JP2019204026A - Microscope system - Google Patents

Abstract

To provide a microscope system capable of acquiring, in real time, an image of a sample serving as an observation object using a simple optical system by a different observation method even when it includes a spinning disk type confocal scanner.SOLUTION: A microscope system 1 for acquiring an image of a sample 11 serving as an observation object comprises: a microscope device 10 including a light-emitting unit 12 which irradiates the sample 11 with first illumination light L1; a light-emitting device 20 which irradiates the sample 11 with second illumination light L2; an optical unit 30 optically coupled to the microscope device 10 and the light-emitting unit 20 and including a scanning unit 31 which scans the sample with the second illumination light L2; and an imaging device 40 which detects observation light containing information on the image of the sample 11. The optical unit 30 includes an optical element 36 for observation arranged on an image-side pupil conjugate surface S2 conjugated with a pupil surface S1 of an object lens 15 of the microscope device 10.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a microscope system.

  A technique related to a microscope system that acquires an image of a sample to be observed by an arbitrary method including a phase difference observation method and a confocal observation method is conventionally known.

  For example, Patent Document 1 discloses a microscope apparatus that employs an external phase difference observation method in order to perform phase difference observation and confocal observation using the same objective lens.

  For example, Patent Document 2 discloses a technique for forming a phase difference image by image processing.

JP 2010-175774 A Japanese translation of PCT publication No. 2002-529689

  For example, a microscope system may include a spinning disk confocal scanner to obtain 3D information of a sample. When such a confocal scanner is used to acquire an image of a sample by different observation methods such as phase contrast observation in addition to confocal observation, in the prior art, the entire microscope system including the microscope apparatus and the confocal scanner is used. The optical system becomes complicated. For example, in the microscope apparatus described in Patent Document 1, in order to perform external phase difference observation, it is necessary to add two relay lenses in the microscope apparatus body to extend the imaging optical path. This complicates the optical system included in the microscope apparatus, and as a result, complicates the optical system of the entire microscope system including the confocal scanner.

  In the technique described in Patent Document 2, it is necessary to capture a plurality of images necessary for image processing while changing the focal plane of the microscope, and real-time phase difference observation is difficult.

  An object of the present disclosure is to provide a microscope system that can acquire an image of a sample to be observed in real time by a different observation method using a simple optical system even when a spinning disk type confocal scanner is provided. To do.

  A microscope system according to some embodiments is a microscope system that acquires an image of a sample to be observed, a microscope apparatus having a light emitting unit that irradiates the sample with first illumination light, and a second that is applied to the sample. Information relating to a light emitting device for irradiating illumination light, an optical unit optically coupled to the microscope device and the light emitting device, and having a scanning unit that scans the sample with the second illumination light, and information about the sample image And an optical device for observation arranged on an image-side pupil conjugate plane conjugate with a pupil plane of an objective lens included in the microscope apparatus. Thereby, even if it has a spinning disk type confocal scanner, a microscope system can acquire the image of the sample used as an observation object in real time by a different observation method using a simple optical system.

  In one embodiment, the observation light includes first observation light including transmitted light and diffracted light from the sample based on the first illumination light, and second light including fluorescence from the sample based on the second illumination light. A phase plate that shifts the phase of one of the transmitted light and the diffracted light included in the first observation light so that mutual interference increases, and And a fluorescent filter that selectively transmits the fluorescence included in the second observation light. Thereby, the microscope system can reduce the shift between the image by confocal observation and the image by phase difference observation using a simple optical system, and can realize phase difference observation in real time.

  In one embodiment, the observation light includes first observation light including transmitted light from the sample based on the first illumination light, and second observation light including fluorescence from the sample based on the second illumination light. The optical element for observation includes an optical path mask that blocks a part of the optical path of at least one of the transmitted light included in the first observation light and the fluorescence included in the second observation light, and the second A fluorescence filter that selectively transmits the fluorescence contained in the observation light. Thus, the microscope system can realize real-time observation while realizing a function corresponding to image processing by an optical action using a simple optical system.

  In one embodiment, the optical path mask blocks an approximately half area of a cross section of the first observation light that is substantially orthogonal to the optical axis of the first observation light. In the optical path mask, a portion that blocks the optical path and other portions May be formed symmetrically with respect to the optical axis of the first observation light. Thereby, the microscope system can acquire the image which emphasized the three-dimensional information of the sample in transmitted light observation.

  In one embodiment, the optical path mask may block at least a center of a cross section of the observation light substantially perpendicular to an optical axis of the observation light. Thereby, the microscope system can acquire an image in which illumination unevenness is suppressed or an image in which a fine structure is emphasized in at least one of transmitted light observation and confocal observation.

  In one embodiment, the optical unit further includes a processing device, and the optical unit includes an optical element switching unit that switches an optical element arranged on the optical path of the observation light to one of the optical elements included in the observation optical element. In addition, the processing device controls switching by the optical element switching unit to any one of the optical elements included in the optical element for observation, and the corresponding optical element is synchronized with the switching so that the corresponding optical element is the light. When arranged on the road, the light output of the light emitting unit and the light emitting device of the microscope device are turned on, and the first observation light and the second observation light are synchronized with the switching, and the imaging device You may make it detect alternately. Thereby, the microscope system can obtain, for example, an image obtained by phase difference observation and an image obtained by confocal observation alternately using a simple optical system. The microscope system can use a simple optical system, for example, to alternately obtain an image by transmitted light observation and an image by confocal observation when the observation light is acted on by an optical path mask.

  In one embodiment, the optical unit further includes a branching optical element that separates the first observation light and the second observation light, and the imaging device captures the first observation light and the second observation light. May be detected in parallel. This increases the imaging throughput.

  In one embodiment, two different imaging devices may be provided, and the first observation light and the second observation light may be detected by the two different imaging devices, respectively. Thereby, in a microscope system, the apparatus suitable for each imaging method is applicable with respect to two imaging devices.

  In one embodiment, one imaging device may be provided, and the first observation light and the second observation light may be detected at different portions on the detection surface of the imaging device. This reduces the cost of manufacturing a microscope system for parallel imaging.

  In one embodiment, the scanning unit of the optical unit is disposed at a condensing disk in which a plurality of condensing optical elements are disposed, and a condensing position of the second illumination light by the plurality of condensing optical elements, respectively. A pinhole disk having a plurality of pinholes, and rotating the pinhole disk, the second illumination light that has been condensed by the condensing optical element and passed through the corresponding pinhole is The sample may be scanned. Accordingly, in the microscope system, the scanning unit can scan the sample at high speed using the second illumination light. Therefore, the time required for confocal observation is shortened, and the efficiency in confocal observation is improved.

  According to the present disclosure, it is possible to provide a microscope system that can acquire an image of a sample to be observed in real time by different observation methods using a simple optical system even when the spinning disk type confocal scanner is provided. .

It is a schematic diagram which shows the structure of the microscope system which concerns on 1st Embodiment. It is a schematic diagram which shows the structure in the case of performing only confocal observation using the optical unit of FIG. It is the schematic diagram which showed only the optical path regarding the confocal observation of the microscope system of FIG. It is the schematic diagram which showed only the optical path regarding the phase difference observation of the microscope system of FIG. It is a schematic diagram which shows the structure of the microscope system which concerns on 2nd Embodiment. It is a schematic diagram which shows the structure of the microscope system which concerns on 3rd Embodiment. It is a schematic diagram which shows the structure of the microscope system which concerns on 4th Embodiment. It is a schematic diagram which shows the example of the image imaged based on the 1st observation light in the microscope system of FIG. It is a schematic diagram which shows the 1st modification of the microscope system of FIG. It is a schematic diagram which shows the example of the image imaged based on the 1st observation light in the microscope system of FIG. FIG. 10 is a schematic diagram illustrating an example of an image when an optical path mask is arranged in the optical path of the second observation light in the microscope system of FIG. 9. It is a schematic diagram which shows the 2nd modification of the microscope system of FIG. It is a schematic diagram which shows the example of the image imaged based on the 1st observation light in the microscope system of FIG. It is a schematic diagram which shows the example of an image when the optical path mask is arrange | positioned in the optical path of 2nd observation light in the microscope system of FIG. It is a schematic diagram which shows the structure of the microscope system which concerns on 5th Embodiment. It is a schematic diagram which shows the structure of the microscope system which concerns on 6th Embodiment.

  Hereinafter, an embodiment of the present disclosure will be mainly described with reference to the accompanying drawings. For convenience of explanation, only the processing apparatus 50 described later is shown in FIGS. 1, 3, and 4. In other drawings, the illustration of the processing device 50 is omitted.

(First embodiment)
When a conventionally known phase difference observation method is used, an observer can observe, for example, colorless and transparent cells to be observed without being stained with a fluorescent reagent. However, in order to apply the phase difference observation method to a microscope apparatus, a dedicated objective lens having a phase plate inside is required. This objective lens dedicated to phase difference observation has a low light transmittance due to the phase plate and ND filter provided therein, and is not suitable for confocal observation for observing weak fluorescence. Accordingly, it is necessary to use different objective lenses for confocal observation and phase difference observation. At this time, there is a problem in that there is a difference between an image obtained by confocal observation and an image obtained by phase difference observation due to differences in magnification and aberration between the two.

  As one conventional technique for solving such a problem, an external phase difference observation method as described in Patent Document 1 is known. In the external phase difference observation method, the phase plate and the ND filter necessary for phase difference observation are arranged on the image conjugate pupil plane on the image side conjugate with the pupil plane of the objective lens, so that the observer can perform confocal observation. Phase difference observation can be performed using the same objective lens. Therefore, the deviation between the image by confocal observation and the image by phase difference observation is reduced.

  However, when a conventional external phase difference observation method as described in Patent Document 1 is used, two relay lenses are newly added between an imaging device such as a camera or an eyepiece and an imaging lens, and a phase plate is provided. It is necessary to form the pupil conjugate plane of the objective lens on which the optical element including the lens is disposed in the microscope apparatus main body. As a result, the imaging optical path in the microscope apparatus main body is extended, and the image quality is deteriorated based on the aberration of each of the two added relay lenses and the transmittance of the observation light.

  As one conventional technique for solving such problems related to the conventional external phase difference observation method, a phase difference image forming technique based on image processing as described in Patent Document 2 is known. In this technique, a plurality of images having different focal planes of a microscope are captured, and an image in which phase information is recovered by performing image operations such as Fourier transform on these images is obtained. Patent Document 2 discloses an example in which an image is picked up while moving a focal plane by moving a microscope stage on which a sample to be observed is placed by a motor or the like. On the other hand, an inverted microscope for observing a sample to be observed from below is generally used in a microscope for observing a living organism. In such an inverted microscope, imaging is performed by moving an objective lens up and down with a motor or the like with respect to an observation target.

  As described above, it is common to observe an image by changing the relative distance between the observation target and the objective lens. An image similar to the image obtained by the phase difference observation method can be acquired by recovering the phase from a plurality of images captured by such a method. In such a phase difference image forming technique by image processing, it is not necessary to use special illumination light and an objective lens unlike the phase difference observation method, and the optical system becomes simple.

  However, in the above-described configuration in the phase difference image forming technology based on image processing, it is necessary to perform imaging while synchronizing a motor that drives a microscope stage on which a sample to be observed is placed and an imaging device such as a camera that captures an image. In addition, the configuration of the imaging system becomes complicated. In addition, a phase difference image is first formed by imaging a plurality of images by changing the relative distance between the observation target and the objective lens, and then performing image processing using these images. Therefore, when the phase difference image forming technology by image processing is used, it is difficult to acquire a phase difference image in real time, and it is necessary to repeat operations related to imaging and image processing.

  Even if the microscope system 1 according to the first embodiment of the present disclosure has a spinning disk type confocal scanner, an image of the sample 11 to be observed can be obtained in real time using different observation methods using a simple optical system. You can get it. More specifically, the microscope system 1 can reduce the shift between the image by confocal observation and the image by phase difference observation using a simple optical system, and can realize phase difference observation in real time. In the microscope system 1, phase difference observation is possible without adding a new optical system to the inside of the microscope apparatus 10 as in the past by using an optical system originally provided in the optical unit 30 described later. In other words, external phase difference observation is facilitated by optically coupling the optical unit 30 to the microscope apparatus 10 without having to arrange a complicated optical system inside the microscope apparatus 10 in order to adapt to the external phase difference observation method. To be done. By performing external phase difference observation, the microscope system 1 does not need to use a dedicated objective lens as in the prior art, and the manufacturing cost is reduced.

  FIG. 1 is a schematic diagram illustrating a configuration of a microscope system 1 according to the first embodiment. FIG. 2 is a schematic diagram showing a configuration when only confocal observation is performed using the optical unit 30 of FIG. FIG. 3 is a schematic diagram showing only an optical path related to confocal observation of the microscope system 1 of FIG. FIG. 4 is a schematic diagram showing only an optical path related to phase difference observation of the microscope system 1 of FIG. The configuration of the microscope system 1 according to the first embodiment will be mainly described with reference to FIGS. 1 to 4.

  Referring to FIG. 1, the microscope system 1 according to the first embodiment includes a microscope device 10, a light emitting device 20, an optical unit 30, an imaging device 40, and a processing device 50 as large components.

  The microscope apparatus 10 includes a light emitting unit 12 that irradiates the sample 11 to be observed with the first illumination light L1. The first illumination light L1 is illumination light for performing phase difference observation on the sample 11. The microscope apparatus 10 includes a ring diaphragm 13 that squeezes the first illumination light L1 emitted from the light emitting unit 12 in a ring shape. The ring diaphragm 13 has a slit 13a formed concentrically. The microscope apparatus 10 includes a condenser lens 14 that condenses the first illumination light L <b> 1 narrowed in a ring shape on the sample 11. The microscope apparatus 10 includes an objective lens 15 and an imaging lens 16. The objective lens 15 and the imaging lens 16 optically act on the first observation light O1 including the transmitted light T1 and the diffracted light D1 from the sample 11 based on the first illumination light L1.

  The microscope apparatus 10 may include one ring diaphragm 13, the objective lens 15, and the imaging lens 16 one by one, or a plurality of each. In any case, the ring diaphragm 13 and the objective lens 15 arranged in the optical paths of the first illumination light L1 and the first observation light O1, respectively, are such that the size of the slit 13a of the ring diaphragm 13 is the magnification of the objective lens 15. And a numerical aperture.

  The light emitting device 20 irradiates the sample 11 with the second illumination light L2 different from the first illumination light L1. The second illumination light L2 is illumination light for performing confocal observation on the sample 11. More specifically, the second illumination light L2 passes through the inside of the optical unit 30, enters the inside of the microscope apparatus 10 from the imaging lens 16, passes through the objective lens 15, and is irradiated on the sample 11. . When the sample 11 is irradiated with the second illumination light L2, the second observation light O2 including the fluorescence F2 from the sample 11 based on the second illumination light L2 is emitted from the sample 11 toward the objective lens 15. Hereinafter, the first observation light O1 and the second observation light O2 described above are simply referred to as “observation light”.

  The optical unit 30 includes, for example, a spinning disk type confocal scanner. The optical unit 30 is optically coupled to the microscope apparatus 10 and the light emitting apparatus 20. For example, the optical unit 30 is attached to the microscope apparatus 10 and the light emitting apparatus 20. The optical unit 30 includes a scanning unit 31 that scans the irradiation position of the second illumination light L <b> 2 on the sample 11. The scanning unit 31 includes a condensing disk 31a having a plurality of condensing optical elements and a pinhole having a plurality of pinholes respectively disposed at the condensing positions of the second illumination light L2 by the plurality of condensing optical elements. And a disk 31b. For example, in the condensing disk 31a, a large number of microlenses are arranged in a spiral shape. In the pinhole disk 31b, pinholes are arranged at the condensing positions of the second illumination light L2 corresponding to the arrangement positions of the microlenses. The scanning unit 31 further includes a motor 31c that rotates the pinhole disk 31b. As the pinhole disk 31b is rotated by the motor 31c, the scanning unit 31 scans the sample 11 with the second illumination light L2 collected by the condensing optical element and passing through the corresponding pinhole.

  By combining the condensing disk 31a and the pinhole disk 31b of the optical unit 30, most of the second illumination light L2 that has passed through the condensing disk 31a passes through the pinhole disk 31b. Irradiation intensity increases. Further, the reflection of the second illumination light L2 at a portion other than the pinhole in the pinhole disk 31b is suppressed. Therefore, the second illumination light L2 is prevented from entering the optical path of the observation light, and noise with respect to the image of the sample 11 is reduced.

  The observation light emitted from the sample 11 passes through the pinhole disk 31b. When phase difference observation is performed, the pinhole disk 31b moves the base on which the motor 31c is installed together with the motor 31c and the condensing disk 31a in a direction perpendicular to the optical axis A of the observation light by a linear actuator or the like. Thus, it may be configured to be retractable from the optical path of the observation light. By retracting the pinhole disk 31b from the optical path of the observation light, the amount of observation light increases.

  The optical unit 30 includes a first relay lens 33 and a second relay lens 34 of an infinity correction optical system that forms an image of the observation light reflected by the beam splitter 32 on the imaging device of the imaging device 40. The detection surface of the sample 11, the pinhole disk 31b, and the imaging device 40 is optically conjugate.

  The optical unit 30 includes a filter wheel 35 between the first relay lens 33 and the second relay lens 34. A plurality of observation optical elements 36 are attached to the filter wheel 35. For example, the observation optical element 36 includes a phase plate 37 and a fluorescent filter 38. The filter wheel 35 selectively switches one phase plate 37 or one fluorescent filter 38 by rotational movement of the observation light with respect to the optical axis A. In other words, the filter wheel 35 functions as an optical element switching unit that switches an optical element arranged on the optical path of the observation light to either the phase plate 37 or the fluorescent filter 38. Although the filter wheel 35 has been described as selectively switching the optical element by the rotational movement of the observation light with respect to the optical axis A, the switching method is not limited to this. The filter wheel 35 may selectively switch the optical element by parallel movement using a linear actuator or the like.

  The phase plate 37 includes a phase ring 37a that shifts the phase of one of the transmitted light T1 and the diffracted light D1 included in the first observation light O1 so that mutual interference increases. For example, in the phase ring 37a, a quarter wavelength plate that shifts the phase of light by a quarter wavelength and an ND filter that absorbs light are integrally formed in a ring shape. In the phase plate 37, portions other than the phase ring 37a are transparent to the first observation light O1.

  The phase plate 37 is disposed on the image side pupil conjugate surface S2 conjugate with the pupil surface S1 of the objective lens 15 included in the microscope apparatus 10. The phase plate 37 is disposed at a position optically conjugate with the ring diaphragm 13. The image side pupil conjugate plane S <b> 2 corresponds to the image side focal plane of the first relay lens 33.

  The size of the first observation light O1 projected onto the phase ring 37a of the phase plate 37 corresponds to the size of the slit 13a of the ring diaphragm 13. The size when the slit 13 a of the ring diaphragm 13 is projected onto the phase plate 37 varies depending on the magnification of the objective lens 15. Therefore, when the microscope apparatus 10 includes a plurality of objective lenses 15, the filter wheel 35 may include a plurality of corresponding phase plates 37. Thereby, phase difference observation is possible for each of the plurality of objective lenses 15.

  The fluorescent filter 38 selectively transmits the fluorescent light F2 included in the second observation light O2. The filter wheel 35 may have a plurality of fluorescent filters 38 corresponding to the type of fluorescent reagent used for the sample 11. Thereby, multi-color imaging corresponding to each of a plurality of types of fluorescent reagents can be performed in confocal observation.

  The imaging device 40 includes an arbitrary imaging element that detects observation light including information related to the image of the sample 11. For example, the imaging device 40 may be a camera including a two-dimensional sensor array, and includes an arbitrary imaging element such as a CCD and a CMOS.

  The processing device 50 includes one or more processors. The processing device 50 is a computer device used by an observer, and includes any device such as a PC (Personal Computer), a tablet PC, a mobile phone such as a smartphone and a feature phone, and a personal digital assistant (PDA). Including. The processing device 50 is connected to each component included in the microscope system 1 and controls the operation of the entire microscope system 1. For example, the processing device 50 controls switching between the phase plate 37 and the fluorescent filter 38 by the filter wheel 35. In synchronization with such switching, the processing device 50 turns on the light output from the light emitting unit 12 and the light emitting device 20 of the microscope device 10 when the corresponding optical element is arranged on the optical path of the observation light. . The processing device 50 causes the imaging device 40 to alternately detect the first observation light O1 and the second observation light O2 in synchronization with such switching.

  More specifically, the processing device 50 includes a phase plate 37 having a phase ring 37a corresponding to the magnification and numerical aperture of the objective lens 15 being used when the light output from the light emitting unit 12 is on. It selects and arrange | positions in the optical path of the 1st observation light O1, and acquires the image by phase difference observation. Similarly, when the light output from the light emitting device 20 is on, the processing device 50 selects the fluorescent filter 38 corresponding to the wavelength of the second illumination light L2 and the fluorescence spectrum of the sample 11 to be observed. Are arranged in the optical path of the second observation light O2, and an image by confocal observation is acquired.

  The configuration and function of the optical unit 30 when only confocal observation is performed using the optical unit 30 of FIG. 1 will be mainly described with reference to FIG.

  The optical unit 30 is originally a unit that performs confocal observation on the sample 11 in combination with the light emitting device 20. At this time, only the at least one fluorescent filter 38 is attached to the filter wheel 35 of the optical unit 30, and the phase plate 37 is not attached. The optical unit 30 transfers the image of the sample 11 formed on the pinhole disk 31b to the detection surface of the imaging device 40 by the two first relay lenses 33 and the second relay lens 34 constituting the infinity correction optical system. To do.

  A microscope system 1 shown in FIG. 1 is an optical system in which the optical unit 30 shown in FIG. 2 and a microscope apparatus 10 for external phase difference observation are combined. As described above, the image-side focal plane of the first relay lens 33 of the optical unit 30 is a conjugate plane of the ring diaphragm 13. Therefore, by installing the phase plate 37 on this conjugate plane, external phase difference observation and confocal observation can be performed only with the optical system originally provided in the microscope apparatus 10 for external phase difference observation and the optical unit 30. It becomes possible. In addition, in the microscope system 1, it is not necessary to perform image processing, and it is not necessary to change the relative distance between the sample 11 to be observed and the objective lens 15. An image is obtained in real time.

  A confocal observation method using the microscope system 1 of FIG. 1 will be described with reference to FIG.

  The second illumination light L2 emitted from the light emitting device 20 passes through the condensing optical element of the condensing disk 31a, the beam splitter 32, the pinhole of the pinhole disk 31b, the imaging lens 16, and the objective lens 15, and passes through the sample. 11 is irradiated. The second observation light O2 based on the second illumination light L2 emitted from the sample 11 passes through the objective lens 15, the imaging lens 16, and the pinhole of the pinhole disk 31b again and is reflected by the beam splitter 32. Thereby, the second observation light O2 is separated from the second illumination light L2. The second observation light O2 passes through the first relay lens 33 and becomes a parallel light beam. The second observation light O2 passes through the fluorescent filter 38 and forms an image on the detection surface of the imaging device 40 by the second relay lens 34.

  A phase difference observation method using the microscope system 1 of FIG. 1 will be described with reference to FIG.

  The first illumination light L <b> 1 irradiated from the light emitting unit 12 of the microscope apparatus 10 is narrowed by the ring diaphragm 13 and is irradiated onto the sample 11 through the condenser lens 14. The first illumination light L1 that has reached the sample 11 is divided into transmitted light T1 that has traveled straight through the sample 11 and diffracted light D1 that has been diffracted and bent by the sample 11 that is a phase object. The diffraction phenomenon occurs at a portion where the refractive index is different. Accordingly, the diffracted light D1 is generated, for example, at the boundary between the microorganism and the solution and the internal structure of the microorganism, and includes shape information of the sample 11 that is a phase object.

  The first observation light O1 passes through the objective lens 15, the imaging lens 16, and the pinhole of the pinhole disk 31b and is reflected by the beam splitter 32. For example, the transmitted light T1 passes through the first relay lens 33 and becomes a ring-shaped parallel light beam. The transmitted light T1 passes through the phase ring 37a of the phase plate 37. Therefore, the phase of the transmitted light T1 is shifted by ¼ wavelength, and the amount of transmitted light T1 is reduced. On the other hand, most of the diffracted light D <b> 1 passes through the transparent portion of the phase plate 37. Therefore, the phase and light quantity of the diffracted light D1 hardly change. The transmitted light T1 and the diffracted light D1 are imaged on the detection surface of the imaging device 40 by the second relay lens.

  According to the microscope system 1 according to the first embodiment, an image of the sample 11 to be observed can be acquired in real time using a simple optical system. If the microscope system 1 according to the first embodiment is used, the observer can observe the living cells without staining colorless and transparent cells by phase difference observation. In the microscope system 1, since the confocal observation and the phase difference observation can be performed with the same objective lens 15, a shift between the image by the confocal observation and the image by the phase difference observation is suppressed. The microscope system 1 can realize the above-described effects by using an optical system originally provided in the microscope apparatus 10 and the optical unit 30.

  In the above description, the optical unit 30 has been described as having the condensing disk 31a, but is not limited thereto. The optical unit 30 may not have the condensing disk 31a. At this time, the manufacturing cost of the optical unit 30 is reduced, and the work process for performing optical adjustment between the condensing disk 31a and the pinhole disk 31b is reduced.

(Second Embodiment)
FIG. 5 is a schematic diagram illustrating a configuration of the microscope system 1 according to the second embodiment. Hereinafter, the configuration and functions of the microscope system 1 according to the second embodiment will be mainly described with reference to FIG. The microscope system 1 according to the second embodiment has two different imaging devices 40a and 40b, and is different from the first embodiment in that confocal observation and phase difference observation can be performed in parallel. The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted. Differences from the first embodiment will be mainly described.

  The fluorescence F2 based on the second illumination light L2 emitted from the sample 11, the transmitted light T1 and the diffracted light D1 based on the first illumination light L1, and the pinhole of the objective lens 15, the imaging lens 16, and the pinhole disk 31b. And is separated from the second illumination light L2 by the beam splitter 32. The fluorescence F2, the transmitted light T1, and the diffracted light D1 pass through the first relay lens 33 and pass through the barrier filter 39a. At this time, noise light mainly including reflected light of the second illumination light L2 reflected by the pinhole disk 31b and the motor 31c is suppressed by the barrier filter 39a. The barrier filter 39a may be switched by the rotation operation of the filter wheel 35a based on the setting of the observer, for example, according to the wavelength that the light emitting device 20 can irradiate.

  The fluorescence F2, the transmitted light T1, and the diffracted light D1 transmitted through the barrier filter 39a reach the branching optical element 39b. At this time, the fluorescence F2 is reflected by the branching optical element 39b. The fluorescence F2 reflected by the branching optical element 39b passes through the fluorescence filter 38 in a state where only light having a target wavelength is selected by the fluorescence filter 38. At this time, the fluorescent filter 38 may be switched by the rotation operation of the filter wheel 35b based on the setting of the observer, for example, according to the fluorescence spectrum of the fluorescent reagent used for the sample 11. The fluorescence F2 is imaged on the detection surface of the imaging device 40b by the second relay lens 34b.

  On the other hand, the transmitted light T1 and the diffracted light D1 are transmitted through the branching optical element 39b. The transmitted light T1 and the diffracted light D1 transmitted through the branching optical element 39b pass through the phase plate 37. At this time, the phase plate 37 may be switched by the rotation operation of the filter wheel 35c based on the setting of the observer according to the objective lens 15 and the ring diaphragm 13, for example. The transmitted light T1 and the diffracted light D1 are imaged on the detection surface of the imaging device 40a by the second relay lens 34a.

  As described above, the optical unit 30 of the microscope system 1 according to the second embodiment includes the branching optical element 39b that separates the first observation light O1 and the second observation light O2, and includes the imaging devices 40a and 40b. The first observation light O1 and the second observation light O2 are detected in parallel.

  According to the microscope system 1 according to the second embodiment, the same effects as those of the first embodiment can be obtained. In addition, in the microscope system 1, the observer can perform imaging by confocal observation and imaging by phase difference observation in parallel. This increases the imaging throughput. That is, the imaging efficiency by confocal observation and phase difference observation is improved. In addition, in the microscope system 1, an apparatus suitable for each imaging method can be applied to the two imaging apparatuses 40a and 40b. For example, the imaging device 40a that captures an image by phase difference observation may be a high-resolution camera, and the imaging device 40b that captures an image by confocal observation may be a high-sensitivity camera.

(Third embodiment)
FIG. 6 is a schematic diagram illustrating a configuration of the microscope system 1 according to the third embodiment. Hereinafter, the configuration and functions of the microscope system 1 according to the third embodiment will be mainly described with reference to FIG. The microscope system 1 according to the third embodiment is different from the first embodiment and the second embodiment in that confocal observation and phase difference observation can be performed in parallel with only one imaging device 40. The same components as those in the first embodiment and the second embodiment are denoted by the same reference numerals, and the description thereof is omitted. Differences from the first and second embodiments will be mainly described.

  The fluorescence F2 reflected by the branching optical element 39b passes through the fluorescence filter 38 in a state where only light having a target wavelength is selected by the fluorescence filter 38. Thereafter, the fluorescence F2 is reflected by the bending mirror 39c, passes through the combining mirror 39d, and forms an image on the detection surface of the imaging device 40 by the second relay lens 34.

  On the other hand, the transmitted light T1 and the diffracted light D1 transmitted through the branching optical element 39b pass through the phase plate 37. Thereafter, the transmitted light T1 and the diffracted light D1 are reflected by the bending mirror 39c, reflected by the combining mirror 39d, and imaged on the detection surface of the imaging device 40 by the second relay lens 34.

  The fluorescence F2, the transmitted light T1, and the diffracted light D1 are branched by the branching optical element 39b, passed through different optical paths, and then combined by the multiplexing mirror 39d. The second observation light O2 including the combined fluorescence F2 and the first observation light O1 including the transmitted light T1 and the diffracted light D1 are detected at different portions on the detection surface of the imaging device 40, respectively.

  According to the microscope system 1 according to the third embodiment, the same effects as those of the first embodiment and the second embodiment can be obtained. In addition, in the microscope system 1 according to the third embodiment, the observer performs imaging by confocal observation and imaging by phase difference observation in a state where the number of imaging devices 40 is reduced as compared to the second embodiment. Can be done in parallel. As a result, the cost of manufacturing the microscope system 1 aiming at parallel imaging is reduced.

(Fourth embodiment)
FIG. 7 is a schematic diagram illustrating a configuration of the microscope system 1 according to the fourth embodiment. FIG. 8 is a schematic diagram illustrating an example of an image captured based on the first observation light O1 in the microscope system 1 of FIG.

  Hereinafter, the configuration and functions of the microscope system 1 according to the fourth embodiment will be mainly described with reference to FIGS. 7 and 8. The microscope system 1 according to the fourth embodiment is different from the first embodiment in that the observation optical element 36 includes an optical path mask 37b instead of the phase plate 37 and the ring diaphragm 13 is removed. The same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted. Differences from the first embodiment will be mainly described.

  Conventionally, for example, when an image shown in FIG. 8 in which the shape of the sample 11 is shaded is captured, a differential interference method and a relief contrast method are known as observation methods. According to the differential interference method, colorless and transparent cells can be observed alive without staining. However, in order to realize the differential interference method, a polarizing element called an analyzer and a polarizer and a Wollaston prism are required, and there is a problem that an optical system becomes complicated. In addition, in the differential interference method, when a resin observation container is used, the phase of illumination light is disturbed by the birefringence of the resin, and a normal differential interference image cannot be obtained.

  As a technique for solving such a problem, a relief contrast method is known. According to the relief contrast method, a colorless and transparent cell can be observed alive without staining it using a resin observation container as in the case of the differential interference method. However, in order to realize the relief contrast method, a dedicated objective lens having a modulator inside, a polarizing plate, and a slit diaphragm are required, and the optical system remains complicated.

  According to the microscope system 1 according to the fourth embodiment, such a problem is solved, and even when the spinning disk type confocal scanner is provided, the sample 11 to be observed using a simple optical system. Images can be obtained in real time by different observation methods. More specifically, the microscope system 1 can realize real-time observation of an image in which the shape of the sample 11 is shaded using a simple optical system.

  In the microscope system 1 according to the fourth embodiment, unlike the first embodiment, the observation light includes first observation light O1 including transmitted light T1 from the sample 11 based on the first illumination light L1, and second illumination light L2. And the second observation light O2 including the fluorescence F2 from the sample 11 based on the above. That is, the first observation light O1 does not include the diffracted light D1 from the sample 11 based on the first illumination light L1. At this time, the observation optical element 36 selectively transmits the optical path mask 37b that blocks a part of the optical path of the transmitted light T1 included in the first observation light O1 and the fluorescence F2 included in the second observation light O2. And a filter 38.

  For example, the processing device 50 controls switching between the optical path mask 37 b and the fluorescent filter 38 by the filter wheel 35. In synchronization with such switching, the processing device 50 turns on the light output from the light emitting unit 12 and the light emitting device 20 of the microscope device 10 when the corresponding optical element is arranged on the optical path of the observation light. . The processing device 50 causes the imaging device 40 to alternately detect the first observation light O1 and the second observation light O2 in synchronization with such switching.

  The optical path mask 37b blocks a substantially half area of the cross section of the transmitted light T1 substantially orthogonal to the optical axis A. In the optical path mask 37b, the portion that blocks the optical path and the other portions are formed symmetrically with respect to the optical axis A. For example, the optical path mask 37b is formed in a substantially circular shape as a whole, and blocks the transmitted light T1 in a substantially half region that forms a substantially semicircle. The transmitted light T1 can pass through the optical path mask 37b only in the portion other than the portion that blocks the optical path in the optical path mask 37b.

  The optical path mask 37b is disposed on the Fourier plane S3 formed at an intermediate position between the first relay lens 33 and the second relay lens 34. At this time, the Fourier transform image of the sample 11 is projected on the position of the optical path mask 37b. That is, a Fourier transform image is formed in which the spatial frequency component in the image of the sample 11 is distributed on a plane orthogonal to the optical axis A around the optical axis A of the transmitted light T1. At this time, the spatial frequency increases as the distance from the optical axis A increases. The optical path mask 37b shaped as shown in FIG. 7 blocks one whole of the spatial frequency region formed symmetrically with the optical axis A in between.

  Referring to FIG. 8, according to the microscope system 1 according to the fourth embodiment, the shape of the sample 11 is shaded as compared with an image (without an optical path mask) when the optical path mask 37 b deviates from the optical path of the transmitted light T <b> 1. It is possible to obtain an image with a mark (with an optical path mask). That is, it is possible to acquire an image obtained by observing transmitted light with the three-dimensional information of the sample 11 enhanced. As described above, according to the microscope system 1 according to the fourth embodiment, the observer can alternately capture an image by normal confocal observation and an image by transmitted light observation in which the three-dimensional information of the sample 11 is emphasized. is there.

  As described above, the microscope system 1 according to the fourth embodiment can acquire an image obtained by observing transmitted light as an image in which the three-dimensional information of the sample 11 is emphasized by applying the optical path mask 37b to the first observation light O1. is there.

  The arrangement of the optical path mask 37b is not limited to the above-described content. The optical path mask 37b may be disposed, for example, at the position of the ring diaphragm 13 in FIG.

  By arranging the optical path masks 37b having various shapes on the Fourier plane S3 described above, the image projected on the imaging device 40 can be converted. A first modification and a second modification of the microscope system 1 according to the fourth embodiment described above will be described below.

  FIG. 9 is a schematic diagram showing a first modification of the microscope system 1 of FIG. FIG. 10A is a schematic diagram illustrating an example of an image captured based on the first observation light O1 in the microscope system 1 of FIG. FIG. 10B is a schematic diagram showing an example of an image when the optical path mask 37b is arranged in the optical path of the second observation light O2 in the microscope system 1 of FIG. FIG. 11 is a schematic diagram showing a second modification of the microscope system 1 of FIG. 12A is a schematic diagram illustrating an example of an image captured based on the first observation light O1 in the microscope system 1 of FIG. FIG. 12B is a schematic diagram illustrating an example of an image when the optical path mask 37b is arranged in the optical path of the second observation light O2 in the microscope system 1 of FIG.

  Referring to FIGS. 9 and 11, the optical path mask 37b of the microscope system 1 according to the first modification and the second modification blocks at least the center of the cross section of the transmitted light T1 substantially orthogonal to the optical axis A of the transmitted light T1. . In the microscope system 1 according to the first modification and the second modification, the areas of the portions that block the transmitted light T1 in the optical path mask 37b are different from each other. When the area of the portion that blocks the transmitted light T1 in the optical path mask 37b is small as in the first modification, the illumination unevenness included in the first illumination light L1 is suppressed. On the other hand, when the area of the portion that blocks the transmitted light T1 in the optical path mask 37b is large as in the second modification, it is possible to perform transmitted light observation that emphasizes the fine structure.

  For example, the optical path mask 37b shown in FIG. 9 is formed in a substantially circular shape as a whole, and blocks the transmitted light T1 at the center thereof. When the optical path mask 37b that blocks the center of the optical path of the transmitted light T1 is disposed on the Fourier plane S3 during the transmitted light observation, an image from which the low frequency components of the spatial frequency are removed is formed in the imaging device 40. Thereby, the illumination nonuniformity contained in the 1st illumination light L1 is suppressed.

  More specifically, as shown in FIG. 10A, an image (with an optical path mask) in which illumination unevenness is suppressed is compared with an image when the optical path mask 37b is out of the optical path of the transmitted light T1 (without an optical path mask). It can be acquired. That is, it is possible to acquire an image obtained by observing transmitted light in which uneven illumination included in the first illumination light L1 is suppressed.

  In the microscope system 1 according to the first modification, the same processing may be performed on an image by confocal observation. At this time, in the microscope system 1 according to the first modification, the observation optical element 36 may further include an optical path mask 37b similar to the above that blocks a part of the optical path of the fluorescence F2 included in the second observation light O2. . For example, the optical path mask 37b acting on the fluorescence F2 and the fluorescence filter 38 may be separately disposed at arbitrary positions where an image can be acquired by confocal observation, or as an optical element 38a integrated with each other. The filter wheel 35 may be attached. That is, the optical element 38a may have the function of the optical path mask 37b that blocks a part of the optical path of the fluorescence F2 and the function of the fluorescence filter 38.

  At this time, according to the microscope system 1 according to the first modified example, as shown in FIG. 10B, illumination unevenness is suppressed as compared with an image (without an optical path mask) when the optical path mask 37 b is out of the optical path of the fluorescence F2. The acquired image (with optical path mask) can be acquired. That is, it is possible to acquire an image by confocal observation in which illumination unevenness included in the second illumination light L2 is suppressed.

  For example, the processing device 50 controls switching between the optical path mask 37b and the optical element 38a by the filter wheel 35. In synchronization with such switching, the processing device 50 turns on the light output from the light emitting unit 12 and the light emitting device 20 of the microscope device 10 when the corresponding optical element is arranged on the optical path of the observation light. . The processing device 50 causes the imaging device 40 to alternately detect the first observation light O1 and the second observation light O2 in synchronization with such switching. The observer can alternately capture an image by transmitted light observation and an image by confocal observation in which uneven illumination is suppressed.

  In addition to the above, the microscope system 1 according to the first modification may be configured to be able to capture an image by normal transmitted light observation and an image by confocal observation in which illumination unevenness is suppressed. At this time, the optical path mask 37b disposed in the optical path of the transmitted light T1 is removed.

  As described above, in the microscope system 1 according to the first modification, the optical path mask 37b is applied to at least one of the first observation light O1 and the second observation light O2, so that the image by the transmitted light observation and the confocal observation are used. At least one of the images may be acquired as an image in which uneven illumination is suppressed.

  For example, the optical path mask 37b shown in FIG. 11 is formed in a substantially circular shape as a whole, and transmits the transmitted light T1 only at its outer edge. When the optical path mask 37b that transmits only the outer edge portion of the optical path of the transmitted light T1 is disposed on the Fourier plane S3 when observing the transmitted light, an image including only the high-frequency component of the spatial frequency is formed in the imaging device 40. The cutoff frequency of the optical path mask 37b in the second modification is higher than the cutoff frequency of the optical path mask 37b in the first modification. Thereby, the transmitted light observation which emphasized the fine structure can be performed.

  More specifically, as shown in FIG. 12A, an image (with an optical path mask) in which the fine structure is emphasized as compared with an image when the optical path mask 37b is out of the optical path of the transmitted light T1 (without an optical path mask). It can be acquired. That is, it is possible to acquire an image obtained by observing transmitted light in which the fine structure is emphasized.

  In the microscope system 1 according to the second modification, the same processing may be performed on an image by confocal observation. At this time, the above description in the microscope system 1 according to the first modification is applied to the configuration of the observation optical element 36, the processing by the processing device 50, and the like. According to the microscope system 1 according to the second modified example, as shown in FIG. 12B, an image in which the fine structure is emphasized as compared with an image in the case where the optical path mask 37 b is out of the optical path of the fluorescence F <b> 2 (no optical path mask). (With optical path mask) can be acquired. That is, it is possible to acquire an image by confocal observation in which the fine structure is emphasized.

  As described above, the microscope system 1 according to the second modification causes the optical path mask 37b to act on at least one of the first observation light O1 and the second observation light O2, similarly to the microscope system 1 according to the first modification. Thus, at least one of the image by transmitted light observation and the image by confocal observation may be acquired as an image in which the fine structure is emphasized.

(Fifth embodiment)
FIG. 13 is a schematic diagram illustrating a configuration of the microscope system 1 according to the fifth embodiment.

  Hereinafter, the configuration and functions of the microscope system 1 according to the fifth embodiment will be mainly described with reference to FIG. In the microscope system 1 according to the fifth embodiment, the phase plate 37 of the microscope system 1 according to the second embodiment is replaced with an optical path mask 37b, and the ring diaphragm 13 is removed. Therefore, in the microscope system 1 according to the fifth embodiment, the phase difference observation method in the second embodiment using the ring diaphragm 13 and the phase plate 37 is changed to the transmitted light observation method in the fourth embodiment using the optical path mask 37b. The image of the sample 11 is acquired by replacement. In the microscope system 1 according to the fifth embodiment, the confocal observation method in the second embodiment using the fluorescent filter 38 is the same as that in the first and second modifications of the fourth embodiment using the optical element 38a. An image of the sample 11 may be acquired instead of the focus observation method. As described above, the description of the second embodiment and the fourth embodiment is applied to the description of the configuration, function, and the like of the microscope system 1 according to the fifth embodiment.

(Sixth embodiment)
FIG. 14 is a schematic diagram illustrating a configuration of the microscope system 1 according to the sixth embodiment.

  Hereinafter, the configuration and functions of the microscope system 1 according to the sixth embodiment will be mainly described with reference to FIG. In the microscope system 1 according to the sixth embodiment, the phase plate 37 of the microscope system 1 according to the third embodiment is replaced with an optical path mask 37b, and the ring diaphragm 13 is removed. Therefore, in the microscope system 1 according to the sixth embodiment, the phase difference observation method in the third embodiment using the ring diaphragm 13 and the phase plate 37 is changed to the transmitted light observation method in the fourth embodiment using the optical path mask 37b. The image of the sample 11 is acquired by replacement. In the microscope system 1 according to the sixth embodiment, the confocal observation method in the third embodiment using the fluorescent filter 38 is used in the first modification and the second modification in the fourth embodiment using the optical element 38a. An image of the sample 11 may be acquired instead of the focus observation method. As described above, the description of the third embodiment and the fourth embodiment is applied to the description of the configuration, function, and the like of the microscope system 1 according to the sixth embodiment.

  It will be apparent to those skilled in the art that the present disclosure can be implemented in other predetermined forms other than the above-described embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the foregoing description is illustrative and not restrictive. The scope of the disclosure is defined by the appended claims rather than by the foregoing description. Some of all changes that fall within the equivalent scope shall be included therein.

  For example, the shape, arrangement, orientation, number, and the like of each component described above are not limited to the contents described above and illustrated in the drawings. The shape, arrangement, orientation, number, and the like of each component may be arbitrarily configured as long as the function can be realized.

DESCRIPTION OF SYMBOLS 1 Microscope system 10 Microscope apparatus 11 Sample 12 Light emission part 13 Ring aperture 13a Slit 14 Condenser lens 15 Objective lens 16 Imaging lens 20 Light emission apparatus 30 Optical unit 31 Scan part 31a Condensing disk 31b Pinhole disk 31c Motor 32 Beam splitter 33 1st Relay lens 34, 34a, 34b Second relay lens 35, 35a, 35b, 35c Filter wheel 36 Observation optical element 37 Phase plate 37a Phase ring 37b Optical path mask 38 Fluorescence filter 38a Optical element 39a Barrier filter 39b Branching optical element 39c Bending Mirror 39d Multiplexing mirror 40, 40a, 40b Imaging device 50 Processing device A Optical axis D1 Diffracted light F2 Fluorescence L1 First illumination light L2 Second illumination light O1 First observation light O2 Second observation light S1 Pupil plane S2 Image side pupil Conjugate surface S3 Fourier surface T1 Transmitted light

Claims (10)

A microscope system for acquiring an image of a sample to be observed,
A microscope apparatus having a light emitting unit for irradiating the sample with the first illumination light;
A light emitting device for irradiating the sample with the second illumination light;
An optical unit optically coupled to the microscope device and the light emitting device, and having a scanning unit that scans the sample with the second illumination light;
An imaging device for detecting observation light including information on an image of the sample;
With
The optical unit includes an observation optical element disposed on an image side pupil conjugate plane conjugate with a pupil plane of an objective lens included in the microscope apparatus.
Microscope system. The observation light is
First observation light including transmitted light and diffracted light from the sample based on the first illumination light;
Second observation light including fluorescence from the sample based on the second illumination light;
Including
The observation optical element is:
A phase plate that shifts the phase of any one of the transmitted light and the diffracted light included in the first observation light so that mutual interference increases;
A fluorescence filter that selectively transmits the fluorescence contained in the second observation light;
including,
The microscope system according to claim 1. The observation light is
First observation light including transmitted light from the sample based on the first illumination light;
Second observation light including fluorescence from the sample based on the second illumination light;
Including
The observation optical element is:
An optical path mask that blocks a part of the optical path of at least one of the transmitted light included in the first observation light and the fluorescence included in the second observation light;
A fluorescence filter that selectively transmits the fluorescence contained in the second observation light;
including,
The microscope system according to claim 1. The optical path mask blocks an area of approximately half of the cross section of the first observation light substantially orthogonal to the optical axis of the first observation light;
In the optical path mask, the part that blocks the optical path and the other part are formed symmetrically with respect to the optical axis of the first observation light.
The microscope system according to claim 3. The optical path mask interrupts at least the center of the cross section of the observation light substantially perpendicular to the optical axis of the observation light;
The microscope system according to claim 3. Further comprising a processing device;
The optical unit further includes an optical element switching unit that switches an optical element arranged on the optical path of the observation light to any one of the optical elements included in the observation optical element,
The processor is
Controlling switching to any optical element included in the observation optical element by the optical element switching unit,
In synchronization with the switching, when the corresponding optical element is disposed on the optical path, the light output from the light emitting unit and the light emitting device of the microscope device is turned on,
In synchronization with the switching, the imaging device alternately detects the first observation light and the second observation light.
The microscope system according to any one of claims 2 to 5. The optical unit further includes a branching optical element that separates the first observation light and the second observation light,
Detecting the first observation light and the second observation light in parallel by the imaging device;
The microscope system according to any one of claims 2 to 5. Two different imaging devices,
Detecting the first observation light and the second observation light by two different imaging devices, respectively.
The microscope system according to claim 7. Comprising the one imaging device;
Detecting the first observation light and the second observation light at different portions on a detection surface of the imaging device, respectively.
The microscope system according to claim 7. The scanning unit of the optical unit is
A condensing disk provided with a plurality of condensing optical elements;
A pinhole disk having a plurality of pinholes respectively disposed at the condensing position of the second illumination light by the plurality of condensing optical elements;
Have
When the pinhole disk rotates, the second illumination light that has been condensed by the condensing optical element and passed through the corresponding pinhole is scanned with respect to the sample.
The microscope system according to any one of claims 1 to 9.