JP2014067064A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To avoid re-acquisition of an image to improve work efficiency.SOLUTION: There is provided a microscope device including: a confocal observation unit and an imaging unit that are capable of acquiring an image of a sample S; a stimulation unit that irradiates the sample S with light to stimulate the sample S; an area specification unit that specifies an ROI 170 to be stimulated by the stimulation unit on the image acquired by the confocal observation unit or the imaging unit; and a visual field range display unit that displays display representing the maximum visual field range of the stimulation unit overlapped on a screen.

Description

  The present invention relates to a microscope apparatus.

  Conventionally, a microscope that is equipped with multiple devices for observation and stimulation, and on the reference image containing a specimen such as a cell acquired by a certain device, a region that stimulates the specimen by another device or an area that observes the specimen Devices are known (see, for example, Patent Document 1 and Patent Document 2). The microscope apparatus described in Patent Literature 1 includes two scanners for observation and stimulation, and can designate an observation area and a stimulation area on a pre-scanned image acquired in advance. In addition, the microscope apparatus described in Patent Document 2 can designate a measurement target region that is subjected to pattern irradiation with illumination light by DMD and spectrally detected by PMT on an image acquired by CCD.

JP 2007-148223 A JP 2006-171024 A

  However, the microscope apparatuses described in Patent Document 1 and Patent Document 2 have the same maximum field of view range for each device. If the maximum field of view varies from device to device, the observation range of devices with a wide maximum field of view is limited to devices with a narrow maximum field of view, and the original field of view cannot be fully utilized. There is.

  That is, when an area is specified on the reference image in a state where the maximum visual field range for each device is different, an area is specified on the reference image when the device that acquires the reference image has a larger maximum visual field range than the device that specifies the area. There may be ranges that cannot. In such a case, the reference image is reacquired in accordance with the desired area, and the work efficiency is poor.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a microscope apparatus that can avoid re-acquisition of an image and improve work efficiency.

In order to solve the above problems, the present invention employs the following means.
The present invention provides an observation optical system that can acquire an image of a specimen, a stimulation optical system that stimulates the specimen by irradiating light on the specimen, and a stimulus that is stimulated by the stimulation optical system on an image acquired by the observation optical system There is provided a microscope apparatus comprising region designating means for designating an area, and visual field range display means for displaying a display representing the maximum visual field range of the stimulation optical system on the image.

  According to the present invention, by designating the stimulation region of the stimulation optical system on the sample image by the region designating unit, the sample can be easily selected and desired stimulation can be performed. In this case, the visual field range display means displays a display representing the maximum visual field range of the stimulation optical system superimposed on the image, so that the image can be acquired after grasping the specifiable stimulation area in advance. it can.

  Therefore, even when the maximum visual field ranges of the observation optical system and the stimulation optical system are different, it is possible to avoid re-acquisition of an image in accordance with a desired stimulation region. As a result, it is possible to fully utilize the individual visual field ranges of the observation optical system and the stimulation optical system and improve the working efficiency.

  In the above invention, a plurality of optical path combining units that combine the optical path of the observation optical system and the optical paths of the plurality of stimulation optical systems, and switching means that can selectively insert and remove the optical path combining unit in the optical path, Offset amount storage means for storing an offset amount between the optical paths for each of the optical path synthesis sections, and the visual field range display means based on the offset amount of the optical path synthesis section switched by the switching means The display position representing the maximum visual field range of the optical system may be corrected.

  With this configuration, even when the optical path synthesis unit is switched by the switching unit and the sample is stimulated by different stimulation optical systems, the display is based on the display representing the maximum visual field range whose position is corrected by the visual field range display unit. The stimulation area can be effectively designated within the maximum visual field range for each stimulation optical system. For example, this is particularly effective when a sample is stimulated by a predetermined optical path from any one of the optical path synthesis units.

In the above-mentioned invention, it is good also as fitting means which makes the acquisition field of the image correspond to the maximum field of view range of the stimulus optical system.
With this configuration, an unnecessary range that cannot be designated as a stimulation region from the image can be removed by the fitting means.

  The present invention provides an observation optical system capable of acquiring an image of a specimen, a plurality of stimulation optical systems that stimulate the specimen by irradiating light, and stimulation by the stimulation optical system on an image acquired by the observation optical system Area designating means for designating a stimulation area to be performed, a plurality of optical path synthesis units for synthesizing the optical path of the observation optical system and the optical path of the stimulation optical system, and the optical path synthesis unit can be selectively inserted into and removed from the optical path A switching unit, an offset amount storage unit that stores an offset amount between the optical paths for each of the optical path combining units, and a common visual field range common to all the stimulation optical systems based on the offset amounts of the optical path combining units There is provided a microscope apparatus provided with a visual field range display means for displaying a display.

According to the present invention, it is possible to acquire an image after grasping in advance a stimulable region that can be designated by the display representing the common visual field range displayed by the visual field range display means. In this case, even when the optical path synthesis unit is switched by the switching unit and the sample is stimulated by different stimulation optical systems, the stimulation region can be designated within the visual field range common to all the stimulation optical systems. For example, this is particularly effective when the specimen is continuously stimulated by different stimulation optical systems while switching the optical path synthesis unit.
In the above-mentioned invention, it is good also as providing the fitting means which makes the acquisition field of the image correspond to the common visual field range.

Moreover, in the said invention, the said visual field range display means is good also as superimposing and displaying on the said image the display showing the magnitude | size of the largest range which can be stimulated at once by the said stimulus optical system.
If the specimen can be stimulated at once, the working efficiency can be improved as compared to the case of stimulating multiple times. Therefore, by displaying a display indicating the size of the maximum range that can be stimulated at once by the visual field range display means, a sample having a size suitable for stimulation can be easily and reliably searched. Redesignation of the area can be avoided.

Moreover, in the said invention, the said visual field range display means is good also as superimposing and displaying on the said image the display showing the magnitude | size of the minimum range which can be stimulated with the said stimulus optical system.
With this configuration, the stimulation area can be specified in detail by the area specifying unit based on the display indicating the size of the minimum range that can be stimulated.

In the above invention, the stimulation optical system includes scanning means for two-dimensionally scanning light on the specimen, and the visual field range display means displays a scanning direction by the scanning means on the image. It is good as well.
With this configuration, when the scanning speed of the scanning unit varies depending on the scanning direction, it is possible to increase the frame rate and reduce the time by setting the stimulation area small in the direction in which the scanning speed is slow. .

  The invention as a reference example of the present invention includes a plurality of observation optical systems capable of acquiring images of the same specimen, and a reference image acquired by any one of the observation optical systems. Area specifying means for specifying an acquisition area for the observation image to be displayed, field-of-view range display means for displaying a display representing the maximum field-of-view range of the other observation optical system on the reference image, and the observation optical system A plurality of optical path combining units that combine the optical paths, a switching unit that can selectively insert / remove the optical path combining unit in the optical path, and an offset amount storage unit that stores an offset amount between the optical paths for each of the optical path combining units And the visual field range display means compensates the position of the display representing the maximum visual field range of the other observation optical system based on the offset amount of the optical path combining unit switched by the switching means. Providing a microscope apparatus for and view.

  According to the invention as a reference example of the present invention, a specimen can be easily obtained by using a plurality of observation optical systems having different observation methods by designating an acquisition area of the observation image on the reference image of the specimen by the area designation means. The desired observation can be performed. In this case, the visual field range display means displays the display representing the maximum visual field range of the observation optical system for acquiring the observation image superimposed on the reference image. The image for reference can be acquired after grasping in advance.

  Therefore, even when the maximum optical field range of the observation optical system for acquiring the reference image and the observation optical system for acquiring the observation image are different, the reference image is acquired in accordance with the desired acquisition region of the observation image. Repair can be avoided. As a result, it is possible to fully utilize the individual visual field ranges of the plurality of observation optical systems and improve work efficiency.

  In the above invention, a plurality of optical path combining units that combine the optical paths of the observation optical system, switching means that can selectively insert / remove the optical path combining unit to / from the optical path, and between the optical paths for each of the optical path combining units Offset amount storage means for storing an offset amount, and the field-of-view range display means, based on the offset amount of the optical path synthesis unit switched by the switching means, the maximum field-of-view range of the other observation optical system Correct the position of the display indicating.

  With this configuration, even when the optical path synthesis unit is switched by the switching unit and an image for observation is acquired by a different observation optical system, the display representing the maximum visual field range whose position is corrected by the visual field range display unit The observation image acquisition area can be effectively designated within the maximum visual field range for each observation optical system. For example, this is particularly effective when a specimen is observed along a predetermined optical path from any of the optical path combining units. The offset amount refers to a shift amount in a direction orthogonal to the optical axis.

In the above invention, a fitting unit that matches the acquisition region of the reference image with the maximum visual field range of the other observation optical system may be provided.
With this configuration, an unnecessary range that cannot be designated as the observation image acquisition region can be removed from the reference image by the fitting unit.

  The invention as another reference example of the present invention includes a plurality of observation optical systems capable of acquiring an image of the same specimen, and another observation optical system on a reference image acquired by any one of the observation optical systems Region specifying means for specifying an observation image acquisition region acquired by the above, a plurality of optical path combining units for combining the optical paths of the observation optical system, and a switching unit capable of selectively inserting and removing the optical path combining unit in the optical path And an offset amount storage means for storing an offset amount between the optical paths for each of the optical path combining units, and a display representing a common visual field range common to all the observation optical systems based on the offset amounts of the optical path combining units. Provided is a microscope apparatus including a visual field range display means for displaying.

According to the invention as another reference example of the present invention, the reference image is acquired after grasping in advance the acquisition region of the specifiable observation image by the display indicating the common visual field range displayed by the visual field range display means. can do. In this case, the observation image acquisition area is specified within the field of view common to all observation optical systems, even when the optical path synthesis unit is switched by the switching means and the observation images are acquired by different observation optical systems. can do. For example, this is particularly effective when the specimen is continuously observed with different observation optical systems while switching the optical path synthesis unit.
In the above-mentioned invention, it is good also as fitting means which makes the acquisition field of the above-mentioned image for reference coincide with the above-mentioned common visual field range.

In the above invention, the field-of-view range display means superimposes and displays a display indicating the maximum size of the observation image that can be acquired at one time by the other observation optical system on the reference image. It is good to do.
If the sample observation image can be acquired at once, the working efficiency can be improved as compared with the case where the sample observation image is acquired a plurality of times. Therefore, by displaying a display indicating the size of the maximum observation image that can be acquired at one time by the visual field range display means, a sample having a size suitable for observation can be easily and reliably searched. It is possible to avoid redesignation of the image acquisition area.

In the above invention, the field-of-view range display means superimposes and displays a display indicating the size of the minimum observation image obtainable by the other observation optical system on the reference image. Also good.
With this configuration, the observation image acquisition area can be specified in more detail based on the display indicating the size of the minimum observation image displayed by the visual field range display unit. For example, this is effective when an observation image acquisition region is designated on a reference image having a particularly high magnification.

In the above invention, the other observation optical system includes scanning means for two-dimensionally scanning light on the specimen, and the visual field range display means indicates the scanning direction by the scanning means for the reference image. It may be displayed above.
With this configuration, when the scanning speed of the scanning unit varies depending on the scanning direction, the frame rate can be increased by setting the observation image acquisition region small in the direction in which the scanning unit has a low scanning speed. Observation time can be shortened.

In the above invention, the other observation optical system includes an imaging device having a plurality of pixels arranged two-dimensionally, and the visual field range display unit displays the arrangement direction of the pixels on the reference image. It is good also as displaying on.
With this configuration, partial transfer (subarray transfer) that sets the observation image acquisition area small in any direction of the pixel arrangement direction and transfers only image data corresponding to pixels in the limited area. By reducing the number of lines, the frame rate can be increased and the observation time can be shortened.

  According to the present invention, there is an effect that it is possible to avoid re-acquisition of an image and improve work efficiency.

It is a figure which shows schematic structure of the microscope apparatus which concerns on one Embodiment of this invention. It is a figure which shows schematic structure of the confocal observation unit of FIG. It is a figure which shows schematic structure of the stimulation unit of FIG. (A) is a figure which shows a mode that the maximum restriction | limiting display was superimposed and displayed on the reference image, (b) is a figure which shows a mode that the maximum size display was superimposed and displayed on the reference image, (C) is a figure which shows a mode that the minimum size display was superimposed and displayed on the reference image. It is a figure which shows a mode that the X direction and the Y direction were displayed on the image for a reference. (A) is a figure which shows a mode that the area | region which acquires an image was shifted, (b) is a figure which shows a mode that the magnification of the image was changed, (c) is an angle of an image around the central axis of a visual field. It is a figure which shows a mode that it changed. It is a figure which shows a mode that the image for a reference was displayed on the monitor. It is a figure which shows a mode that the maximum restriction | limiting display was superimposed and displayed on the reference image of a monitor. It is a figure which shows a mode that the image for a reference of a monitor was rotated to the surroundings of the central axis of a visual field. It is a figure which shows a mode that it shifted, expanding the reference image of a monitor. It is a figure which shows a mode that ROI was designated on the reference image of a monitor. (A) shows a state in which a maximum restriction display is superimposed and displayed on a reference image when a predetermined optical path synthesis unit is arranged, and (b) shows a case where an optical path synthesis unit different from (a) is arranged. It is a figure which shows a mode that the maximum restriction | limiting display is superimposed and displayed on a different position on the reference image. It is the figure which showed a mode that the maximum restriction | limiting display was displayed on the whole reference image. It is a figure which shows a mode that the maximum restriction | limiting display was displayed on the whole reference image of a monitor. It is a figure which shows a mode that the maximum restriction | limiting display was superimposed and displayed on the reference image using resonance GS. (A) shows a state in which a common restriction display is superimposed and displayed on a reference image when a predetermined optical path synthesis unit is arranged in a microscope apparatus according to a modification of the present embodiment, and (b) shows (a). It is a figure which shows a mode that a common restriction | limiting display is superimposed and displayed on the reference image when the optical path synthetic | combination part different from FIG.

Hereinafter, a microscope apparatus according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the microscope apparatus 100 according to the present embodiment includes a microscope 10 that visually observes a sample S, a confocal observation unit (observation optical system) 50 that confocally observes the sample S, and light the sample S. A stimulation unit (stimulation optical system) 70 that stimulates, an imaging unit (observation optical system) 110 that acquires a two-dimensional image of the specimen S, a microscope 10, the confocal observation unit 50, the stimulation unit 70, and the imaging unit 110. And a microscope connecting device 120 for optical connection. Hereinafter, the confocal observation unit 50, the stimulation unit 70, and the imaging unit 110 are simply referred to as “units 50, 70, 110”.

  Further, the microscope apparatus 100 displays a laser unit (not shown) that generates laser light, a PC 154 that performs control, information processing and storage, image construction, and the like of the entire apparatus, and information and images processed by the PC 154. A monitor 156 is provided. The laser unit includes single mode fibers 141 and 142 for guiding laser light to the confocal observation unit 50 and the stimulation unit 70, and laser unit collimating lenses 143 and 144 (see FIGS. 2 and 3).

  The microscope 10 includes a halogen lamp 11 that emits illumination light for transmission observation, and illumination light that is emitted from the halogen lamp 11 and reflected by the microscope first reflecting mirror 13A through a field stop (FS) 15 and an aperture stop (AS) 17. And a plurality of objective lenses 23 that collect the transmitted light that has passed through the sample S when irradiated with the illumination light collected by the capacitor 19. ing. The objective lens 23 is supported by a revolver 25.

  Further, the microscope 10 visually observes an image formed by the image forming lens 27 that forms the light from the objective lens 23 and the image forming lens 27 and is divided by the prism 29 through the second microscope reflecting mirror 13B. The eyepiece 31 is provided. Reference numeral 33 denotes a microscope shutter, and reference numeral 35 denotes an aiming unit for aiming.

  Further, the microscope 10 emits illumination light for epi-illumination observation, and the illumination light emitted from the microscope mercury lamp 37 is reflected to the objective lens 23 to irradiate the specimen S, while A microscope beam splitter 39 that transmits observation light and enters the imaging lens 27 is provided. Hereinafter, the beam splitter is referred to as “BS”.

  The microscope BS39 is provided in the cube turret 41 for switching the spectroscopic method. The microscope BS39 may have a wedge shape whose thickness gradually changes along the longitudinal direction, and the back surface reflected light may be formed to have an angle with respect to the optical axis.

  Further, the microscope 10 is provided with an external connection port 43 to which the microscope connection device 120 is connected. A microscope switching mirror 45 is detachably disposed between the imaging lens 27 and the microscope second reflecting mirror 13B. In a state where the microscope switching mirror 45 is removed from the optical path of the microscope 10, a primary image of the sample S is formed on the eyepiece 31 side, and in a state where the microscope switching mirror 45 is arranged in the optical path of the microscope 10, 1 of the sample S is obtained. The next image is formed on the microscope connecting device 120 side (primary image position K in FIG. 1) via the external connection port 43.

  As shown in FIG. 2, the confocal observation unit 50 includes an observation unit connection unit 51 connected to the microscope connection device 120. Further, the confocal observation unit 50 includes an observation unit first reflection mirror 53A and an observation unit first BS 55A that reflect the laser light guided from the laser unit by the single mode fiber 141 and converted into a parallel light beam by the collimator lens 143, and these An observation unit galvano scanner (scanning means) 57 that two-dimensionally scans the laser beam reflected by the specimen 10 on the specimen S of the microscope 10 and a secondary image position of the specimen S obtained by scanning the laser light scanned by the observation unit galvano scanner 57 And an observation unit pupil projection lens 59 that condenses light to L. Hereinafter, the galvano scanner is referred to as “GS”.

  Further, the confocal observation unit 50 reflects the laser light reflected by the observation unit first reflection mirror 53A or the observation unit first BS 55A to the observation unit GS57, and is generated in the sample S irradiated with the laser light. An observation unit excitation DM 61 that transmits fluorescence descanned by the GS 57 is provided. The observation unit GS57 can be switched to a resonance GS (scanning means) 58.

  Further, the confocal observation unit 50 is reflected by the spectroscopic DMs 63A and 63B and the observation unit second reflecting mirror 53B, which reflect the fluorescence transmitted through the observation unit excitation DM61 and reflected by the observation unit second BS 55B for each wavelength. Confocal pinholes 67A, 67B, and 67C that partially pass the fluorescence condensed by the focusing lenses 65A, 65B, and 65C, and a PMT that detects fluorescence that has passed through the confocal pinholes 67A, 67B, and 67C (photoelectron enhancement) Double tube) 69A, 69B, 69C.

The spectroscopic DMs 63A and 63B are provided in the observation unit filter turrets 64A and 64B, and can be switched according to the wavelength characteristics to be split.
The PMTs 69A, 69B, and 69C output a detection signal for each pixel obtained by detecting fluorescence to the PC 154. As a result, a two-dimensional image is constructed based on the detection signal in the PC 154 and displayed on the monitor 156.

  As shown in FIG. 3, the stimulation unit 70 includes a stimulation unit connection unit 71 connected to the microscope connection device 120. The stimulation unit 70 also includes a stimulation unit mercury lamp 73 that emits light, a stimulation unit shutter 75 that passes or blocks light emitted from the stimulation unit mercury lamp 73, and light of a predetermined wavelength that has passed through the stimulation unit shutter 75. An excitation filter 77 that transmits light, and a stimulation unit DMD (micro deflection element array) 83 that deflects light that has passed through the excitation filter 77 and is collected by the stimulation unit condensing lens 79 via the stimulation unit first reflection mirror 81A. A stimulation unit relay optical system 85 that condenses the light deflected by the stimulation unit DMD 83 at the secondary image position M of the sample S.

The stimulation unit mercury lamp 73 is detachably provided on the frame of the stimulation unit 70.
The excitation filter 77 is provided in the stimulation unit filter turret 78 and can be switched according to the wavelength characteristics.

  The stimulation unit DMD 83 includes a plurality of swingable micromirrors (not shown) that are two-dimensionally arranged at positions conjugate to the image position of the imaging lens 27 relayed by the stimulation unit relay optical system 85. For the micromirror, the ON region / OFF region may be arbitrarily selected by the PC 154. By this micromirror, multipoint simultaneous stimulation and range designation stimulation of the specimen S can be performed.

  Further, the stimulation unit 70 includes a stimulation unit second reflection mirror 81B, a stimulation unit first BS 87A, and a stimulation unit third reflection mirror 81C that reflect the laser light guided from the laser unit by the single mode fiber 142 and transmitted through the collimator lens 144. The stimulation unit GS89 that scans the laser beam reflected by these two-dimensionally on the sample S of the microscope 10 via the stimulation unit connection unit 71, and the laser beam scanned by the stimulation unit GS89 is the secondary of the sample S. A stimulating unit pupil projection lens 91 for condensing light at the image position M.

  The laser beam scanned by the stimulation unit GS89 can be combined with the optical path of the light deflected by the stimulation unit DMD83 by the stimulation unit second BS 87B. The stimulation unit second BS 87B has a characteristic of transmitting the light deflected by the stimulation unit DMD83 and reflecting the laser beam scanned by the stimulation unit GS89.

  The stimulation unit second BS 87B may be arranged to be switched to the stimulation unit switching mirror 95 by stimulation unit switching means 93 such as a filter turret. The stimulation unit switching mirror 95 is disposed in the optical path when stimulating the specimen S with light emitted from the stimulation unit mercury lamp 73, and is removed from the optical path when stimulating the specimen S with laser light guided from the laser unit. And it is sufficient.

  The imaging unit 110 includes a CCD (imaging device) 111 having a plurality of pixels (not shown) arranged two-dimensionally, and an image of the specimen S imaged at the secondary image position N by the imaging lens 27. The CCD 111 can take a picture. The two-dimensional image acquired by the CCD 111 is displayed on the monitor 156.

  Since a two-dimensional imaging device such as a CCD detects light on its surface, it has a higher frame rate than when confocal observation is performed. Therefore, the CCD 111 can catch a fast reaction such as calcium ions. The CCD 111 has a field of view that is, for example, relatively rotated about 3 ° around the central axis of the field of view of the confocal observation unit 50.

  Also, the mounting mount of the CCD camera is usually standardized, and the distance (flange back) from the mounting surface to the light receiving surface is determined. Therefore, the CCD 111 is connected to the microscope connection device 120 via the camera adapter 113 that converts the connection portion and the CCD camera mount. In addition, the camera adapter 113 may function as a scaling adapter or a filter turret, instead of simply changing the mount and increasing the distance.

  The microscope connection device 120 includes a microscope connection port 121 connected to the external connection port 43 of the microscope 10, a first connection port 123 </ b> A to which the camera adapter 113 of the imaging unit 110 is connected, and a stimulation unit connection portion 71 of the stimulation unit 70. Are connected, and a third connection port 123C to which the observation unit connection part 51 of the confocal observation unit 50 is connected. Hereinafter, the first connection port 123A, the second connection port 123B, and the third connection port 123C are referred to as unit connection ports 123A, 123B, and 123C. These unit connection ports 123A, 123B, and 123C may all have the same shape.

  The microscope connection device 120 also includes a connection unit optical system 125 that optically connects the microscope 10 and the imaging unit 110, the stimulation unit 70, or the confocal observation unit 50, and a DM that combines the optical paths of the connection unit optical system 125. And optical path combining units 127A and 127B.

  The connection unit optical system 125 relays the primary image of the sample S imaged by the imaging lens 27 to the confocal observation unit 50, the stimulation unit 70, and the imaging unit 110. The connection unit optical system 125 may form a substantially parallel light beam.

  In addition, the connection unit optical system 125 includes a first relay lens 129A that makes light incident from the microscope 10 through the microscope connection port 121 into a substantially parallel light beam, and light that has passed through the first relay lens 129A. The second relay lens 129B that condenses through 123A, the third relay lens 129C that condenses the light that has passed through the first relay lens 129A through the second connection port 123B, and the first relay lens 129A. A fourth relay lens 129D that condenses the light via the third connection port 123C.

  The first relay lens 129A and the second relay lens 129B relay the primary image of the sample S to the secondary image position N in the photographing unit 110, and the first relay lens 129A and the third relay lens 129C The primary image is relayed to the secondary image position M in the stimulation unit 70, and the first relay lens 129A and the fourth relay lens 129D convert the primary image of the sample S into the secondary image position L in the confocal observation unit 50. To relay.

  Between the first relay lens 129A and the second relay lens 129B, a first optical path combining unit 127A that transmits light captured by the CCD 111 and reflects light of other wavelengths is disposed. On the reflection side of the first optical path combining unit 127A, a second optical path combining unit 127B that reflects light from the first optical path combining unit 127A to the third relay lens 129C and transmits light of other wavelengths is disposed. Has been. A connection unit reflecting mirror 131 that reflects the light transmitted through the second optical path combining unit 127B to the fourth relay lens 129D is disposed on the transmission side of the second optical path combining unit 127B.

  Since these optical path combining units 127A and 127B are arranged on a substantially parallel light beam, it is possible to prevent image deterioration due to astigmatic difference. The astigmatic difference refers to a shift in the light collecting position in two directions perpendicular to the optical axis when light is collected. In addition, the connection unit optical system 125 forms a non-parallel light beam, so that it is possible to prevent light from being reflected from the back surface in the optical path combining units 127A and 127B, and to prevent interference fringes from occurring and image deterioration.

  The optical path synthesizing units 127A and 127B are provided in disk-shaped connection unit filter turrets (switching means) 128A and 128B that can be rotated by a motor (not shown). Depending on the wavelength characteristics, the optical path synthesizing units 127C, 127D, and 127E are provided. Switching is possible. The optical path combining units 127A and 127B may include a plurality of connection units BS, mirrors, and glass (not shown), and each connection unit BS may be detachably provided on the connection unit filter turrets 128A and 128B.

  The secondary images of the specimen S relayed by the connection unit optical system 125 may be formed at positions separated from each unit connection port 123A, 123B, 123C by an equal distance. That is, all of the distance from the first connection port 123A to the secondary image position N, the distance from the second connection port 123B to the secondary image position M, and the distance from the third connection port 123C to the secondary image position L are all included. May be equal. In this way, the confocal observation unit 50, the stimulation unit 70, and the imaging unit 110 can be freely combined and connected to any of the unit connection ports 123A, 123B, and 123C, and variations in observation methods are increased. be able to.

  The laser unit includes, for example, a laser oscillator, a wavelength selection unit, a laser intensity dimming unit, etc., and the wavelength and intensity suitable for observation of the laser light oscillated by the laser oscillator by the wavelength selection unit and the laser intensity dimming unit. It is good also as adjusting and outputting to.

  The PC 154 captures an image (hereinafter, “an image for reference”) acquired by either the confocal observation unit 50 or the imaging unit 110 (hereinafter, referred to as “reference image”) by another imaging unit 110 or the confocal observation unit 50. ROI (acquisition area) ”, an area designation unit (area designation means) 161 for designating an ROI (stimulation area) stimulated by the stimulation unit 70, and a reference as shown in FIG. A field-of-view range display unit (field-of-view range display means) 163 that superimposes and displays a maximum restriction display F1 representing the maximum field-of-view range of the imaging unit 110, confocal observation unit 50, or stimulation unit 70 ing. Note that the maximum visual field range is larger in the order of the confocal observation unit 50, the imaging unit 110, and the stimulation unit 70.

  When the optical path synthesis units 127A and 127B are switched to the optical path synthesis units 127C, 127D, 127E, and the like by the connection unit filter turrets 128A, 128B, the visual field range display unit 163 displays the optical path synthesis units 127C, 127D, Based on the offset amount such as 127E, the position of the maximum restriction display F1 representing the maximum visual field range of the units 50, 70, 110 is corrected.

  In addition, as shown in FIG. 4B, the visual field range display unit 163 can be stimulated at once by the maximum observation image size that can be acquired by the CCD 110 or the PMTs 69A, 69B, and 69C and the stimulation unit DMD83. The maximum size display G representing the size of the maximum range can be displayed superimposed on the reference image 171. In addition, as shown in FIG. 4C, the visual field range display unit 163 has a minimum size of an image for observation that can be acquired by the CCD 110 or the PMTs 69A, 69B, and 69C, and a minimum size that can be stimulated by the stimulation unit DMD83. The minimum size display H representing the height can be displayed superimposed on the reference image 171.

  Further, as shown in FIG. 5, the visual field range display unit 163 displays the scanning direction (X direction and Y direction) of the observation unit GS57 and the pixel array direction (X direction and Y direction) of the CCD 111 on the reference image 171. It can be displayed. Here, when the scanning speed of the observation unit GS57 is different between the X direction and the Y direction, the faster scanning speed is the X direction, and the slower scanning speed is the Y direction.

  Further, the PC 154 is a control unit that controls the swing angle range, swing angle, scanning timing, etc. of the observation unit GS57 in the confocal observation unit 50 and switching of the optical path combining units 127A and 127B by the connection unit filter turrets 128A and 128B. Fitting means) 165, a memory (offset amount storage means) 167 for storing reference information for each of the units 50, 70, and 110, and an image construction unit 169 for constructing an image.

  The control unit 165 adjusts the swing angle range of the observation unit GS57, shifts the area for acquiring an image as shown in FIG. 6A (hereinafter referred to as “pan control”), and swings the observation unit GS57. The angle is adjusted, the magnification of the image is changed as shown in FIG. 6B (hereinafter referred to as “zoom control”), the two-dimensional scanning timing of the observation unit GS57 is adjusted, and FIG. The image can be rotated around the central axis of the field of view (hereinafter referred to as “rotation control”).

  The memory 167 includes, as reference information, the positional relationship between the units 50, 70, 110, the size of the maximum visual field range of each unit 50, 70, 110, the size restriction of the ROI, and each of the optical path synthesis units 127A, 127B. The offset amount between the optical paths is stored.

The operation of the microscope apparatus 100 according to the present embodiment configured as described above will be described below.
Using the microscope apparatus 100 according to the present embodiment, the reference image 171 of the sample S is acquired by the confocal observation unit 50, and the sample S is stimulated by the stimulation unit 70 on the reference image 171 or the sample by the imaging unit 110. A case will be described in which stimulation or observation is performed by designating an ROI for acquiring an S observation image.

  First, the connection unit filter turrets 128 </ b> A and 128 </ b> B are controlled by the PC 154, and the first optical path synthesis unit 127 </ b> A and the second optical path synthesis unit 127 </ b> B are disposed between the optical paths between the microscope 10 and the confocal observation unit 50.

  In the confocal observation unit 50, the laser light guided from the laser unit is reflected to the observation unit GS57 by the observation unit excitation DM61 via the observation unit first reflection mirror 53A or the observation unit first BS 55A. The laser light is scanned by the observation unit GS57, condensed by the observation unit pupil projection lens 59, passes through the third connection port 123C, and enters the microscope connection device 120.

  The laser light incident on the microscope connection device 120 passes through the fourth relay lens 129D to become a substantially parallel light beam, and is reflected by the connection unit reflection mirror 131. The laser light passes through the second optical path synthesis unit 127B, is reflected by the first optical path synthesis unit 127A, is condensed by the first relay lens 129A, passes through the microscope connection port 121, and enters the microscope 10.

  The laser light incident on the microscope 10 passes through the microscope BS 39 via the microscope switching mirror 45 and the imaging lens 27 and is irradiated onto the sample S by the objective lens 23. When fluorescence is generated in the specimen S by being irradiated with laser light, the fluorescence is condensed by the objective lens 23, then transmitted through the imaging lens 27, reflected by the microscope switching mirror 45, and passed through the microscope connection port 121. Then, the light enters the microscope connection device 120.

  The fluorescence incident on the microscope connection device 120 passes through the first relay lens 129A and is reflected by the first optical path combining unit 127A. Then, the fluorescence passes through the second optical path combining unit 127B, is reflected by the connection unit reflection mirror 131, is collected by the fourth relay lens 129D, passes through the third connection port 123C, and enters the confocal observation unit 50. The

  The fluorescence incident on the confocal observation unit 50 is descanned by the observation unit GS57, passes through the observation unit excitation DM61, and is dispersed by the spectral DMs 63A and 63B through the observation unit second BS 55B according to the wavelength characteristics. The split fluorescence is condensed by the confocal lenses 65A, 65B, and 65C, passes through the confocal pinholes 67A, 67B, and 67C, and enters the PMTs 69A, 69B, and 69C.

  In the PMTs 69A, 69B, and 69C, a detection signal obtained for each pixel by detecting fluorescence is output to the PC 154. Then, a two-dimensional image of the specimen S is constructed by the image construction unit 169 of the PC 154 based on the input detection signal. Thereby, for example, as shown in FIG. 7, a screen including the reference image 171 (“LSM Image” in the drawing) of the sample S is displayed on the monitor 156.

  Here, as shown in FIG. 8, when an item for displaying the maximum restriction display F1 indicating the maximum field of view of the photographing unit 110 on the monitor 156 (in the figure, “Overlay-ROI Editable Area” of “CCD”) is checked. By the operation of the visual field range display unit 163, the maximum limit display F <b> 1 is superimposed and displayed on the reference image 171 based on the size of the maximum visual field range of the photographing unit 110 stored in the memory 167. Thus, it is possible to acquire the reference image 171 by confirming that the desired specimen S exists in the maximum restriction display F1.

  On the reference image 171, a minimum size display H of the observation image as shown in FIG. 4C may be superimposed and displayed. In this way, the ROI of the observation image can be specified in more detail. This is particularly effective when the ROI of the observation image is specified with the magnification of the reference image 171 increased.

  As shown in FIG. 9, when a predetermined numerical value is input to an item for controlling rotation of the observation unit GS57 on the monitor 156 (“Setting-Rot” in “LSM” in the figure), the control unit 165 controls the observation unit GS57. The scanning timing is controlled, and the reference image 171 rotated around the central axis of the visual field of the confocal observation unit 50 can be displayed. In this case, the maximum limit display F1 is updated and displayed in accordance with the rotated reference image 171 by the operation of the visual field range display unit 163.

  Further, as shown in FIG. 10, when a predetermined numerical value is input to an item for performing zoom control and pan control of the observation unit GS57 on the monitor 156 (“Setting-Zoom, Pan X, Pan Y” of “LSM” in the figure). The control unit 165 controls the swing angle and swing angle range of the observation unit GS57, changes the magnification of the reference image 171 (enlarges in the figure), and shifts and displays the region for acquiring the reference image 171. can do. In this case, by the operation of the visual field range display unit 163, the maximum limit display F1 is updated and displayed in accordance with the reference image 171 after the shift.

  Then, as shown in FIG. 11, when an item for designating the ROI of the observation image on the monitor 156 (“ROI-Rectangle” in “CCD” in the figure) is clicked, the region designation unit 161 takes an image taken by the CCD 111. The ROI of the work image (see reference numeral 170 in the figure) is designated on the reference image 171.

  In this case, the visual field range display unit 163 may display the arrangement direction (X direction, Y direction) of the pixels of the CCD 111 on the reference image 171. In this way, for example, the ROI 170 of the observation image is set small in the Y direction, and the number of lines in the partial transfer (subarray transfer) of the CCD 111 is reduced, thereby increasing the frame rate and reducing the observation time. Can do.

  The ROI designation by the stimulation unit 70 is also performed while viewing the reference image 171 on the monitor 156. Also in this case, the observation unit GS57 may be rotated, zoomed, or panned by the control unit 165. Further, the visual field range display unit 163 may display the maximum size display G and the minimum size display H superimposed on the reference image 171. In this case, the minimum size display H may be a display corresponding to the size of each micromirror of the stimulation unit DMD83, for example.

Next, the case where the stimulation unit 70 stimulates the specimen S in the ROI 170 designated on the reference image 171 will be described.
First, the connection unit filter turrets 128 </ b> A and 128 </ b> B are controlled by the PC 154, and the first optical path synthesis unit 127 </ b> C and the second optical path synthesis unit 127 </ b> D having appropriate wavelength characteristics are disposed between the optical paths between the microscope 10 and the stimulation unit 70. .

  In this case, the position of the maximum restriction display F1 is corrected by the operation of the visual field range display unit 163 based on the offset amount of the optical path combining units 127C and 127D stored in the memory 156. Thereby, the maximum restriction display F1 when the optical path synthesis units 127A and 127B as shown in FIG. 12A are arranged, and the optical path synthesis units 127C and 127D as shown in FIG. 12B are arranged. When the position of the maximum restriction display F1 on the reference image 171 is different when compared with the maximum restriction display F1 at the time, the ROI 170 can be effectively designated within the maximum visual field range of the stimulation unit 70. it can.

  In the stimulation unit 70, the light emitted from the stimulation unit mercury lamp 73 is incident on the stimulation unit DMD 83 via the stimulation unit shutter 75, the excitation filter 77, the stimulation unit condensing lens 79, and the stimulation unit first reflection mirror 81A. The The light incident on the stimulation unit DMD 83 is reflected by the micro mirror in the ON region, passes through the stimulation unit relay optical system 85 and the stimulation unit second BS 87B, passes through the second connection port 123B, and is incident on the microscope connection device 120.

  The light incident on the microscope connection device 120 passes through the third relay lens 129C to become a substantially parallel light beam, and is reflected by the second optical path synthesis unit 127D and the first optical path synthesis unit 127C. The light reflected by the first optical path combining unit 127C is collected by the first relay lens 129A, passes through the microscope connection port 121, and enters the microscope 10. The light incident on the microscope 10 is applied to the sample S in the ROI 170 designated by the region designation unit 161 via the microscope switching mirror 45, the imaging lens 27, and the objective lens 23. This stimulates the specimen S.

Next, a case where an image for observation of the specimen S in the ROI 170 designated on the reference image is acquired by the imaging unit 110 will be described.
First, the connection unit filter turrets 128 </ b> A and 128 </ b> B are controlled by the PC 154, and the first optical path combining unit 127 </ b> E having an appropriate wavelength characteristic is disposed between the optical paths between the microscope 10 and the imaging unit 110. Also in this case, the position of the maximum restriction display F1 is corrected based on the offset amount of the optical path synthesis unit 127E stored in the memory 156 by the operation of the visual field range display unit 163.

  In the photographing unit 110, the excitation light emitted from the microscope mercury lamp 37 is irradiated to the specimen S in the ROI 170 of the observation image designated by the region designation unit 161 through the microscope BS 39 and the objective lens 23. When fluorescence is generated in the specimen S by being irradiated with the excitation light, the fluorescence passes through the microscope BS 39 and the imaging lens 27 and is reflected by the microscope switching mirror 45, passes through the microscope connection port 121, and enters the microscope connection device 120. Incident.

  The fluorescence incident on the microscope connection device 120 passes through the first relay lens 129A, the first optical path synthesis unit 127E, and the second relay lens 129B, passes through the first connection port 123A, and enters the imaging unit 110. The fluorescence incident on the photographing unit 110 is photographed by the CCD 111 via the camera adapter 113. An observation image of the specimen S acquired by the CCD 111 is displayed on the monitor 156.

  As described above, according to the microscope apparatus 100 according to the present embodiment, on the reference image 171 acquired by the confocal observation unit 50, the observation image is acquired by the ROI 170 or the imaging unit 110 that is stimulated by the stimulation unit 70. By specifying the ROI 170 to be performed, it is possible to easily select the specimen S and perform desired light stimulation or observation. In this case, the visual field range display unit 163 displays the maximum restriction display F1 representing the maximum visual field range of the stimulation unit 70 or the imaging unit 110 superimposed on the reference image 171. The reference image 171 can be acquired after grasping.

  Therefore, even when the maximum visual field ranges of the units 50, 70, and 110 are different, it is possible to avoid re-acquisition of the reference image 171 in accordance with the desired ROI 170. As a result, the visual field range of each unit 50, 70, 110 can be fully utilized and work efficiency can be improved.

  In the present embodiment, the control unit 165 may function as a fitting unit that matches the size of the maximum visual field range of the stimulation unit 70 or the imaging unit 110 with the region from which the reference image 171 is acquired. In this way, as shown in FIG. 13, the maximum restriction display F1 can be displayed on the entire reference image 171. In this case, as shown in FIG. 14, by clicking, for example, “Fitting-Device B” of “LSM” on the monitor 156, the control unit 165 stores the maximum of the stimulation unit 70 or the imaging unit 110 stored in the memory 167. The scanning condition of the observation unit GS57 may be controlled based on the size of the visual field range. By displaying the maximum restriction display F1 on the entire reference image 171 in this way, it is possible to exclude an unnecessary range where the ROI cannot be specified from the reference image 171.

  In the present embodiment, the case where the image for observation of the sample S is acquired by the imaging unit 110 has been described. For example, the image for observation of the sample S is acquired using the resonance GS58 of the confocal observation unit 50. It is good. In this case, first, the reference image 171 is acquired using the observation unit GS57 of the confocal observation unit 50, and then the observation unit GS57 is switched to the resonance GS58, and the field-of-view range display unit 163 uses the resonance GS58. The maximum restriction display F1 indicating the maximum field of view of the confocal observation unit 50 may be displayed so as to be superimposed on the reference image 171.

  Since the resonance GS58 can be pan-controlled only in the Y direction and cannot be panned in the X direction, for example, as shown in FIG. 15, there is no limit on the maximum visual field range in the Y direction, but the X direction can be acquired at once. The maximum image size (maximum size display G) is the size of the maximum visual field range (maximum limit display F1). The visual field range in the Y direction when using the resonance GS 58 is the same as that when using the observation unit GS57 and can be offset by pan control within the range, but the visual field range in the X direction uses the observation unit GS57. Narrower than the case.

When the maximum size display G of the observation image is displayed on the reference image 171 by the operation of the visual field range display unit 163, the range enclosed by dotted lines in the X direction and the Y direction in FIG. The size display G is displayed. If the observation image of the sample S can be acquired at once, the working efficiency can be improved as compared with the case where the image is acquired multiple times. Therefore, by displaying the maximum size display G, the sample S having a size suitable for observation can be easily and reliably searched, and redesignation of the ROI of the observation image can be avoided.
Note that the control unit 165 may set the ROI small in the Y direction. By doing so, it is possible to shorten the time by increasing the frame rate.

  In the present embodiment, the case where the sample S is stimulated using the DMD stimulation unit DMD83 of the stimulation unit 70 has been described. However, for example, the sample S may be stimulated using the stimulation unit GS89.

Further, the present embodiment can be modified as follows.
For example, in the present embodiment, the field-of-view range display unit 163 displays the maximum restriction display F1 representing the maximum field-of-view range of the units 110, 50, and 70 on the reference image 171. For example, as shown in FIGS. 16A and 16B, the display unit 163 is common to all the units 50, 70, and 110 based on the offset amounts of the optical path combining units 127A, 127B, 127C, 127D, and 127E. It is good also as displaying the common restriction | limiting display F2 showing a common visual field range.

  By doing in this way, the reference image 171 can be acquired after grasping in advance the specifiable stimulation region and observation image acquisition region by the common restriction display F2 displayed by the visual field range display unit 163. . In this case, for example, when the common restriction display F2 when the optical path synthesis units 127A and 127B shown in FIG. 16A are arranged and the optical path synthesis units 127C and 127D shown in FIG. 16B are arranged. As shown in the common restriction display F2, a constant common restriction display F2 can always be superimposed on the reference image 171 even when the optical path combining units 127A, 127B, 127C, 127D, and 127E are switched. it can. For example, this is particularly effective when the sample S is continuously stimulated or observed while switching the optical path synthesis units 127A, 127B, 127C, 127D, and 127E.

  In this modification, the visual field range display unit 163 may display the maximum size display G and the minimum size display H on the reference image 171 together with the common restriction display F2, or may display the observation unit GS57. The scanning direction (X direction and Y direction) and the arrangement direction of the pixels of the CCD 111 (X direction and Y direction) may be displayed on the reference image 171. The control unit 165 may function as a fitting unit that matches the size of the common visual field range common to all the units 50, 70, and 110 with the region from which the reference image 171 is acquired. In this way, the common restriction display F2 can be displayed on the entire reference image 171. The PC 154 causes the field-of-view range display unit 163 to display the maximum limit display F1 that represents the maximum field-of-view range for each of the units 50, 70, and 110, or the common limit that represents the common field-of-view range common to the units 50, 70, and 110. It may be possible to arbitrarily select whether to display the display F2.

As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.
For example, the maximum restriction display F1 or the common restriction display F2 may be displayed or not displayed on the reference image 171 by switching ON / OFF.
In the above embodiment, the confocal observation unit 50, the stimulation unit 70, and the imaging unit 110 are connected to the microscope connection device 120. However, the microscope connection device 120 includes two unit connection ports, and the microscope 10 includes An observation optical system or a stimulation optical system may be connected. Further, the microscope connection device 120 may include three or more unit connection ports, and three or more observation optical systems and stimulation optical systems may be connected to the microscope 10.

50 Confocal observation unit (observation optical system)
57 Observation unit GS (scanning means)
58 Resonant GS (scanning means)
70 Stimulation unit (stimulation optical system)
110 Imaging unit (observation optical system)
111 CCD (imaging device)
127A, 127B, 127C, 127D, 127E Optical path combiner 128A, 128B Connection unit filter turret (switching means)
161 Area designation section (area designation means)
163 Field-of-view range display (field-of-view range display means)
167 Memory (offset amount storage means)
170 ROI (acquisition area, stimulation area)
F1 Maximum limit display (display showing the maximum field of view)
F2 Common restriction display (display showing common visual field range)
S specimen

Claims (8)

An observation optical system capable of acquiring an image of the specimen;
A stimulation optical system for irradiating the specimen with light to stimulate the specimen;
Area designating means for designating a stimulation area to be stimulated by the stimulation optical system on an image acquired by the observation optical system;
A microscope apparatus comprising: a field-of-view range display unit that superimposes and displays a display representing the maximum field-of-view range of the stimulation optical system on the image. A plurality of optical path combining units that combine the optical path of the observation optical system and the optical paths of the plurality of stimulation optical systems;
Switching means capable of selectively inserting and removing the optical path combining unit in the optical path;
An offset amount storage means for storing an offset amount between the optical paths for each of the optical path combining units;
The microscope apparatus according to claim 1, wherein the visual field range display unit corrects a display position representing the maximum visual field range of the stimulation optical system based on the offset amount of the optical path synthesis unit switched by the switching unit. .   The microscope apparatus according to claim 1, further comprising a fitting unit that matches an acquisition area of the image with the maximum visual field range of the stimulation optical system. An observation optical system capable of acquiring an image of the specimen;
A plurality of stimulation optical systems for stimulating the sample by irradiating with light;
Area designating means for designating a stimulation area to be stimulated by the stimulation optical system on an image acquired by the observation optical system;
A plurality of optical path synthesis units for synthesizing the optical path of the observation optical system and the optical path of the stimulation optical system;
Switching means capable of selectively inserting and removing the optical path combining unit in the optical path;
An offset amount storage means for storing an offset amount between the optical paths for each of the optical path combining units;
A microscope apparatus comprising: a visual field range display unit configured to display a display representing a common visual field range common to all the stimulation optical systems based on an offset amount of each of the optical path combining units.   The microscope apparatus according to claim 4, further comprising a fitting unit that matches the image acquisition region with the common visual field range.   The said visual field range display means superimposes and displays the display showing the magnitude | size of the largest range which can be stimulated at once by the said stimulus optical system on the said image. Microscope device.   The microscope apparatus according to any one of claims 1 to 6, wherein the visual field range display unit displays a display representing a size of a minimum range that can be stimulated by the stimulation optical system on the image. The stimulation optical system includes scanning means for scanning light two-dimensionally on the specimen,
The microscope apparatus according to any one of claims 1 to 7, wherein the visual field range display unit displays a scanning direction of the scanning unit on the image.

Images