the first housing by operating the operation device. In this case, parts replacement work and time are not required.

  As a result, it is possible to perform a plurality of types of highly reliable fluorescence observation easily and in a short time without using a darkroom.

  (2) The operating device is operated by a user to command the first and second observation modes, respectively, and the control unit is generated by the pattern generation unit in response to the first observation mode command from the operating device. By controlling the light modulation element based on the formed pattern, the measurement light irradiating device irradiates the object with the first measurement light, and the sectioning image based on the plurality of image data generated at the plurality of phases of the pattern The image data generation unit is controlled so as to generate data, and the object is irradiated with the second measurement light from the measurement light irradiation device in response to the second observation mode command by the operating device, and the normal of the object The image data generation unit may be controlled to generate image data indicating the observation image.

  In this case, the user can command the first observation mode from the outside of the first housing by operating the operating device. In the first housing, in response to the first observation mode command, the first measurement light is automatically irradiated onto the object, and sectioning image data is generated. Similarly, the user can command the second observation mode from the outside of the first housing by operating the operation device. In the first casing, in response to the second observation mode command, the second measurement light is automatically irradiated onto the object, and image data indicating a normal observation image is generated. Thus, the user can easily obtain desired image data without removing the first housing.

  (3) The operation device is operated by the user to further switch between the first or second observation mode and the third observation mode for observing the object using the third measurement light, and the microscope device is A third measurement light source that generates third measurement light in a third observation mode; and a third measurement light optical system that irradiates an object with the third measurement light generated by the third light source; The light receiving unit receives the third measurement light transmitted through the object, outputs a light reception signal indicating the amount of received light, and the control unit displays a transmission observation image of the object in the third observation mode. The image data generation unit may be controlled to generate the image data shown.

  In this case, the user can switch between the first or second observation mode and the third observation mode from the outside of the first housing by operating the operation device. Therefore, parts replacement work and time are not required.

  In the third observation mode, image data indicating a transmission observation image of the object is generated based on the third measurement light transmitted through the object. Therefore, the object observation method is diversified.

  (4) The microscope apparatus further includes an imaging visual field position moving unit that moves the imaging visual field position of the object by moving the stage in a direction that intersects the optical axis of the light receiving unit. The operating device is operated by a user to command the operation of the imaging visual field position moving unit, and the control unit controls the imaging visual field position moving unit in response to the command from the operating device. Also good.

  In this case, the imaging visual field position moving unit moves the stage in a direction intersecting the optical axis of the light receiving unit. Thereby, the imaging visual field position of the object is moved. The user can command the operation of the imaging visual field position moving unit from the outside of the first housing by operating the operating device. The imaging visual field position moving unit is controlled in response to a command from the operating device. Thereby, the user can adjust the imaging visual field position of the object easily and in a short time without removing the first housing.

  (5) The measurement light irradiation device has a condensing optical system having an observation axis, and the condensing optical system is generated as the first measurement light generated by the light modulation element and the second measurement light by the light source device. The microscope apparatus includes an objective lens configured to be able to collect the collected light on the object, and the microscope apparatus moves the stage and the objective lens relative to each other by moving at least one of the stage and the objective lens along the observation axis. A focal position moving unit that moves the focal position of the objective lens with respect to the object by changing a specific distance, the light receiving optical system and the focal position moving unit are housed in the first darkroom casing, It is operated by a user to command the operation of the focal position moving unit, and the control unit may control the focal position moving unit in response to a command from the operating device.

  In this case, the first measurement light generated by the light modulation element and the light generated as the second measurement light by the light source device can be condensed on the object by the objective lens of the condensing optical system. At least one of the stage and the objective lens is moved along the observation axis by the focal position moving unit. Thereby, the relative distance between the object and the objective lens changes. As a result, the focal position of the objective lens with respect to the object changes.

  The user can command the operation of the focal position moving unit from the outside of the first housing by operating the operating device. The focal position moving unit is controlled in response to a command from the operating device. Accordingly, the user can easily and quickly adjust the focal position of the objective lens with respect to the object without removing the first housing.

  (6) The condensing optical system has a plurality of objective lenses, and the first measurement generated by the light modulation element is performed by selectively positioning one of the objective lenses on the observation axis. Including a first switching device for condensing the light and the light generated as the second measurement light by the light source device onto the object through one objective lens, and the operation device selects one of the plurality of objective lenses In response to the selection of the one objective lens by the operation device, the controller operates the first switching device so that the selected one objective lens is positioned on the observation axis. You may control.

  In this case, the user can select one of the plurality of objective lenses from the outside of the first housing by operating the operating device. In response to the selection by the operating device, the first switching device is controlled so that the selected one objective lens is positioned on the observation. Thereby, the user can easily and quickly switch the objective lens without removing the first housing.

  (7) The measurement light irradiation device further includes a filter optical system, and the filter optical system includes the first measurement light generated by the light modulation element and the light generated as the second measurement light by the light source device. A light modulation element including a plurality of filter members configured to transmit light in a partial wavelength region on the observation axis, and selectively positioning one of the plurality of filter members on the observation axis Including a second switching device for irradiating the object with the first measurement light generated by the light source device or the light generated as the second measurement light by the light source device, and the operation device includes a plurality of filter members. In response to the selection of the one filter member by the operating device, the control unit positions the selected one filter member on the observation axis. May control the second switching device so that.

  In this case, light in a part of the wavelength region of the first measurement light generated by the light modulation element or the light generated as the second measurement light by the light source device passes through one of the plurality of filter members to the object. Irradiated. The user can select one of the plurality of filter members from the outside of the first housing by operating the operating device. In response to the selection by the operating device, the second switching device is controlled so that the selected one filter member is positioned on the observation. Thus, the user can easily and quickly switch the filter member without removing the first housing.

  (8) In the first and second observation modes, the control unit does not continuously change the measurement condition after the sectioning image data or the image data indicating the normal observation image of the object is generated. In addition, at least one of the light source device and the light modulation element may be controlled so that the object is not irradiated with the first and second measurement lights.

  In this case, it is possible to shorten the time during which the object is irradiated with the first and second measurement lights. Thereby, the fading of the fluorescent reagent applied to the object is suppressed.

  (9) The first housing has an opening for maintenance, and the microscope apparatus further includes a lid member that opens and closes the opening, and an open / close detector that detects an open / closed state of the lid member. Further, at least one of the light source device and the light modulation element may be controlled based on the detection of the open / close detector so that the first and second measurement lights do not reach the opening when the lid member is opened.

  In this case, during the maintenance of the microscope apparatus, the first and second measurement lights are prevented from being emitted from the inside of the first casing to the outside of the first casing through the opening. As a result, the first and second measurement lights are prevented from entering the eyes of the user.

  According to the present invention, it is possible to perform a plurality of types of highly reliable fluorescence observation easily and in a short time without using a darkroom.

1 is an external perspective view of a microscope apparatus according to an embodiment of the present invention. It is a figure for demonstrating the hardware constitutions of the microscope apparatus of FIG. It is a schematic diagram which shows the optical path in a measurement part and a measurement light supply part. It is a block diagram which shows the control system of the microscope apparatus of FIG. It is a block diagram which shows the structure of CPU. It is a schematic diagram showing fluorescence emitted from a measuring object when measuring light is irradiated to a measuring object. It is a figure which shows an example of the fluorescence observation image obtained by irradiating uniform measurement light to the predetermined observation range in a measurement object. It is a figure which shows an example of the sectioning observation method. It is an external appearance perspective view of a pattern provision part. It is an external appearance perspective view which shows the state of the measurement part at the time of replacement | exchange work of a measuring object, an objective lens, and a filter cube. It is a figure which shows the example of a display of a display part. It is a figure which shows the example of the observation image displayed on the image display area of FIG. It is a figure which shows the example of the observation image displayed on the image display area of FIG. It is a figure which shows the example of the observation image displayed on the image display area of FIG. It is a figure which shows the example of the observation image displayed on the image display area of FIG. It is a longitudinal cross-sectional view which shows the 1st structural example of the pattern generation part which concerns on other embodiment. It is a longitudinal cross-sectional view which shows the 2nd structural example of the pattern generation part which concerns on other embodiment. It is a longitudinal cross-sectional view which shows the 3rd structural example of the pattern generation part which concerns on other embodiment. It is a longitudinal cross-sectional view which shows the 4th structural example of the pattern generation part which concerns on other embodiment. It is a longitudinal cross-sectional view which shows the 5th structural example of the pattern generation part which concerns on other embodiment. It is a longitudinal cross-sectional view which shows the 6th structural example of the pattern generation part which concerns on other embodiment.

(1) Configuration of Microscope Device A microscope device according to an embodiment of the present invention will be described. In the microscope apparatus according to the present embodiment, it is possible to perform fluorescence observation of the measurement object. Further, in the microscope apparatus, bright field observation, dark field observation, phase difference observation, differential interference observation, declination observation, and polarization observation using light transmitted through the measurement object can be performed. In the following description, bright field observation, dark field observation, phase difference observation, differential interference observation, declination observation, and polarization observation using light transmitted through a measurement object are collectively referred to as transmission observation.

  FIG. 1 is an external perspective view of a microscope apparatus according to an embodiment of the present invention. As shown in FIG. 1, the microscope apparatus 500 of this example mainly includes a measurement unit 100, a PC (personal computer) 200, a measurement light supply unit 300, and a display unit 400. Measuring unit 100 and PC 200 are connected by wiring WR1. The measurement unit 100 and the measurement light supply unit 300 are connected by the wiring WR2 and the light guide member 330. In the example of FIG. 1, a single notebook personal computer is used as a configuration in which the PC 200 and the display unit 400 are integrated. As the wiring WR1, a LAN (local area network) cable, a USB (universal serial bus) cable, or the like is used. As the wiring WR2, a cable in which a power supply line and a signal line are integrated is used.

  FIG. 2 is a diagram for explaining a hardware configuration of the microscope apparatus 500 of FIG. In FIG. 2, longitudinal sections of the measurement unit 100 and the measurement light supply unit 300 are schematically shown.

  As shown in FIG. 2, the measurement light supply unit 300 includes a housing 301, a power supply device 310, a light projecting unit 320, and a heat exhaust device 340. The power supply device 310, the light projecting unit 320, and the heat exhaust device 340 are accommodated in the housing 301. The power supply device 310 supplies power to the light projecting unit 320 and also supplies power to the measuring unit 100 via the wiring WR2.

  An opening 309 for exhaust heat is formed in the housing 301 of the measurement light supply unit 300. Exhaust heat device 340 includes an exhaust heat fan and a motor that drives the exhaust heat fan, and exhausts heat generated by power supply device 310 and light projecting unit 320 to the outside of housing 301 through opening 309.

  The measuring unit 100 includes an external housing 101, a plurality of support columns 106, a base 107, a pattern applying unit 110, a light receiving unit 120, a transmitted light supply unit 130, a stage 140, a stage driving device 141, a filter unit 150, a lens unit 160, and a control. A substrate 170 is included.

  The external housing 101 includes a pattern applying unit 110, a light receiving unit 120, a transmitted light supply unit 130, a stage 140, a stage driving device 141, a filter unit 150, a lens unit 160, a control board 170, and driving circuits (not shown) for each component. Accommodate. The external housing 101 includes a front surface portion 101a, a back surface portion 101b, one side surface portion 101c (FIG. 1), another side surface portion 101d (FIG. 1), and an upper surface portion 101e. An upper surface lid 102 is attached to the upper surface portion 101e. A front cover 103 is attached to the center of the front part 101a. With the top cover 102 and the front cover 103 closed, the space including the stage 140 and the light receiving unit 120 is in a dark room state.

  In the external casing 101, a base frame 91, a stage frame 92, and an upper frame 93 are provided so as to be aligned vertically and parallel to a horizontal plane.

  The base frame 91, the stage frame 92, and the upper frame 93 support the pattern applying unit 110, the light receiving unit 120, the transmitted light supply unit 130, the stage 140, the stage driving device 141, the filter unit 150, the lens unit 160, and the control board 170. A part of the support is formed, and the support is supported by a plurality of support columns 106 and a base 107.

  The stage frame 92 is located above the base frame 91, and the upper frame 93 is located above the stage frame 92. The base frame 91 and the stage frame 92 are formed with openings for allowing light used for observing the measuring object S to pass therethrough.

  An opening is also formed at the center of the stage 140. The stage 140 is integrally attached to the upper surface of the stage frame 92 so that the openings of the stage frame 92 and the stage 140 overlap each other. Further, a stage driving device 141 is attached to the upper surface of the stage frame 92 together with the stage 140.

  On the stage 140, the measuring object S is placed using a petri dish or a preparation. A plane on the stage 140 on which the measurement object S is placed is called a placement surface. In this example, the placement surface is maintained parallel to the horizontal plane.

  In the following description, as indicated by arrows in FIG. 2, two directions orthogonal to each other on the placement surface are defined as an X direction and a Y direction, and a direction perpendicular to the placement surface is defined as a Z direction. The X direction corresponds to the horizontal direction of the measurement unit 100, the Y direction corresponds to the front-rear direction of the measurement unit 100, and the Z direction corresponds to the vertical direction of the measurement unit 100. The stage 140 is an XY stage and is configured to be movable in the X direction and the Y direction. The stage driving device 141 moves the stage 140 in the X direction and the Y direction in response to a signal given from the control board 170.

  In this example, the measuring object S is a biological specimen containing various proteins. When performing fluorescence observation, the measurement object S is coated with a fluorescent reagent that is fused to a specific protein. Fluorescent reagents include, for example, GFP (green fluorescent protein), Texas Red (Texas Red), and DAPI (diamidinophenylindole). GFP absorbs light near a wavelength of 490 nm and emits light near a wavelength of 510 nm. Texas Red absorbs light near a wavelength of 596 nm and emits light near a wavelength of 620 nm. DAPI absorbs light near a wavelength of 345 nm and emits light near a wavelength of 455 nm.

  The pattern applying unit 110, the filter unit 150, and the lens unit 160 are attached to the upper surface of the base frame 91, and the light receiving unit 120 is provided below the base frame 91.

  On the base frame 91, the filter unit 150 is disposed at a position in front of the pattern applying unit 110. A transmitted light supply unit 130 is attached to the upper surface of the upper frame 93.

  FIG. 3 is a schematic diagram illustrating optical paths in the measurement unit 100 and the measurement light supply unit 300. In FIG. 3, the details of each component of the measurement unit 100 and the measurement light supply unit 300 are shown along with the optical path. In FIG. 3, as in FIG. 2, the X direction, the Y direction, and the Z direction are indicated by arrows.

  As shown in FIG. 3, the light projecting unit 320 of the measurement light supply unit 300 includes a measurement light source 321, a light reduction mechanism 322, a light shielding mechanism 323, and an optical connector 324. One end of the light guide member 330 is connected to the optical connector 324. In this example, the light guide member 330 is a liquid light guide. The light guide member 330 may be, for example, a glass fiber or a quartz fiber.

  The measurement light source 321 is, for example, a metal halide lamp. The measurement light source 321 may be another light source such as a mercury lamp, a xenon lamp, or a white LED (light emitting diode). The measurement light source 321 is used as a light source for fluorescence observation of the measurement object S. Hereinafter, the light emitted from the measurement light source 321 is referred to as measurement light.

  The light shielding mechanism 323 is, for example, a mechanical shutter. The light shielding mechanism 323 is disposed so as to be positioned on the optical path of the measurement light emitted from the measurement light source 321. When the light shielding mechanism 323 is in the light guide state, the measurement light passes through the light shielding mechanism 323 and enters one end of the light guide member 330. On the other hand, when the light blocking mechanism 323 is in the light blocking state, the measurement light is blocked and does not enter one end of the light guide member 330. The light blocking mechanism 323 may include a light modulation element that can switch between passing and blocking of the measurement light. For example, the light shielding mechanism 323 is in a light guiding state when fluorescence observation of the measurement object S is performed, and is in a light shielding state when transmission observation of the measurement object S is performed. Further, the light shielding mechanism 323 is in a light shielding state, for example, at the time of exchanging an objective lens and a filter cube described later and during processing of an observation image.

  The dimming mechanism 322 includes a plurality of ND (Neutral Density) filters having different transmittances. The dimming mechanism 322 is arranged so that any one of the plurality of ND filters is positioned on the optical path of the measurement light that has passed through the light shielding mechanism 323. By selectively switching the ND filter located on the optical path of the measurement light, the intensity of the measurement light passing through the dimming mechanism 322 can be adjusted. The dimming mechanism 322 may include a light modulation element capable of adjusting the intensity of the measurement light instead of the plurality of ND filters.

  The pattern applying unit 110 of the measuring unit 100 includes an inner housing 10, a light guide component support case 11, a light projecting lens 12, an optical connector 111, a light modulation element 112, and a plurality (two in this example) of mirrors 113.

  The inner housing 10 has a box shape. A light projecting lens 12 is provided on the front surface portion of the inner housing 10. A light modulation element 112 is attached to the inner side of the back surface portion of the inner housing 10. The light modulation element 112 is located on the optical axis of the light projecting lens 12. A light guide component support case 11 having a substantially cylindrical shape is attached to the lower surface portion of the inner housing 10. The lower end portion of the light guide component support case 11 is closed. Two mirrors 113 and an optical connector 111 are provided in the light guide component support case 11.

  The optical connector 111 is provided near the lower end of the light guide component support case 11. The other end of the light guide member 330 is connected to the optical connector 111 from the rear of the measurement unit 100 through the back surface 101b. The optical connector 111 supports the other end of the light guide member 330 so that measurement light guided by the light guide member 330 during fluorescence observation is emitted from the rear of the measurement unit 100 toward the front.

  One mirror 113 is provided in front of the optical connector 111 so as to be inclined with respect to the XY plane. Further, the other mirror 113 is provided above the one mirror 113 so as to be inclined with respect to the XY plane. In this state, the light modulation element 112 is positioned behind the two mirrors 113 and above the two mirrors 113. The measurement light emitted from the other end of the optical connector 111 is reflected by the one mirror 113 so as to be directed upward from below the measurement unit 100. Further, the measurement light reflected by one mirror 113 is reflected by the other mirror 113 so as to go from diagonally forward to diagonally upward and enter the light modulation element 112.

  The light modulation element 112 is, for example, a DMD (digital micromirror device). The light modulation element 112 may be an LCOS (Liquid Crystal on Silicon). The light incident on the light modulation element 112 is converted into a preset pattern and a preset intensity (brightness) by a pattern generation unit 212 described later, and is reflected so as to go from the rear to the front of the measurement unit 100. The The measurement light reflected by the light modulation element 112 enters the filter unit 150 through the light projecting lens 12.

  As described above, in this example, a reflection type device such as DMD or LCOS is used as the light modulation element 112. However, the light modulation element 112 may be a transmissive device such as an LCD (liquid crystal display) instead of the reflective device.

  The filter unit 150 includes a plurality of filter cubes 151, a filter turret 152, and a filter turret driver 153. Each filter cube 151 includes a frame 151a, an excitation filter 151b, a dichroic mirror 151c, and an absorption filter 151d. The frame 151a is a cubic member that supports the excitation filter 151b, the dichroic mirror 151c, and the absorption filter 151d.

  The plurality of filter cubes 151 respectively correspond to a plurality of types of fluorescent reagents used for fluorescence observation of the measurement object S. For example, when the above GFP, Texas Red, and DAPI are used as the fluorescent reagent, three types of filter cubes 151 corresponding to GFP, Texas Red, and DAPI are provided in the filter unit 150.

  In this case, the excitation filter 151b of the filter cube 151 corresponding to GFP passes light near the wavelength of 490 nm, and the absorption filter 151d passes light near the wavelength of 510 nm. The dichroic mirror 151c reflects light having a wavelength of about 490 nm and transmits light having a wavelength of about 510 nm.

  In addition, the excitation filter 151b of the filter cube 151 corresponding to Texas Red passes light near 596 nm, and the absorption filter 151d passes light near 620 nm. The dichroic mirror 151c reflects light having a wavelength of about 596 nm and transmits light having a wavelength of about 620 nm.

  Further, the excitation filter 151b of the filter cube 151 corresponding to DAPI passes light near 345 nm, and the absorption filter 151d passes light near 455 nm. The dichroic mirror 151c reflects light having a wavelength of about 345 nm and allows light having a wavelength of about 455 nm to pass therethrough.

  The filter turret 152 has a disk shape. The filter turret 152 is provided with four through holes at an angular interval of 90 ° with respect to the central axis. The filter turret 152 is rotatably supported around the central axis of the filter turret 152 on the base frame 91 of FIG. When the filter turret driving unit 153 drives the filter turret 152, the filter turret 152 rotates.

  Each of the plurality of filter cubes 151 is provided on the filter turret 152 such that the excitation filter 151b is directed in a direction opposite to the central axis of the filter turret 152 and overlaps one of the four through holes.

  In the present embodiment, three filter cubes 151 respectively corresponding to the three types of fluorescent reagents are attached on the filter turret 152 so as to overlap the three through holes of the filter turret 152.

  The user can select a desired filter cube 151 by rotating the filter turret 152 during fluorescence observation. The selected filter cube 151 is arranged on the observation axis OA parallel to the Z direction passing through the measurement object S on the stage 140 so that the measurement light is incident on the excitation filter 151b. The observation axis OA is the optical axis of the light receiving unit 120 extending in the Z direction. When the measurement light is incident on the excitation filter 151b, a part of the wavelength region of the measurement light passes through the excitation filter 151b. The light that has passed through the excitation filter 151b is reflected upward by the dichroic mirror 151c.

  As described above, in this embodiment, three filter cubes 151 are attached to the three through holes of the filter turret 152, and the filter cube 151 is not attached to the remaining one through hole. Therefore, it is possible to perform bright field observation without using the filter cube 151 by arranging a through hole to which the filter cube 151 is not attached on the observation axis OA (optical path of measurement light).

  The lens unit 160 includes a plurality of objective lenses 161, a lens turret 162, a focal length adjustment mechanism 163, a lens support plate 163p, a lens turret drive unit 164, and a focal length adjustment drive unit 165. The plurality of objective lenses 161 have different magnifications.

  A focal length adjustment mechanism 163 is mounted on the base frame 91 of FIG. The focal length adjustment mechanism 163 supports the lens support plate 163p so as to be parallel to the XY plane and movable in the Z direction.

  A lens turret 162 and a lens turret driving unit 164 are provided on the lens support plate 163p. The lens turret 162 has a disk shape. The lens turret 162 is formed with six through holes at an angular interval of 60 ° with the central axis as a reference. The lens turret 162 is supported so as to be rotatable around the central axis of the lens turret 162. When the lens turret driving unit 164 drives the lens turret 162, the lens turret 162 rotates.

  In the present embodiment, six objective lenses 161 having different magnifications are mounted on the lens turret 162 so as to overlap the six through holes of the lens turret 162. In this state, the six objective lenses 161 are located below the stage 140 and above the filter unit 150.

  The user can select one objective lens 161 used for observation of the measuring object S by rotating the lens turret 162. The selected objective lens 161 is disposed on the observation axis OA.

  The focal length adjustment driving unit 165 drives the focal length adjustment mechanism 163 to move the lens support plate 163p in the Z direction. Thereby, the relative distance in the Z direction between the measuring object S on the stage 140 and the selected objective lens 161 is adjusted.

  During fluorescence observation of the measurement object S, the measurement light reflected by the dichroic mirror 151c of the filter cube 151 passes through the opening of the stage 140 while being condensed by the selected objective lens 161, and is irradiated on the measurement object S. The When the measurement object S is irradiated with measurement light, fluorescence is emitted from the fluorescent reagent applied to the measurement object S. The fluorescence emitted below the measuring object S passes through the selected objective lens 161, the filter cube 151 located below the objective lens 161, and the opening formed in the base frame 91, and enters the light receiving unit 120. Incident.

  The transmitted light supply unit 130 attached on the upper frame 93 of FIG. 2 includes a fixed unit 130A and a swinging unit 130B. The fixing unit 130A includes a transmission light source 131, a diaphragm adjustment unit 132, and a transmission optical system 133, and is fixed on the upper surface of the upper frame 93 (FIG. 2).

  The transmissive light source 131 is, for example, a white LED. The transmissive light source 131 may be another light source such as a halogen lamp. The transmission light source 131 is used as a light source for transmission observation of the measurement object S. Hereinafter, the light emitted from the transmissive light source 131 is referred to as transmitted light. The aperture adjustment unit 132 includes an aperture and a phase difference slit, and is used for adjusting the brightness of the transmitted light irradiated on the measuring object S and for performing phase difference observation using the transmitted light. The transmissive optical system 133 includes a relay lens and guides the transmitted light emitted from the transmissive light source 131 to the front of the fixed portion 130A. The relay lens is arranged such that a conjugate relationship is maintained between the diaphragm and phase difference slit included in the diaphragm adjustment unit 132 and the front focal position of the condenser lens 135.

  The swing part 130B includes a mirror 134, a condenser lens 135, and a swing mechanism (not shown). The swinging portion 130B is configured to be swingable about an axis parallel to the X direction in a state where the upper surface lid 102 of FIG. 2 is opened. As a result, the swinging unit 130B has a normal position where the condenser lens 135 faces the measurement object S on the stage 140 in the Z direction and a separation position where the condenser lens 135 does not face the measurement object S on the stage 140 in the Z direction. Move between.

  At the time of transmission observation of the measuring object S, the rocking part 130B is manually moved to the normal position by the user. In a state where the swinging part 130B is in the normal position, the transmitted light guided forward from the fixed part 130A is reflected downward by the mirror 134 and passes through the measuring object S on the stage 140 through the condenser lens 135.

  As described above, in the filter unit 150, the through hole of the filter turret 152 that is not provided with the filter cube 151 is selected during transmission observation. In this case, the transmitted light that has passed through the measuring object S enters the light receiving unit 120 through the objective lens 161 selected by the user, the through hole of the filter turret 152, and the opening formed in the base frame 91.

  The light receiving unit 120 includes a camera 121, a color filter 122, and an imaging lens 123. The camera 121 is, for example, a CCD (charge coupled device) camera including an image sensor. The image sensor is, for example, a monochrome CCD. The imaging device may be another imaging device such as a CMOS (complementary metal oxide semiconductor) image sensor.

  The fluorescence or transmitted light incident on the light receiving unit 120 is collected and imaged by the imaging lens 123 and then received by the camera 121. Thereby, the image of the measuring object S is obtained. From each pixel of the image sensor of the camera 121, an analog electric signal (hereinafter referred to as a light reception signal) corresponding to the amount of received light is output to the control board 170.

  Unlike a color CCD, a monochrome CCD does not need to be provided with pixels that receive red wavelength light, pixels that receive green wavelength light, and pixels that receive blue wavelength light. Therefore, the measurement resolution of the monochrome CCD is higher than the resolution of the color CCD. Further, unlike a color CCD, a monochrome CCD does not require a color filter for each pixel. Therefore, the sensitivity of the monochrome CCD is higher than that of the color CCD. For these reasons, the camera 121 in this example is provided with a monochrome CCD. On the other hand, when the color CCD has sufficient resolution and sensitivity, the image sensor may be a color CCD.

  On the control board 170, an A / D converter (analog / digital converter) and a FIFO (First In First Out) memory (not shown) are mounted. The light reception signal output from the camera 121 is sampled at a constant sampling period by the A / D converter and converted into a digital signal based on control by the PC 200. Digital signals output from the A / D converter are sequentially stored in the FIFO memory. The digital signals stored in the FIFO memory are sequentially transferred to the PC 200 as pixel data.

(2) Control System of Microscope Device FIG. 4 is a block diagram showing a control system of the microscope device 500 of FIG. In the measurement unit 100, the control board 170 performs operations of the pattern applying unit 110, the light receiving unit 120, the transmitted light supply unit 130, the stage driving device 141, the filter unit 150, and the lens unit 160 in response to a command given from the PC 200. Control. Further, the control board 170 transfers a digital signal based on the light reception signal output from the camera 121 (FIG. 3) to the PC 200 as described above. Further, the control board 170 controls the operations of the power supply device 310 and the light projecting unit 320 of the measurement light supply unit 300 in response to a command from the PC 200.

  The PC 200 includes a CPU (Central Processing Unit) 210, a ROM (Read Only Memory) 220, a RAM (Random Access Memory) 230, a storage device 240, and an operation unit 250. The operation unit 250 is configured to be operated by a user to give a command to the control board 170 of the measurement unit 100, and includes a keyboard and a pointing device. A mouse or a joystick is used as the pointing device.

  The display unit 400 is configured by, for example, an LCD panel or an organic EL (electroluminescence) panel. The ROM 220 stores a system program. The RAM 230 stores various data and functions as a work area for the CPU 210. The storage device 240 is composed of a hard disk or the like. The storage device 240 stores a control program for the microscope apparatus 500 and an image processing program. The storage device 240 is used for storing various data such as pixel data provided from the measurement unit 100.

  FIG. 5 is a block diagram showing the configuration of the CPU 210. As illustrated in FIG. 5, the CPU 210 includes an image data generation unit 211, a pattern generation unit 212, and a control unit 213. The image data generation unit 211 generates image data based on the pixel data provided from the measurement unit 100 by executing the control program and the image processing program. Image data is a set of a plurality of pixel data.

  The pattern generation unit 212 generates a pattern to be irradiated on the measurement object as the pattern of measurement light emitted from the light modulation element 112 in FIG. The control unit 213 irradiates the measurement object S with measurement light having a predetermined pattern by controlling the light modulation element 112 via the control board 170 in FIG. 2 based on the pattern generated by the pattern generation unit 212. While shifting the phase of the pattern.

  Further, the control unit 213 gives instructions to the control board 170 to control the operations of the light receiving unit 120, the transmitted light supply unit 130, the stage 140, the filter unit 150, the lens unit 160, and the light projecting unit 320 via the control board 170. Control. Further, the control unit 213 performs various processes on the generated image data using the RAM 230 and causes the display unit 400 to display an image based on the image data.

(3) Function of pattern imparting unit FIG. 6 is a schematic diagram illustrating fluorescence emitted from the measurement object S when the measurement object S is irradiated with measurement light. As shown in FIG. 6, when the observation target portion sp1 of the measurement target S is arranged at the focal point of the objective lens 161, the measurement light passing through the objective lens 161 is the observation target portion of the measurement target S as indicated by a dotted line. Condensed toward sp1. Thereby, when a fluorescent reagent is present in the observation target portion sp1 of the measurement object S, fluorescence is emitted from the observation target portion sp1. A part of the fluorescence enters the light receiving unit 120 (FIG. 3) through the objective lens 161 as indicated by a thick arrow.

  When the measurement light is focused on the observation target portion sp1 of the measurement target S by the objective lens 161, the fluorescent reagent existing at a position out of the focus of the objective lens 161 also receives the measurement light and emits fluorescence. . In the example of FIG. 6, fluorescence is also emitted from portions sp2 and sp3 other than the observation target portion sp1. When the portions sp2 and sp3 are located at positions away from the depth of field DF of the objective lens 161, the fluorescence emitted from the portions sp2 and sp3 enters the light receiving unit 120 as stray light.

  FIG. 7 is a diagram illustrating an example of a fluorescence observation image obtained by irradiating a predetermined observation range on the measurement object S with uniform measurement light. For the above reason, when the measurement light is uniformly irradiated to the predetermined observation range in the measurement object S, as shown in FIG. 7, the fluorescence emitted from the position outside the depth of field DF of the objective lens 161 is stray light. Is incident on the light receiving unit 120 to reduce the overall contrast of the fluorescence observation image.

  Therefore, in the measurement unit 100 of the present example, fluorescence observation using patterned measurement light is performed in order to obtain a fluorescence observation image with high contrast. In the following description, fluorescence observation using uniform measurement light is referred to as normal observation, and fluorescence observation using patterned measurement light is referred to as sectioning observation.

  In sectioning observation, the measurement object S is irradiated with measurement light having a predetermined pattern, and the spatial phase of the predetermined pattern is moved by a certain amount. The measurement light having a predetermined pattern has an intensity that periodically changes in one direction (for example, the Y direction) or two directions (for example, the X direction and the Y direction) on the XY plane, for example. Hereinafter, measurement light having a pattern is referred to as pattern measurement light.

  The pattern of the pattern measurement light and its phase are controlled by the light modulation element 112. Here, the portion of the pattern measurement light having an intensity equal to or higher than a predetermined value is called a bright portion, and the portion of the pattern measurement light having an intensity smaller than the predetermined value is called a dark portion.

  The pattern measurement light includes, for example, a plurality of linear bright portions that are parallel to one direction (for example, the X direction) and arranged at substantially equal intervals in another direction (for example, the Y direction) orthogonal to the one direction. Striped measurement light having a cross section including a plurality of linear dark portions between the portions can be used.

  FIG. 8 is a diagram illustrating an example of the sectioning observation method. In sectioning observation, as shown in FIGS. 8A to 8D, the bright part of the pattern measurement light (in this example, the striped measurement light) is irradiated at least once on all parts of the measurement object S. The phase of the pattern measurement light pattern is shifted by a certain amount. Further, every time the phase of the pattern is moved by a certain amount, the fluorescence emitted from the measuring object S is detected. Thereby, a plurality of image data of the measuring object S is generated.

  Hereinafter, the image data obtained when the measurement object S is irradiated with the pattern measurement light is referred to as pattern image data. An image based on the pattern image data is called a pattern image.

  In each pattern image data, pixel data corresponding to a bright portion of the pattern measurement light has a high value (luminance value), and pixel data corresponding to a dark portion of the pattern measurement light has a low value (luminance value). Therefore, as indicated by arrows in FIGS. 8A to 8D, in each pattern image, the pixel portion corresponding to the bright portion of the pattern measurement light is bright and the pixel portion corresponding to the dark portion of the pattern measurement light. Is dark.

  Here, each pattern image is affected by stray light. In order to obtain image data from which the influence of stray light has been removed, the following processing is executed.

  First, a component (hereinafter referred to as a focusing component) representing the degree of contrast is calculated from the plurality of pattern image data using the values of the plurality of pixel data for each pixel. Image data is generated by stitching together pixels having in-focus components. The image data generated in this way is called sectioning image data. An image based on the sectioning image data is called a sectioning image.

  In the pattern image data generated using the above-described striped measurement light, the focus component is, for example, the difference between the maximum value (maximum luminance value) and the minimum value (minimum luminance value) of the pixel data, or the pixel data The standard deviation of values.

  In this example, for each pixel, the difference between the pixel data of the pattern image data when the bright part and the dark part of the pattern measurement light are irradiated is calculated as a focusing component. Sectioning image data is generated by connecting the focus components for all the calculated pixels. For each pixel, the value of the pixel data of the pattern image data when the dark portion of the pattern measurement light is irradiated corresponds to the stray light component. Therefore, sectioning image data from which the influence of stray light is removed can be obtained. As a result, as shown in FIG. 8E, it is possible to obtain a fluorescence observation image (sectioning image) with high contrast from which the influence of stray light is removed.

  In the following description, for the sectioning image data and the sectioning image, image data obtained in normal observation is referred to as normal image data, and a fluorescence observation image based on the normal image data is referred to as a normal image.

  As the pattern measurement light, for example, sine wave measurement light having a cross section including a pattern that is parallel to the X direction and whose intensity changes in a sine wave shape in the Y direction can be used instead of the striped measurement light. In pattern image data generated using sinusoidal measurement light, the focus component is, for example, the amplitude (peak to peak) of pixel data. Further, as the pattern measurement light, instead of the striped measurement light, dot-shaped measurement light having a cross section including a plurality of dot-like bright portions arranged at substantially equal intervals in the X direction and the Y direction can be used. In the pattern image data generated using the dot-shaped measurement light, the focus component is, for example, the difference between the maximum value (maximum luminance value) and the minimum value (minimum luminance value) of the pixel data, or the value of the pixel data. Standard deviation.

(4) Structure of Internal Housing The internal housing 10 of the pattern applying unit 110 in FIG. 3 will be described. FIG. 9 is an external perspective view of the pattern imparting unit 110. FIG. 9 shows a state in which a part of the inner housing 10 is cut away.

  As shown in FIG. 9, the internal housing 10 includes a front surface portion 21, a back surface portion 22, one side surface portion 23, another side surface portion 24, an upper surface portion 25, and a lower surface portion 26. The front surface portion 21, the back surface portion 22, the one side surface portion 23, the other side surface portion 24, the upper surface portion 25, and the lower surface portion 26 are rectangular plate-like members, and are connected to each other.

  An emission opening 10 a is formed in the central portion of the front portion 21. A light projecting lens 12 is attached to the exit opening 10a. An incident opening 10 b is formed in the central portion of the lower surface portion 26. The light guide component support case 11 is attached to the lower surface portion 26 so as to close the incident opening 10b. In FIG. 9, the measurement light incident on the light modulation element 112 through the light guide member 330 in FIG. 3 is indicated by a thick arrow L0.

  At the time of sectioning observation, the light modulation element 112 reflects the measurement light guided from the light guide member 330 toward the emission opening 10a as indicated by a thick arrow L1 in FIG. Thereby, a bright portion of the pattern measurement light is generated. Further, the light modulation element 112 reflects the measurement light guided from the light guide member 330 toward a position away from the emission opening 10a as indicated by a thick arrow L2 in FIG. Thereby, a dark portion of the pattern measurement light is generated. Light reflected at a position deviating from the emission opening 10a becomes unnecessary light. Unnecessary light is reflected from the inner surface of the inner casing 10, thereby preventing the unnecessary light from being directly emitted from the emission opening 10 a to the outside of the inner casing 10.

  In this case, unnecessary light is prevented from leaking out of the internal housing 10. Thereby, unnecessary light does not reach the space including the stage 140 and the light receiving unit 120. Therefore, unnecessary light is prevented from entering the light receiving unit 120, and a reduction in contrast of the sectioning image due to stray light is prevented.

  Further, as described above, in the pattern applying unit 110, the exit opening 10 a and the entrance opening 10 b of the internal housing 10 are connected to the light projecting lens 12 and the light guide in a state where the light modulation element 112 is accommodated in the internal housing 10. They are respectively closed by the component support cases 11. Therefore, even if dust enters the outer casing 101, the dust is prevented from adhering to the light modulation element 112. Thereby, dust is prevented from appearing in the sectioning image and the normal image of the measuring object S.

  As a result, an image of the measuring object S having high reliability can be obtained. Furthermore, since it is not necessary to carry out dustproofing of the entire external housing 101, an increase in the size and cost of the measuring unit 100 is suppressed.

  As described above, as the light modulation element 112, a transmissive device such as an LCD may be used instead of a reflective device such as DMD or LCOS.

(5) Exchange Operation of Measurement Object, Objective Lens, and Filter Cube FIG. 10 is an external perspective view showing the state of the measurement unit 100 during the exchange operation of the measurement object S, the objective lens 161, and the filter cube 151. As shown in FIG. 10A, an upper opening OP1 is formed in the outer casing 101 of the measurement unit 100. An upper surface lid 102 capable of opening and closing the upper opening OP1 is attached to a part of the upper surface portion 101e of the external housing 101 via a hinge 102H (FIG. 2). When the measurement object S placed on the stage 140 is exchanged, the upper surface lid 102 is opened as shown by a thick arrow in FIG. Thereafter, as indicated by a thick arrow in FIG. 10B, the swinging part 130B of the transmitted light supply part 130 is manually swung from the normal position np1 to the separation position cp1. Thereby, the user can easily perform the exchange operation of the measuring object S from the front of the measuring unit 100 through the upper opening OP1.

  A swing part detector S1 is provided in the vicinity of the fixed part 130A. The swing part detector S1 is a magnetic sensor including, for example, a magnet and a Hall element. The swing part detector S1 detects whether or not the swing part 130B is at the normal position np1, and gives a detection result to the control board 170 (FIG. 2). When the swinging part 130B is not in the normal position np1, the control board 170 puts the light shielding mechanism 323 (FIG. 3) of the light projecting part 320 (FIG. 3) into a light shielding state and transmits the light source 131 (FIG. 3) from the power supply device 310. ) Stop supplying power to. Accordingly, it is possible to prevent the measurement light from being irradiated onto the stage 140 and the transmitted light from being emitted to the front of the measurement unit 100 from the fixed unit 130A.

  The control board 170 controls the light modulation element 112 of the pattern applying unit 110 instead of putting the light shielding mechanism 323 (FIG. 3) in a light shielding state when the swinging unit 130B is not in the normal position np1. The measurement light may be prevented from being irradiated toward the stage 140.

  The outer casing 101 is further formed with a front opening OP2. A front lid 103 capable of opening and closing the front opening OP2 is attached to a part of the front surface portion 101a of the external housing 101 via a hinge 103H (FIG. 2).

  When the objective lens 161 attached to the lens turret 162 in FIG. 3 is exchanged, the front cover 103 is manually opened as shown by a thick dotted line arrow in FIG. In addition, the front lid 103 is also manually opened when the filter cube 151 attached to the filter turret 152 of FIG. 3 is replaced. In these cases, the user can easily replace the objective lens 161 and the filter cube 151 from the front of the measurement unit 100 through the front opening OP2.

  In the vicinity of the front cover 103, a front cover detector S2 is provided. The front lid detector S2 is a magnetic sensor including, for example, a magnet and a Hall element, detects the open / closed state of the front lid 103, and gives the detection result to the control board 170. The control board 170 switches the light shielding mechanism 323 (FIG. 3) of the light projecting unit 320 (FIG. 3) from the light guiding state to the light shielding state when the front cover 103 is switched from the closed state to the open state based on the detection result. . Further, the control board 170 stops supplying power from the power supply device 310 to the transmissive light source 131 (FIG. 3). This prevents the measurement light from being emitted from the pattern applying unit 110 (FIG. 3) toward the filter unit 150 and the transmitted light from being emitted from the transmitted light supply unit 130 (FIG. 3) to the filter unit 150. The

  The control board 170 controls the light modulation element 112 of the pattern applying unit 110 instead of putting the light shielding mechanism 323 (FIG. 3) in the light shielding state when the front cover 103 is switched from the closed state to the open state. Thus, the measurement light may be prevented from being emitted toward the filter unit 150.

  In this way, the measurement light and the transmitted light are prevented from leaking out of the external housing 101. Therefore, the measurement light and the transmitted light are prevented from entering the eyes of the user.

  The top cover 102 and the front cover 103 are manually closed by the user. In this case, light is prevented from entering the measurement object S and the light receiving unit 120 on the stage 140 from the outside of the external housing 101. Thereby, as described above, the space including the measurement object S and the light receiving unit 120 placed on the placement surface of the stage 140 is in a dark room state. Therefore, only fluorescence emitted from the measuring object S can be incident on the light receiving unit 120 during fluorescence observation.

(6) Display contents of display unit and operation of measurement unit Transmitted light emitted from the transmitted light supply unit 130 of FIG. 2 passes through the measurement target S and is received by the light receiving unit 120, whereby the measurement target S Image data is generated. Hereinafter, the image data of the measuring object S generated using the transmitted light is referred to as transmitted image data. An image based on the transmission image data is called a transmission image.

  FIG. 11 is a diagram illustrating a display example of the display unit 400. As shown in FIG. 11, the display unit 400 is provided with an image display area 410 and a setting display area 420 arranged side by side. In the image display area 410, various images such as a pattern image, a sectioning image, a normal image, or a transmission image are displayed. In the image display area 410, a plurality of images can be superimposed and displayed.

  In the setting display area 420, for example, a filter selection column 430, a measurement condition setting column 440, a shooting analysis column 450, and a shooting type selection column 490 are displayed. In the setting display area 420, a plurality of tabs are displayed. The plurality of tabs include a focus position adjustment tab 470. The user can give various commands to the CPU 210 and the control board 170 of FIG. 4 by operating a GUI (Graphical User Interface) displayed on the display unit 400 using the operation unit 250 of the PC 200 of FIG. .

  In the filter selection field 430, a plurality of (three in this example) filter selection buttons 431, 432, 433, and 434 are displayed. The three filter selection buttons 431 to 433 correspond to the three filter cubes 151 provided in the filter turret 152, respectively, and the filter selection button 434 corresponds to the through hole of the filter turret 152 where the filter cube 151 is not provided.

  Any one of the filter selection buttons 431 to 434 is selected by the user. 3 is arranged so that the through hole of the filter cube 151 or the filter turret 152 corresponding to the selected filter selection button is located on the observation axis OA (the optical axis of the light receiving unit 120) in FIG. The drive unit 153 is driven. Thus, the user can switch the filter cube 151 easily and in a short time from the outside of the external housing 101 by operating the operation unit 250 of FIG.

  Further, the control board 170 has a pattern so that the measurement object S is irradiated with the measurement light and the measurement object S is not irradiated with the transmitted light when any of the filter selection buttons 431 to 433 is selected by the user. The application unit 110, the transmitted light supply unit 130, and the light projecting unit 320 are controlled. Furthermore, when the filter selection button 434 is selected by the user, the control board 170 is configured so that the measurement object S is irradiated with the transmitted light and the measurement object S is not irradiated with the measurement light. The light supply unit 130 and the light projecting unit 320 are controlled.

  The measurement condition setting field 440 displays a sectioning observation button 441, a normal observation button 442, and a plurality of setting buttons for setting measurement conditions. The plurality of setting buttons include, for example, a button for setting the shape of the pattern imparted to the measurement light and a button for setting the exposure time.

  With one of the three filter selection buttons 431 to 433 selected, the sectioning observation button 441 is operated by the user. In this case, the control board 170 controls the light modulation element 112 so as to give a pattern to the measurement light in response to a command from the PC 200. Thereby, the measurement object S is irradiated with the pattern measurement light. On the other hand, the normal observation button 442 is operated by the user while any one of the three filter selection buttons 431 to 433 is selected. In this case, the control board 170 controls the light modulation element 112 so as not to give a pattern to the measurement light in response to a command from the PC 200. Thereby, the measurement object S is irradiated with uniform measurement light. In this way, the user can easily switch between sectioning observation and normal observation from the outside of the external housing 101 in a short time by operating the operation unit 250 of FIG.

  With the focus position adjustment tab 470 selected from the plurality of tabs, an objective lens selection field 471, a focus position adjustment field 472, and a stage position adjustment field 473 are displayed in the setting display area 420.

  In the objective lens selection field 471, a plurality (six in this example) of objective lens selection buttons 471a, 471b, 471c, 471d, 471e, 471f are displayed. The six objective lens selection buttons 471a to 471e correspond to the six objective lenses 161, respectively.

  Any of the objective lens selection buttons 471a to 471f is selected by the user. The control board 170 drives the lens turret driving unit 164 so that the objective lenses 161 corresponding to the selected objective lens selection buttons 471a to 471f are positioned on the observation axis OA. As described above, the user can switch the objective lens 161 easily and in a short time from the outside of the external housing 101 by operating the operation unit 250 of FIG.

  In the focus position adjustment field 472, a focus position adjustment bar 472a, an initial distance button 472b, and an autofocus button 472c are displayed. The focal position adjustment bar 472a has a slider that can move in the vertical direction. The position of the slider of the focal position adjustment bar 472a corresponds to the distance between the measuring object S and the objective lens 161.

  By moving the slider of the focal position adjustment bar 472a, the distance between the measuring object S and the objective lens 161 is adjusted. The control board 170 controls the focal length adjustment driving unit 165 in FIG. 3 so that the distance between the measurement object S and the objective lens 161 is the distance adjusted by the slider.

  When the initial distance button 472b is operated, the control board 170 controls the focal length adjustment driving unit 165 so that the distance between the measurement object S and the objective lens 161 becomes a distance set in advance as an initial condition. . When the autofocus button 472c is operated, the control board 170 controls the focal length adjustment drive unit 165 so that the objective lens 161 is focused on the central portion of the measurement object S.

  Thus, the user can adjust the distance between the measuring object S and the objective lens 161 from the outside of the external housing 101 easily and in a short time by operating the operation unit 250 of FIG. .

  In the stage position adjustment field 473, stage movement buttons 473a, 473b, 473c, 473d and an initial position button 473e are displayed. When the stage movement buttons 473a and 473b are operated, the control board 170 controls the stage driving unit so that the stage 140 moves in one direction and the opposite direction in the X direction.

  When the stage moving buttons 473c and 473d are operated, the control board 170 controls the stage driving device 141 of FIG. 4 so that the stage 140 moves in one direction and the opposite direction in the Y direction. When the initial position button 473e is operated, the control board 170 controls the stage driving device 141 so that the position of the measuring object S moves to a position set in advance as an initial condition.

  As described above, the user operates the operation unit 250 in FIG. 4 to easily and quickly move the measurement object S on the stage 140 from the outside of the external housing 101 in the X direction and the Y direction. The range can be adjusted.

  In the shooting analysis field 450, a single shooting button 451, a multi shooting button 452, and an analysis button 453 are displayed. A single shooting button 451 is operated by the user. In this case, the control board 170 irradiates the measurement object S with the measurement light passing through the currently selected filter cube 151 or the transmission light passing through the through-hole of the filter turret 152, and generates a light reception signal output from the camera 121. Based pixel data is transferred to the PC 200. Thereby, in the PC 200, image data based on a plurality of pixel data is generated, and the generated image data is stored in the storage device 240. An image based on the generated image data is displayed in the image display area 410.

  On the other hand, the multi shooting button 452 is operated by the user. In this case, the control board 170 irradiates the measurement object S with the measurement light or the transmitted light while switching the filter cube 151 by controlling the filter turret driving unit 153. Further, every time the filter cube 151 is switched, the control board 170 transfers pixel data based on the light reception signal output from the camera 121 to the PC 200.

  Thereby, in the PC 200, image data corresponding to each of the plurality of filter cubes 151 and image data obtained by transmission observation are generated, and the generated plurality of image data are stored in the storage device 240. Further, by superimposing a plurality of generated image data, an image in which a plurality of types of fluorescence observation images and transmission observation images are superimposed is displayed in the image display area 410.

  When operating the multi-photograph button 452, a plurality of observation channels are set. The user designates an observation method in each observation channel using an operation button (not shown). For example, when four observation channels are set, the user sets sectioning observation using GFP as the first observation channel. The user also sets sectioning observation using Texas Red for the second observation channel. Further, the user sets normal observation using DAPI and phase difference observation by transmitted light to the third and fourth observation channels, respectively.

  In this case, by switching the filter cube 151, sectioning observation by GFP, sectioning observation by Texas Red, normal observation by DAPI, and phase difference observation by transmitted light are automatically switched in this order. In addition, the generated two sectioning image data, normal image data, and transmission image data are superimposed, and the two sectioning images, the normal image, and the transmission image are superimposed and displayed on the image display area 410.

  The analysis button 453 is operated by the user. In this case, analysis processing of image data stored in the storage device 240 of FIG. 4 is executed in the PC 200, for example. At this time, the control board 170 switches the light shielding mechanism 323 (FIG. 3) of the light projecting unit 320 (FIG. 3) from the light guiding state to the light shielding state. This prevents the measurement light from being emitted from the pattern applying unit 110 (FIG. 3) toward the filter unit 150. Therefore, it is possible to reduce unnecessary discoloration of the fluorescent reagent due to the measurement object S being continuously irradiated with the measurement light during the analysis of the image data.

  Note that the control board 170 performs measurement from the pattern applying unit 110 to the filter unit 150 instead of switching the light shielding mechanism 323 (FIG. 3) of the light projecting unit 320 (FIG. 2) from the light guiding state to the light shielding state when analyzing the image data. The light modulation element 112 may be controlled so that light is not emitted. Alternatively, the control board 170 may temporarily shut off the power source of the measurement light source 321.

  In the shooting type selection field 490, a Z stack shooting button 491, a linked shooting button 492, and an omnifocal shooting button 493 are displayed.

  The Z stack shooting button 491 is operated by the user. In this case, the control board 170 is based on the output of the light receiving unit 120 while changing the distance between the selected objective lens 161 and the measuring object S, for example, by controlling the focal length adjustment driving unit 165 of FIG. Transfer the pixel data to the PC 200. Thereby, in the PC 200, image data is generated at each position of the focal point of the objective lens 161. The plurality of generated image data is stored, for example, in the storage device 240 of FIG.

  In this way, when the measurement object S has a three-dimensional structure, the user can observe a plurality of sectioning images that are observed in a state where the focus of the objective lens 161 is positioned at a plurality of portions of the measurement object S. A normal image or a transmission image can be obtained.

  The linked shooting button 492 is operated by the user. In this case, the control board 170 moves the observation range of the measuring object S by the light receiving unit 120 in the X direction or the Y direction, for example, by controlling the stage driving device 141 in FIG. Further, every time the observation range of the measuring object S moves by a predetermined amount, pixel data based on the output of the light receiving unit 120 is transferred to the PC 200. Thereby, in the PC 200, a plurality of image data corresponding to a plurality of different observation ranges are generated. The plurality of generated image data are connected to each other. The connected image data is stored in the storage device 240 of FIG. Accordingly, the user can obtain a high-magnification fluorescence observation image or transmission image over a wide range.

  The omnifocal shooting button 493 is operated by the user. In this case, the control board 170 changes the pixel data based on the output of the light receiving unit 120 while changing the distance between the selected objective lens 161 and the measuring object S, as in the case where the Z stack photographing button 491 is operated. Are transferred to the PC 200. Thereby, in the PC 200, image data is generated at each position of the focal point of the objective lens 161.

  When the measuring object S has a three-dimensional structure, the objective lens 161 is not focused on the other part of the measuring object S even if the objective lens 161 is focused on a part of the measuring object S. . Accordingly, a part of the pixel data of the image data at a certain focal position is obtained in a state where the measurement object S is in focus, and the other part of the pixel data is in focus on the measurement object S. Obtained without. Hereinafter, pixel data obtained in a state where a part of the measuring object S is in focus is referred to as focused pixel data.

  Out of a plurality of image data obtained at a plurality of focal positions, the focused pixel data is synthesized, thereby generating image data obtained in a state where the entire measuring object S is in focus. Hereinafter, the image data obtained in a state where the entire measurement object S is in focus is referred to as omnifocal image data. An image based on the omnifocal image data is referred to as an omnifocal image. The generated omnifocal image data is stored in the storage device 240 of FIG.

  As described above, when the measurement object S has a three-dimensional structure, the user can obtain an omnifocal image in which the objective lens 161 is focused on a plurality of portions of the measurement object S.

(7) Example of observation image FIGS. 12-15 is a figure which shows the example of the observation image displayed on the image display area 410 of FIG. FIG. 12A shows an example of a sectioning image when the measurement object S is irradiated with measurement light having an absorption wavelength of GFP, and FIG. 12B shows measurement light having an absorption wavelength of Texas Red. An example of a sectioning image when the object S is irradiated is shown. FIG. 13 (a) shows an example of a normal image when the measurement object S is irradiated with measurement light having a DAPI absorption wavelength, and FIG. 13 (b) shows a phase difference observation using transmitted light. An example of a transmitted image is shown.

  As described above, when the multi shooting button 452 in FIG. 11 is operated, for example, two or more of the images in FIGS. 12A and 12B and FIGS. 13A and 13B are superimposed. The displayed image is displayed in the image display area 410.

  In the example of FIG. 14A, the sectioning images of FIGS. 12A and 12B are superimposed and displayed. In the example of FIG. 14B, the transmission image of FIG. 13B is further superimposed on the image of FIG. In the example of FIG. 15, the normal image of FIG. 13A is further superimposed on the image of FIG.

  When the measurement object S is a biological specimen, it is effective to superimpose a sectioning image and a transmission image (for example, an image obtained by observing the phase difference) when observing the shape of the cell of the measurement object S. is there. Thereby, the user can easily recognize the part of the measuring object S where the fluorescence is generated only when the light having a specific wavelength is irradiated by the composition of the protein. As a result, the nucleus, cell membrane or DNA (deoxyribonucleic acid) in the cell can be easily identified.

(8) Example of observation procedure of measurement object The user observes the measurement object S by the following procedure, for example. The user first performs transmission observation of the measuring object S in a state where the measuring object S to which a plurality of fluorescent reagents are applied is placed on the stage 140. Accordingly, the user confirms the shape of the measurement object S and sets various measurement conditions for performing fluorescence observation. The measurement conditions include, for example, the observation range, the filter cube 151 to be used, the objective lens 161 to be used, the pattern of measurement light to be irradiated on the measurement object S, the brightness of the measurement light, the exposure time, and the like.

  Thereafter, the user performs sectioning observation or normal observation. When the sectioning observation or the normal observation is started, the light shielding mechanism 323 in FIG. 3 is maintained in the light guide state, and the measurement object S is irradiated with the measurement light.

  When the measurement conditions are not changed, it is not necessary to irradiate the measurement object S with the measurement light for a long time. Therefore, the control board 170 controls the light modulation element 112 of the pattern applying unit 110 when the measurement conditions are not changed continuously for a predetermined period after the sectioning image data or the normal image data is generated. The measurement light irradiation to the object S is blocked. Thereby, unnecessary fading of the fluorescent reagent due to the measurement object S being continuously irradiated with the measurement light can be reduced.

  Thereafter, the user analyzes sectioning image data or normal image data obtained by sectioning observation or normal observation. When the analysis of the sectioning image data or the normal image data is started, the light shielding mechanism 323 in FIG. 3 is switched from the light guiding state to the light shielding state, and the incidence of the measurement light to the light modulation element 112 is stopped.

  As described above, the light shielding mechanism 323 in FIG. 3 is maintained in a light shielding state during analysis of sectioning image data or normal image data, or during transmission observation. Thereby, the deterioration of the light modulation element 112 due to the incidence of the measurement light is suppressed, and the life of the light modulation element 112 is extended.

(9) Other Functions In the microscope apparatus 500 according to the present embodiment, the following operation may be automatically performed when the user operates the operation unit 250.

  (9-a) For example, normal observation of the measuring object S is performed, and a normal image is displayed on the image display area 410 of FIG. In this state, the user designates an arbitrary part in the normal image using the operation unit 250. Accordingly, the control board 170 may control the stage driving device 141 of FIG. 4 so that the designated portion moves on the observation axis OA in response to a command from the operation unit 250. Further, the control board 170 may control the focal length adjustment driving unit 165 of FIG. 3 so that the objective lens 161 is focused on a designated portion.

  Further, the control board 170 generates sectioning image data in a state where the portion designated by the user is located on the observation axis OA and the objective lens 161 is focused on the designated portion, and the sectioning image data is generated. The based sectioning image may be displayed on the image display area 410.

  (9-b) For example, when the lens unit 160 includes a plurality of objective lenses 161 having different magnifications, normal observation of the measuring object S is performed using one objective lens 161, and the image display area of FIG. A normal image is displayed on 410.

  In this state, the user designates an arbitrary part in the normal image using the operation unit 250. Thereby, the control board 170 controls the stage driving device 141 in FIG. 4 so that the specified portion moves on the observation axis OA, and the lens turret driving unit in FIG. 3 so that the objective lens 161 is switched. 164 may be controlled. When the objective lens 161 is switched, for example, another objective lens 161 having a magnification higher than that of the one objective lens 161 is positioned on the observation axis OA.

  Further, the control board 170 generates sectioning image data in a state where the portion designated by the user is positioned on the observation axis OA and the other objective lens 161 is positioned on the observation axis OA, and the sectioning image is generated. A sectioning image based on the data may be displayed on the image display area 410.

  (9-c) For example, when the omnifocal photographing button 493 of FIG. 11 is operated by the user during transmission observation, omnifocal image data based on the transmitted light is generated. Thereby, an omnifocal image based on the generated in-focus pixel data is displayed on the image display area 410 in FIG.

  In this state, the user designates an arbitrary part in the omnifocal image obtained by transmission observation by the operation unit 250. Accordingly, the control board 170 may control the focal length adjustment driving unit 165 in FIG. 3 so that the objective lens 161 is focused on a portion designated by the user.

  Further, the control board 170 generates sectioning image data in a state where the objective lens 161 is focused on a portion designated by the user, and displays a sectioning image based on the sectioning image data on the image display area 410. May be.

  (9-d) For example, normal observation of the measuring object S is performed, and a normal image is displayed on the image display area 410 of FIG. In this state, the user designates an arbitrary area in the normal image using the operation unit 250. In this case, the control unit 213 in FIG. 5 may set the designated region as the ROI (region of interest). The control board 170 may control the light modulation element 112 so that the measurement light is irradiated only on the portion of the measurement object S corresponding to the ROI and the measurement light is not irradiated on the other portions. Thereby, pattern image data or normal image data can be generated at high speed.

(10) Effect In the microscope apparatus 500 described above, the light modulation element 112 is controlled based on the pattern generated by the pattern generation unit 212 during sectioning observation. Thereby, pattern measurement light is generated by the light modulation element 112, and the generated pattern measurement light is irradiated onto the measurement object S while the spatial phase is sequentially moved by a predetermined amount. At this time, the fluorescence emitted from the measuring object S is received by the light receiving unit 120 and a light reception signal is output. A plurality of pattern image data is generated based on the received light signal. Sectioning image data is generated based on the plurality of pattern image data.

  During normal observation, the measurement object S is irradiated with uniform measurement light having no pattern. At this time, the fluorescence emitted from the measuring object S is received by the light receiving unit 120 and a light reception signal is output. Normal image data is generated based on the received light signal.

  Inside the outer casing 101, the space including the stage 140 and the light receiving unit 120 is in a dark room state with the top cover 102 and the front cover 103 closed. In this case, the light from the outside of the external housing 101 does not enter the measurement object S and the light receiving unit 120 during sectioning observation and normal observation. Therefore, it is not necessary to install the microscope apparatus 500 in the darkroom.

  In addition, the light modulation element 112 is accommodated in the internal housing 10 in the external housing 101. During sectioning observation, unnecessary light other than the pattern measurement light is generated when the pattern measurement light is generated by the light modulation element 112. Even in such a case, unnecessary light other than the pattern measurement light generated by the light modulation element 112 is not directly emitted from the emission opening 10 a of the internal housing 10 to the outside of the internal housing 10. Thereby, unnecessary light does not reach the measuring object S and the light receiving unit 120. Therefore, highly reliable fluorescence observation can be performed.

  Furthermore, according to the above configuration, the user operates the sectioning observation button 441 and the normal observation button 442 in FIG. 11 using the operation unit 250, so that the observation method by the measurement unit 100 can be performed from the outside of the external housing 101. It is possible to switch between sectioning observation and normal observation. In this case, parts replacement work and time are not required.

  As a result, it is possible to perform a plurality of types of highly reliable fluorescence observation easily and in a short time without using a darkroom.

  In the microscope apparatus 500 described above, the transmitted light is irradiated onto the measurement object S by the transmitted light supply unit 130 during transmission observation. At this time, transmitted light that passes through the measuring object S is received by the light receiving unit 120, and a light reception signal is output. Transmission image data is generated based on the received light signal.

  The user can switch the observation method by the measurement unit 100 from the outside of the external housing 101 to sectioning observation or normal observation and transmission observation by operating the filter selection button 434 in FIG. 11 using the operation unit 250. . Even in this case, the part replacement work and the replacement time are not required. In this case, the method for observing the measuring object S is diversified.

(11) Other Embodiments (11-a) In the above embodiment, the light modulation element 112 is provided in the inner casing 10 of the pattern applying unit 110, and the emission opening 10 a formed in the inner casing 10. A projection lens 12 is attached to the projector. A light guide component support case 11 is attached to the incident opening 10 b formed in the inner housing 10. Two mirrors 113 are provided in the light guide component support case 11. In addition to this, in the inner housing 10, other optical members may be provided in addition to the light modulation element 112. In addition to the two mirrors 113 or in place of the two mirrors 113, another optical member may be provided in the light guide component support case 11.

  FIG. 16 is a longitudinal cross-sectional view showing a first configuration example of a pattern applying unit 110 according to another embodiment. In the example of FIG. 16, an optical member 114 is provided in the inner housing 10 in addition to the light modulation element 112 of FIG. 3. The optical member 114 includes, for example, at least one of a collector lens, a relay lens, a diaphragm, a mirror, a light modulation element for adjusting the intensity of measurement light, and a light shielding filter. In the example of FIG. 16, an optical member 115 is provided in the light guide component support case 11 instead of the mirror 113 of FIG. The optical member 115 includes, for example, at least one of a collector lens, a relay lens, a diaphragm, a light modulation element for adjusting the intensity of measurement light, and a light shielding filter.

  Also in this example, since the light modulation element 112 is provided in the inner housing 10, the same effect as that in the above embodiment can be obtained.

  (11-b) In the above embodiment, the light projecting unit 320 including the measurement light source 321 is provided separately from the measurement unit 100. Not limited to this, the measurement light source 321 may be provided in the internal housing 10 of the pattern applying unit 110. FIG. 17 is a longitudinal sectional view showing a second configuration example of the pattern imparting unit 110 according to another embodiment.

  In the example of FIG. 17, optical members 114 and 115 are provided in the inner housing 10 together with the light modulation element 112 of FIG. 3. The optical members 114 and 115 of this example include at least one of, for example, a collector lens, a relay lens, a diaphragm, a mirror, a light modulation element for adjusting the intensity of measurement light, and a light shielding filter.

  Also in this example, since the light modulation element 112 is provided in the inner housing 10, the same effect as that in the above embodiment can be obtained. Further, in this example, the measurement light source 321 is accommodated in the internal housing 10 so that light generated from the measurement light source 321 does not directly enter the light projecting lens 12. Therefore, unnecessary light generated from the measurement light source 321 is prevented from entering the measurement object S and the light receiving unit 120 during sectioning observation and normal observation. As a result, it is possible to perform multiple types of fluorescence observation with high reliability.

  (11-c) In the above embodiment, the light projecting unit 320 including the measurement light source 321 is provided separately from the measurement unit 100. Not limited to this, the measurement light source 321 may be provided in the light guide component support case 11 of the pattern applying unit 110. FIG. 18 is a longitudinal sectional view showing a third configuration example of the pattern imparting unit 110 according to another embodiment.

  In the example of FIG. 18, an optical member 114 is provided in the internal housing 10 together with the light modulation element 112 of FIG. 3. In addition, an optical member 115 and a measurement light source 321 are provided in the light guide component support case 11 instead of the mirror 113 of FIG. The optical members 114 and 115 of this example include at least one of, for example, a collector lens, a relay lens, a diaphragm, a mirror, a light modulation element for adjusting the intensity of measurement light, and a light shielding filter.

  Also in this example, since the light modulation element 112 is provided in the inner housing 10, the same effect as that in the above embodiment can be obtained. Further, in this example, the measurement light source 321 is accommodated in the light guide component support case 11 so that light generated from the measurement light source 321 does not directly enter the light projecting lens 12. Therefore, unnecessary light generated from the measurement light source 321 is prevented from entering the measurement object S and the light receiving unit 120 during sectioning observation and normal observation. As a result, it is possible to perform multiple types of fluorescence observation with high reliability.

  (11-d) In the above embodiment, in the pattern applying unit 110, the pattern measurement light used during sectioning observation and the uniform measurement light used during normal observation pass through the common optical path to the filter unit 150. Emitted. Not limited to this, in the pattern imparting unit 110, the optical path of the pattern measurement light and the optical path of the uniform measurement light may be provided separately. FIG. 19 is a longitudinal sectional view showing a fourth configuration example of the pattern imparting unit 110 according to another embodiment.

  In the example of FIG. 19, mirrors 116 a, 116 b, 116 c, mirror driving units 117 a, 117 b, and optical members 118 a, 118 b, 118 c, 118 d are provided in the internal housing 10 together with the light modulation element 112 of FIG. Further, a measurement light source 321 is provided in the inner housing 10.

  The mirrors 116a, 116b, and 116c are total reflection mirrors. The mirror driving units 117a and 117b move the mirrors 116a and 116b, respectively, as indicated by arrows m in FIG. 19, thereby changing the optical path of the measurement light generated from the measurement light source 321 to the first optical path LL1 and the second optical path LL1. Switch to the optical path LL2. Each of the optical members 118a to 118d includes at least one of, for example, a collector lens, a relay lens, a diaphragm, a mirror, a light modulation element for adjusting the intensity of measurement light, and a light shielding filter.

  In this example, the first optical path LL1 is formed by the light modulation element 112, the mirror 116b, and the optical members 118a and 118b. In the first optical path LL1, measurement light generated from the measurement light source 321 first enters the light modulation element 112 through the optical member 118a. The light modulation element 112 generates pattern measurement light by reflecting the measurement light. The generated pattern measurement light travels toward the mirror 116b through the optical member 118b. The pattern measurement light is reflected by the mirror 116 b and travels toward the light projecting lens 12. The first optical path LL1 is used during sectioning observation.

  On the other hand, in this example, the second optical path LL2 is formed by the mirrors 116a and 116c and the optical members 118c and 118d. In the second optical path LL2, the measurement light generated from the measurement light source 321 is first reflected by the mirror 116a. The reflected measurement light is directed to the mirror 116c through the optical member 118c. The measurement light is reflected again by the mirror 116c and travels toward the light projecting lens 12 through the optical member 118d. In the second optical path LL2, no pattern is given to the measurement light. Thereby, uniform measurement light is emitted toward the filter unit 150. The second optical path LL2 is used during normal observation.

  In the example of FIG. 19, a total reflection mirror may be used instead of the reflective light modulation element 112. In this case, one of the optical members 118a and 118b has a transmissive light modulation element. Thereby, pattern measurement light is generated by the transmissive light modulation element.

  In the example of FIG. 19, one of the mirrors 116 a and 116 b may be a half mirror, and the half mirror may be fixed to the internal housing 10. In this case, it is not necessary to provide a plurality of mirror driving units in the internal housing 10.

  (11-e) FIG. 20 is a longitudinal sectional view showing a fifth configuration example of the pattern imparting unit 110 according to another embodiment. The fifth configuration example differs from the fourth configuration example in FIG. 19 in the following points.

  As shown in FIG. 20, in this example, the mirrors 116 a and 116 b are half mirrors and are fixed to the internal housing 10. Therefore, the mirror driving units 117a and 117b in FIG.

  On the other hand, a shutter sha and a shutter drive unit 119a are provided in the first optical path LL1, and a shutter shb and a shutter drive unit 119b are provided in the second optical path LL2. The shutter driving units 119a and 119b move the shutters sha and shb, respectively, as indicated by arrows n in FIG. 20, thereby guiding the measurement light passing through the first optical path LL1 to the light projecting lens 12 during sectioning observation. The measurement light passing through the second optical path LL2 is shielded. Further, during normal observation, the measurement light passing through the second optical path LL2 is guided to the light projecting lens 12, and the measurement light passing through the first optical path LL1 is shielded. Thereby, the measurement light irradiated to the measuring object S is switched.

  (11-f) FIG. 21 is a longitudinal sectional view showing a sixth configuration example of the pattern imparting unit 110 according to another embodiment. The sixth configuration example is different from the fifth configuration example in FIG. 20 in the following points.

  In the internal housing 10 of this example, a measurement light source 321a that generates measurement light passing through the first optical path LL1 and a measurement light source 321b that generates measurement light passing through the second optical path LL2 are individually provided. Thereby, the mirror 116a of FIG. 20 becomes unnecessary. In this example, a light source device is configured by the measurement light sources 321a and 321b.

  (11-g) In the above embodiment, in the lens unit 160, one objective lens 161 used for observation is selected from the plurality of objective lenses 161. As the lens turret 162 rotates, the selected objective lens 161 is positioned on the observation axis OA. Not limited to this, the lens unit 160 may be provided with a zoom lens and its driving device instead of the plurality of objective lenses 161, the lens turret 162 and the lens turret driving unit 164. In this case, for example, the user operates the operation unit 250 in FIG. 4 to specify the magnification. Thereby, the control board 170 adjusts the magnification of the zoom lens so that the designated magnification is obtained.

  (11-h) In the above embodiment, the focal length adjustment driving unit 165 adjusts the distance between the measurement object S and the objective lens 161 by moving the objective lens 161 in the Z direction. Not limited to this, the focal distance adjustment driving unit 165 may adjust the distance between the measurement object S and the objective lens 161 by moving the stage 140 in the Z direction.

  (11-i) In the above embodiment, the measurement object S can be observed with a plurality of different magnifications by switching the plurality of objective lenses 161. However, the measurement unit 100 may be provided with only one objective lens 161. In this case, the objective lens 161 may be configured to be detachable from the measuring unit 100. Thereby, size reduction and simplification of the configuration of the measurement unit 100 are realized.

  Similarly, in the above-described embodiment, by switching the plurality of filter cubes 151, it is possible to perform fluorescence observation of the measurement object S with measurement light in different wavelength regions. Not only this but the measurement part 100 may be provided with only one filter cube 151. In this case, the filter cube 151 may be configured to be detachable from the measuring unit 100. Thereby, size reduction and simplification of the configuration of the measurement unit 100 are realized.

  (11-j) In the above embodiment, the light projecting lens 12 is provided so as to block the emission opening 10 a formed in the internal housing 10. Not limited to this, the light projecting lens 12 may be provided inside the internal housing 10 or may be provided outside the internal housing 10. In these cases, the emission opening 10 a of the inner housing 10 is not blocked by the light projecting lens 12. Therefore, a light transmissive member that transmits the measurement light may be provided in the emission opening 10a. This further prevents dust from entering the inside of the inner housing 10 from the emission opening 10a.

(12) Correspondence relationship between each constituent element of claim and each part of the embodiment Hereinafter, an example of correspondence between each constituent element of the claim and each constituent element of the embodiment will be described. It is not limited to examples.

  In the above embodiment, the measurement object S is an example of the object, the stage 140 is an example of the stage, the pattern measurement light having a pattern is an example of the first measurement light, and sectioning observation is the first. 1 is an example of an observation mode, uniform measurement light having no pattern is an example of a second measurement light, normal observation is an example of a second observation mode, and the operation unit 250 is an example of an operation device. The measurement light sources 321, 321 a, and 321 b are examples of light source devices, the light modulation element 112 is an example of a light modulation element, the constituent elements of the pattern applying unit 110 excluding the measurement light sources 321, 321 a, and 321 b, The lens unit 160 is an example of a measuring light irradiation device.

  In addition, the light receiving unit 120 is an example of a light receiving unit, the image data generation unit 211 is an example of an image data generation unit, the pattern generation unit 212 is an example of a pattern generation unit, and the control board 170 and the control unit 213 are controlled. The external casing 101 is an example of a first casing, the internal casing 10 is an example of a second casing, and the microscope apparatus 500 is an example of a microscope apparatus.

  Further, the transmission observation is an example of the third observation mode, the transmitted light is an example of the third measurement light, the transmitted light source 131 is an example of the third measurement light light source, and the transmitted light supply unit 130 is swung. The moving unit 130B is an example of a third measurement light optical system, the stage driving device 141 is an example of an imaging visual field position moving unit, the observation axis OA is an example of an observation axis, and the lens unit 160 is a condensing optical system. The objective lens 161 is an example of an objective lens, the focal length adjustment mechanism 163, the lens support plate 163p, and the focal length adjustment drive unit 165 are examples of a focal position moving unit, and the lens turret 162 and the lens turret The drive unit 164 is an example of a first switching device.

  The filter unit 150 is an example of a filter optical system, the filter cube 151 is an example of a filter member, the filter turret 152 and the filter turret driving unit 153 are examples of a second switching device, and the front opening OP2 is It is an example of an opening, the front lid 103 is an example of a lid member, and the front lid detector S2 is an example of an open / close detector.

  As each constituent element in the claims, various other constituent elements having configurations or functions described in the claims can be used.

  The present invention can be effectively used for a microscope apparatus.

DESCRIPTION OF SYMBOLS 10 Internal housing | casing 10a Outgoing opening 10b Incident opening 11 Light guide component support case 12 Projection lens 21, 101a Front part 22, 101b Rear part 23, 101c One side part 24, 101d Other side part 25, 101e Upper surface part 26 Lower surface part 91 Base frame 92 Stage frame 93 Upper frame 100 Measurement unit 101 External housing 102 Top cover 102H, 103H Hinge 103 Front cover 106 Post 107 Base 110 Pattern applying unit 111,324 Optical connector 112 Optical modulation element 113, 116a, 116b, 116c , 134 Mirrors 114, 115, 118a, 118b, 118c, 118d Optical members 117a, 117b Mirror driving units 119a, 119b Shutter driving units 120 Light receiving units 121 Cameras 122 Color filters 12 Imaging lens 130 Transmitted light supply unit 130A Fixed unit 130B Oscillating unit 131 Transmitted light source 132 Adjusting unit 133 Transmitting optical system 135 Condenser lens 140 Stage 141 Stage driving device 150 Filter unit 151 Filter cube 151a Frame 151b Excitation filter 151c Dichroic mirror 151d Absorption Filter 152 Filter turret 153 Filter turret drive unit 160 Lens unit 161 Objective lens 162 Lens turret 163 Focal length adjustment mechanism 163p Lens support plate 164 Lens turret drive unit 165 Focal length adjustment drive unit 170 Control board 200 PC
210 CPU
211 Image data generation unit 212 Pattern generation unit 213 Control unit 220 ROM
230 RAM
240 Storage Device 250 Operation Unit 300 Measurement Light Supply Unit 301 Case 309 Opening 310 Power Supply Device 320 Light Projecting Units 321, 321 a, 321 b Measurement Light Source 322 Light Reduction Mechanism 323 Light Shading Mechanism 330 Light Guide Member 340 Heat Exhaust Device 400 Display Unit 410 Display area 420 Setting display area 430 Filter selection field 431, 432, 433, 434 Filter selection button 440 Measurement condition setting field 441 Sectioning observation button 442 Normal observation button 450 Imaging analysis field 451 Single shooting button 452 Multiple shooting button 453 Analysis button 470 Focus Position adjustment tab 471 Objective lens selection field 471a, 471b, 471c, 471d, 471e, 471f Objective lens selection button 472 Focus position adjustment field 472a Focus position adjustment bar 472b Initial distance Tan 472c Autofocus button 473 Stage position adjustment column 473a, 473b, 473c, 473d Stage movement button 473e Initial position button 490 Shooting type selection column 491 Z stack shooting button 492 Linked shooting button 493 All-focus shooting button 500 Microscope device cp1 Separated position DF Depth of field L0, L1, L2, m, n Arrows LL1, LL2 Optical path np1 Normal position OA Observation axis OP1 Upper opening OP2 Front opening S Measurement object S1 Swing part detector S2 Front cover detector sha, shb Shutter sp1 observation target part sp2, sp3 part WR1, WR2 wiring

Claims (9)

A stage on which the object is placed;
Used to switch between a first observation mode for observing an object using first measurement light having a pattern and a second observation mode for observing the object using second measurement light having no pattern An operating device operated by a person,
A light source device for generating light;
Including a light modulation element that generates first measurement light from light generated by the light source device, and generates first measurement light by the light modulation element from light generated by the light source device in a first observation mode. And measuring light irradiation device that irradiates the object with the first measurement light generated and irradiates the object with the light generated by the light source device as the second measurement light in the second observation mode;
A light receiving unit that receives fluorescence emitted from the object and outputs a light reception signal indicating the amount of light received;
An image data generation unit that generates image data based on a light reception signal output from the light reception unit;
A pattern generation unit that generates a pattern of first measurement light to be applied to the object while the spatial phase is sequentially moved by a predetermined amount;
In the first observation mode, the light modulation element is controlled based on the pattern generated by the pattern generation unit, and sectioning image data is generated based on a plurality of image data generated at a plurality of phases of the pattern. Controlling the image data generation unit so as to control the image data generation unit so as to generate image data indicating a normal observation image of the object in the second observation mode;
A first housing that houses the stage, the measuring light irradiation device, and the light receiving unit;
A second housing that houses the light modulation element in the first housing,
The first housing is configured to bring a measurement space including an object on the stage and the light receiving unit into a dark room state,
The operating device is provided outside the first housing,
The second housing is a microscope apparatus configured such that light other than the first measurement light generated by the light modulation element does not reach the measurement space from the light modulation element. The operating device is operated by a user to command the first and second observation modes, respectively.
The control unit controls the light modulation element based on the pattern generated by the pattern generation unit in response to a command of the first observation mode by the operation device, thereby controlling the object from the measurement light irradiation device. Irradiating the first measurement light to the image data and controlling the image data generation unit to generate sectioning image data based on a plurality of image data generated at a plurality of phases of the pattern, In response to the command of the second observation mode, the measurement light irradiation device irradiates the object with the second measurement light, and generates the image data indicating the normal observation image of the object. The microscope apparatus according to claim 1, wherein the microscope apparatus is controlled. The operation device is operated by a user to further switch between the first or second observation mode and the third observation mode for observing the object using the third measurement light,
A third measuring light source for generating third measuring light in the third observation mode;
A third measurement light optical system for irradiating the object with the third measurement light generated by the third light source;
The light receiving unit receives third measurement light that passes through the object, and outputs a light reception signal indicating the amount of light received;
The microscope apparatus according to claim 1, wherein the control unit controls the image data generation unit to generate image data indicating a transmission observation image of the target object in the third observation mode. An imaging field position moving unit that moves the imaging field position of the object by moving the stage in a direction intersecting the optical axis of the light receiving unit;
The imaging visual field position moving unit is housed in the first housing,
The operation device is operated by a user to command the operation of the imaging visual field position moving unit,
The microscope device according to claim 1, wherein the control unit controls the imaging visual field position moving unit in response to a command from the operation device. The measurement light irradiation device has a condensing optical system having an observation axis,
The condensing optical system includes an objective lens configured to condense the first measurement light generated by the light modulation element and the light generated as the second measurement light by the light source device onto an object. ,
The focal position of the objective lens relative to the object is moved by changing the relative distance between the object and the objective lens by moving at least one of the stage and the objective lens along the observation axis. A focus position moving part,
The light receiving optical system and the focal position moving unit are accommodated in the first darkroom casing,
The operating device is operated by a user to command the operation of the focal position moving unit,
The microscope device according to claim 1, wherein the control unit controls the focal position moving unit in response to a command from the operation device. The condensing optical system has a plurality of the objective lenses,
By selectively positioning one objective lens among the plurality of objective lenses on the observation axis, the first measurement light generated by the light modulation element and the second measurement light generated by the light source device are generated. A first switching device that focuses the collected light onto the object through the one objective lens;
The operating device is operated by a user to select one of the plurality of objective lenses,
The control unit controls the first switching device so that the selected one objective lens is positioned on the observation axis in response to selection of the one objective lens by the operation device. 5. The microscope apparatus according to 5. The measurement light irradiation device further includes a filter optical system,
The filter optical system transmits, on the observation axis, light in a part of the wavelength region of the first measurement light generated by the light modulation element and the light generated as the second measurement light by the light source device. Including a plurality of filter members configured to be possible,
By selectively positioning one of the plurality of filter members on the observation axis, the first measurement light generated by the light modulation element or the second measurement light generated by the light source device is generated. A second switching device that irradiates the object with the emitted light through the one filter member;
The operating device is operated by a user to select one of the plurality of filter members,
The control unit controls the second switching device so that the selected one filter member is positioned on the observation axis in response to selection of the one filter member by the operation device. 5. The microscope apparatus according to 5 or 6. In the first and second observation modes, the control unit, when the measurement conditions are not changed continuously for a predetermined period after the sectioning image data or the image data indicating the normal observation image of the object is generated, The microscope apparatus according to claim 1, wherein at least one of the light source device and the light modulation element is controlled so that the object is not irradiated with the first and second measurement lights. A maintenance opening is formed in the first casing,
A lid member for opening and closing the opening;
An open / close detector that detects an open / closed state of the lid member;
The control unit includes at least one of the light source device and the light modulation element based on the detection of the open / close detector so that the first and second measurement lights do not reach the opening when the lid member is open. The microscope apparatus according to claim 1, wherein one is controlled.

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