JP2012013995A - Scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress deterioration of a specimen at the time of fluorescence observation by optical stimulation.SOLUTION: When a specimen 17 is observed, stimulation light emitted from a stimulation laser unit 15 is reflected in a double-sided mirror 52A and deflected by a scanning unit 53. On the other hand, excitation light emitted from an excitation laser unit 16 is reflected in a side of the double-sided mirror 52A different from the side where the stimulation light is reflected and deflected by a scanning unit 54. The stimulation light and the excitation light are combined on a half mirror 55A and irradiated to the specimen 17. Reflecting the stimulation light and the excitation light in different sides of the double-sided mirror 52A and then combining the both light on the half mirror 55A in this way allows the stimulation light and the excitation light to be brought from different point light sources. The present invention can be applied to a scanning confocal microscope.

Description

  The present invention relates to a scanning microscope capable of suppressing deterioration of a specimen when performing fluorescence observation with light stimulation.

  2. Description of the Related Art Conventionally, there is known a scanning microscope that acquires an observation image of a specimen by scanning the specimen with excitation light while stimulating the specimen to be observed with stimulation light (see, for example, Patent Document 1).

  In this scanning microscope, the stimulation light and the excitation light separated by the dichroic mirror are combined by the subsequent dichroic mirror via different scanners and irradiated on the specimen. Thereby, the scan of the stimulation light and the scan of the excitation light can be performed independently.

  For example, in such observation using a scanning microscope, a fluorescent reagent called PA-GFP (Photo Activatable-Green Fluorescent Protein) is introduced into a cell as a specimen, and a strong light amount of 405 nm is stimulated to a part of the cell. Light is irradiated. Then, the appearance of changes in the periphery of the site stimulated by the stimulation light in the cell can be captured at high speed while being excited by the excitation light of 488 nm.

  On the other hand, a technique called FRAP (Fluorescence Recovery After Photobleaching) is known as a technique for capturing the diffusion phenomenon of fluorescent protein GFP introduced into cells. This FRAP is a technique for observing fluorescence by irradiating a portion of cells with intense stimulation light of 488 nm and exciting the state of the surroundings changed by the stimulation light with excitation light of the same wavelength, 488 nm.

International Publication No. WO2008 / 004336

  However, when attempting to observe a specimen using FRAP with the scanning microscope described above, the laser light of 488 nm from the same light source is separated by a half mirror into excitation light and stimulation light. The amount of light could not be adjusted independently. Therefore, if you increase the amount of stimulation light and give a strong stimulus to the cell, the amount of excitation light for observing the reaction due to the stimulation also increases, and it can be applied to parts other than the part you want to stimulate in the cell. Strong light will be irradiated. If it does so, extra fading will arise and a cell will deteriorate.

  The present invention has been made in view of such a situation, and is intended to suppress the deterioration of a specimen when performing fluorescence observation while performing light stimulation.

  The scanning microscope of the present invention is incident from a first scanning unit that scans a specimen to be observed with incident light, a second scanning unit that scans the specimen with incident light, and a first point light source. The first light is reflected by a reflection surface and is incident on the first scanning unit, and the second light incident from a second point light source different from the first point light source is the reflection surface. Reflecting by another different reflecting surface and entering the second scanning unit, and transmitting at least a part of the first light incident from the first scanning unit and the second scanning unit The apparatus includes a combining unit that irradiates the sample with the first light and the second light by reflecting at least a part of the second light incident from the scanning unit.

  According to the present invention, it is possible to suppress the deterioration of a specimen when performing fluorescence observation while performing light stimulation.

It is a figure which shows the structural example of one Embodiment of the observation system to which this invention is applied. It is a figure which shows the optical characteristic of a dichroic mirror. It is a figure which shows the other structural example of an observation system. It is a figure which shows the other structural example of an observation system.

  Embodiments to which the present invention is applied will be described below with reference to the drawings.

<First Embodiment>
[Configuration of observation system]
FIG. 1 is a diagram showing a configuration example of an embodiment of an observation system to which the present invention is applied. This observation system includes a computer 11, a controller 12, a microscope 13, a confocal head 14, a stimulation laser unit 15, and an excitation laser unit 16.

  A confocal head 14 for confocal observation is connected to the microscope 13, so that the microscope 13 functions as a scanning confocal microscope, and a specimen 17 placed on a stage (not shown) is used. It is possible to observe fluorescence while stimulating light.

  The confocal head 14 includes a stimulation laser unit 15 that emits stimulation light for light stimulation and an excitation laser unit 16 that emits excitation light for fluorescence observation through an optical fiber 18 and an optical fiber 19. It is connected. The user can operate the computer 11 to control the controller 12 to operate the confocal head 14 to the excitation laser unit 16.

  The stimulation laser unit 15 is provided with a laser light source 31 and a laser light source 32 for emitting laser light as stimulation light. The stimulation light emitted from the laser light source 31 is reflected by a mirror 33, and further, a dichroic mirror. 34 is reflected. On the other hand, the stimulation light emitted from the laser light source 32 passes through the dichroic mirror 34 as it is. The laser light source 31 and the laser light source 32 emit stimulation light having different wavelengths, and it is possible to emit stimulation light having one or both wavelengths.

  When laser light having the same wavelength is emitted from the laser light source 31 and the laser light source 32, a half mirror is disposed on the optical path of the laser light instead of the dichroic mirror 34, and the laser light source 31 and the laser light source 32 are used. Are combined in the same optical path.

  Further, the stimulating light from the dichroic mirror 34 is appropriately wavelength-selected and intensity-modulated by the acousto-optic filter 35, and is guided to the optical fiber 18 by the collimator lens 37 through the shutter 36. Then, the stimulation light incident on the optical fiber 18 enters the confocal head 14 through the optical fiber 18.

  Similarly, the excitation laser unit 16 includes a laser light source 41, a laser light source 42, a mirror 43, a dichroic mirror 44, an acoustooptic filter 45, a shutter 46, and a collimating lens 47. These laser light source 41 to collimating lens 47 correspond to the laser light source 31 to collimating lens 37 provided in the stimulation laser unit 15 and have the same functions as these members.

  Also in the excitation laser unit 16, excitation light having different wavelengths is emitted from the laser light source 41 and the laser light source 42, and excitation light having either one or both wavelengths is emitted. It is possible. The excitation light condensed by the collimator lens 47 is guided to the optical fiber 19 and enters the confocal head 14.

  Stimulation light incident on the confocal head 14 via the optical fiber 18 from the stimulation laser unit 15 enters the optical path selection unit 52 through the collimator lens 51. That is, the connection portion between the optical fiber 18 and the confocal head 14 becomes a point light source, and the stimulation light from this point light source is collected by the collimator lens 51 and enters the optical path selection unit 52.

  The optical path selection unit 52 is provided on the optical path of the stimulation light and the excitation light, and selects the optical path of the incident light. That is, the optical path selection unit 52 includes a turret that is rotationally driven by a motor, and an optical member that is disposed on the optical path of stimulation light and excitation light and is held by the turret.

  The turret of the optical path selection unit 52 holds, as optical members, a double-sided mirror 52A in which both the front surface and the back surface are mirrors and a hollow hollow block 52B that allows incident light to pass as it is. Light such as stimulation light and excitation light that has entered the optical path selection unit 52 is guided to the scanning unit 53 or the scanning unit 54 by the wavelength of the light and an optical member disposed on the optical path of the light.

  For example, in the example of FIG. 1, since the double-sided mirror 52A is arranged on the optical path of the stimulation light, the stimulation light incident on the optical path selection unit 52 from the collimating lens 51 is reflected by the double-sided mirror 52A and scanned. Is incident on. In addition, the optical member held by the turret of the optical path selection unit 52 may be a dichroic mirror, a base glass, a half mirror, or the like.

  As described above, if the double-sided mirror 52A is arranged on the optical path, the laser light whose wavelength and light amount are independently controlled can be introduced into the optical path of the confocal head 14 from the outside of the confocal head 14 and synthesized. it can.

  The stimulation light that has entered the scanning unit 53 is deflected by the scanning unit 53 and enters the optical path selection unit 55. For example, the scanning unit 53 includes two galvano scanners that deflect stimulation light.

  The optical path selection unit 55 has the same configuration as the optical path selection unit 52, and the optical path selection unit 55 holds a half mirror 55A and a dichroic mirror 55B. In the example of FIG. 1, since the half mirror 55A is disposed on the optical path of the stimulation light, a part of the stimulation light incident on the optical path selection unit 55 is transmitted through the half mirror 55A, and the imaging lens 56 and The specimen 17 is irradiated through the objective lens 57.

  Thereby, the sample 17 is stimulated by the stimulation light. Note that the turret of the optical path selection unit 55 may also hold a mirror, an elemental glass, a hollow block, or the like as an optical member. Further, the collimating lens 51 is movable in the optical path direction of the stimulation light, that is, the optical axis direction of the collimating lens 51. When the collimating lens 51 is moved in the optical axis direction, the condensing of the stimulation light in the specimen 17 is performed. The position changes in the optical axis direction of the objective lens 57. Thereby, it becomes possible to light-stimulate the arbitrary position of the sample 17 in the depth direction (the optical axis direction of the objective lens 57) with the stimulation light.

  Further, the excitation light incident on the confocal head 14 via the optical fiber 19 from the excitation laser unit 16 enters the dichroic mirror 59 through the collimator lens 58. That is, the connection portion between the optical fiber 19 and the confocal head 14 becomes a point light source, and the excitation light from this point light source is collected by the collimator lens 58, reflected by the dichroic mirror 59, and incident on the optical path selection unit 52.

  In the example of FIG. 1, since the double-sided mirror 52A is disposed on the optical path of the excitation light, the excitation light that has entered the optical path selection unit 52 is reflected by the double-sided mirror 52A and enters the scanning unit 54. Here, the stimulation light and the excitation light are incident on different reflecting surfaces of the double-sided mirror 52A and reflected.

  The excitation light incident on the scanning unit 54 is deflected by the scanning unit 54 and enters the optical path selection unit 55. For example, the scanning unit 54 includes a galvano scanner that deflects excitation light and a resonant scanner. According to the scanning unit 54, scanning can be performed at a higher speed than the scanning unit 53.

  Further, part of the excitation light incident on the optical path selection unit 55 from the scanning unit 54 is reflected by the half mirror 55A of the optical path selection unit 55, and is irradiated on the specimen 17 through the imaging lens 56 and the objective lens 57.

  When the sample 17 is irradiated with excitation light, fluorescence is generated from the sample 17, and the fluorescence enters the optical path selection unit 55 through the objective lens 57 and the imaging lens 56. In the example of FIG. 1, since the half mirror 55A is arranged on the optical path for detecting fluorescence, half of the fluorescence is reflected by the half mirror 55A, and further descanned by the scanning unit 54 to the optical path selection unit 52. Incident.

  Further, the fluorescence incident on the optical path selection unit 52 is reflected by the double-sided mirror 52A of the optical path selection unit 52, passes through the dichroic mirror 59, and is collected by the condenser lens 60. The dichroic mirror 59 reflects light having the excitation light wavelength and transmits light having the fluorescence wavelength.

  Further, the fluorescence condensed by the condenser lens 60 enters the photodetector 62 through the pinhole 61 and is received. The pinhole 61 is disposed at the focal position of the objective lens 57, that is, at a position conjugate with the observation surface of the sample 17, so that only the fluorescence condensed at the position of the pinhole 61 enters the photodetector 62. Has been made.

  The photodetector 62 receives incident fluorescence and photoelectrically converts it, thereby converting the fluorescence into an electric signal indicating the received light intensity of the fluorescence. An electrical signal obtained by the photoelectric conversion is supplied from the photodetector 62 to the controller 12. Based on the electrical signal supplied from the photodetector 62, the controller 12 generates an observation image that is an image of the observation surface of the specimen 17 and supplies the observation image to the computer 11. In addition, the computer 11 displays the observation image supplied from the controller 12 on the display.

[Explanation 1 of operation of observation system]
Next, the operation of the observation system in FIG. 1 will be described.

  First, a case where the specimen 17 is scanned with the excitation light having the wavelength of 488 nm while the specimen 17 is stimulated with the stimulation light having the wavelength of 488 nm, and the specimen 17 is fluorescently observed will be described.

  In this case, when the user operates the computer 11 to instruct the start of observation of the specimen 17, the controller 12 operates the optical path selection unit 52 and the optical path selection unit 55 in accordance with the instruction from the computer 11. The optical path selection unit 52 rotates the turret based on the control of the controller 12, and places the double-sided mirror 52A on the optical paths of the stimulation light and the excitation light. Further, the optical path selection unit 55 rotates the turret based on the control of the controller 12, and arranges the half mirror 55A on the optical paths of the stimulation light and the excitation light.

  In addition, the controller 12 causes the stimulation laser unit 15 and the excitation laser unit 16 to emit stimulation light and excitation light having a wavelength of 488 nm, and controls the acoustooptic filter 35 and the acoustooptic filter 45 to generate stimulation light and excitation light. Adjust the amount of excitation light. Thus, in the observation system, the light amount adjustment of the stimulation light and the excitation light having the same wavelength can be performed independently. Therefore, it is possible to prevent the light applied to the specimen 17 from becoming stronger than necessary, and as a result, it is possible to suppress the deterioration of the specimen 17 due to fading or the like.

  Stimulation light emitted from the stimulation laser unit 15 passes through the optical fiber 18 and the collimating lens 51, is reflected by the double-sided mirror 52 A, and enters the scanning unit 53. Then, the stimulation light from the double-sided mirror 52A is deflected by the scanning unit 53, passes through the half mirror 55A, the imaging lens 56, and the objective lens 57, and is irradiated onto the sample 17. At this time, the scanning unit 53 scans the observation surface of the specimen 17 with the stimulation light by deflecting the stimulation light.

  Further, the excitation light emitted from the excitation laser unit 16 is reflected by the dichroic mirror 59 through the optical fiber 19 and the collimator lens 58, and enters the double-sided mirror 52A. The excitation light incident on the double-sided mirror 52A is reflected on a surface different from the surface on which the stimulation light of the double-sided mirror 52A is reflected, and is further deflected by the scanning unit 54. The excitation light deflected by the scanning unit 54 is reflected by the half mirror 55A, passes through the imaging lens 56 and the objective lens 57, and irradiates the sample 17. At this time, the scanning unit 54 scans the observation surface of the specimen 17 with the excitation light by deflecting the excitation light.

  As described above, in the observation system, the stimulation light and the excitation light incident from different point light sources are reflected by different surfaces of the double-sided mirror 52A, are incident on the scanning unit 53 and the scanning unit 54, and are synthesized by the half mirror 55A. Thus, the light amount adjustment of the stimulation light and the excitation light can be performed independently.

  Further, in the observation system, the stimulation light and the excitation light are scanned by different scanning units, so that a desired part of the specimen 17 can be stimulated and at the same time, the excitation light can be irradiated to a part different from the part to be stimulated. Thereby, at the time of the light stimulation which irradiates the specific part of the specimen 17 with the stimulation light, it is not necessary to irradiate the entire specimen 17 with the extra laser light, so that the specimen 17 can be prevented from fading. In addition, the degree of freedom of observation can be improved.

  In this way, when the sample 17 is irradiated with excitation light, fluorescence is generated from the sample 17, and this fluorescence passes through the objective lens 57 and the imaging lens 56 and is reflected by the half mirror 55 </ b> A, and further the scanning unit 54. Is descanned. Then, the fluorescence emitted from the scanning unit 54 is reflected by the double-sided mirror 52A, passes through the dichroic mirror 59 through the pinhole 61, and is received by the photodetector 62.

  When the fluorescence is photoelectrically converted, an electrical signal corresponding to the amount of received fluorescence is supplied from the photodetector 62 to the controller 12, so the controller 12 generates an observation image from this electrical signal and sends it to the computer 11. Supply. As a result, the user can observe the specimen 17 by looking at the observation image displayed on the computer 11.

  In the above description, the example in which the optical path selection unit 55 arranges the half mirror 55A on the optical path has been described. In this example, the amount of fluorescent light from the sample 17 is reduced to half in the half mirror 55A. Therefore, in order to reduce the loss of the amount of fluorescent light, the optical path selection unit 55 may arrange the dichroic mirror 55B on the optical path.

  For example, assuming that the wavelength of the fluorescence is 500 nm or more, the dichroic mirror 55B transmits half of the incident light having a wavelength of less than 490 nm and reflects the remaining half as shown in FIG. Thus, it has a property of reflecting light having a wavelength of 500 nm or more. If such a dichroic mirror 55B is arranged on the optical path, most of the fluorescence incident from the specimen 17 is reflected by the dichroic mirror 55B, and a decrease in the amount of fluorescent light can be suppressed.

  In FIG. 2, the vertical axis indicates the reflectance of light on the reflecting surface of the dichroic mirror 55B, and the horizontal axis indicates the wavelength of light. In the example of FIG. 2, the reflectance of the light having a wavelength between 490 nm and 500 nm is higher as the wavelength is longer.

[Description of operation of observation system 2]
Further, when the sample 17 is irradiated with only the excitation light without performing optical stimulation, the controller 12 is arranged on the optical path depending on which of the scanning unit 54 and the scanning unit 53 is used to scan the excitation light. The optical member for selecting the optical path is switched.

  For example, when scanning of the excitation light is performed by the scanning unit 54, the controller 12 causes the optical path selection unit 52 to place a double-sided mirror 52A or a mirror (not shown) on the optical path of the excitation light, and also to the optical path selection unit 55. A mirror (not shown) is arranged on the optical path of the excitation light.

  Then, the excitation light emitted from the excitation laser unit 16 is reflected by the dichroic mirror 59 through the optical fiber 19 and the collimator lens 58 and enters the optical path selection unit 52. Then, the excitation light incident on the optical path selection unit 52 is reflected by the mirror of the optical path selection unit 52, deflected by the scanning unit 54, and enters the optical path selection unit 55. Further, the excitation light incident on the optical path selection unit 55 is reflected by the mirror of the optical path selection unit 55, and is irradiated on the specimen 17 through the imaging lens 56 and the objective lens 57.

  Further, the fluorescence generated in the specimen 17 enters the dichroic mirror 59 through an optical path opposite to the excitation light. That is, the fluorescence is reflected by the mirror of the optical path selection unit 55, descanned by the scanning unit 54, and further reflected by the mirror of the optical path selection unit 52. Then, the fluorescence is received by the photodetector 62 through the dichroic mirror 59 through the pinhole 61.

  Further, for example, when the scanning of the excitation light is performed by the scanning unit 53, the controller 12 causes the optical path selection unit 52 to place the hollow block 52B on the optical path of the excitation light, and the optical path selection unit 55 also includes a hollow block (not shown). Are arranged on the optical path of the excitation light.

  Then, the excitation light emitted from the excitation laser unit 16 is reflected by the dichroic mirror 59 and then passes through the hollow block 52B and enters the scanning unit 53 as it is. This excitation light is deflected by the scanning unit 53 and irradiated on the specimen 17 through the hollow block of the optical path selection unit 55, the imaging lens 56, and the objective lens 57.

  Further, the fluorescence generated in the specimen 17 enters the dichroic mirror 59 through an optical path opposite to the excitation light. That is, the fluorescence passes through the hollow block of the optical path selection unit 55, is descanned by the scanning unit 53, and further passes through the hollow block 52B. Then, the fluorescence that has passed through the hollow block 52B is received by the photodetector 62 through the dichroic mirror 59 through the pinhole 61.

  Further, for example, the laser beam may be emitted only from the excitation laser unit 16, and the optical path selection unit 52 and the optical path selection unit 55 may arrange the half mirror and the half mirror 55A on the optical path. In this case, using the scanning unit 53 and the scanning unit 54, it is possible to scan the specimen 17 with laser beams having different optical paths having the same wavelength.

  For example, in such a case, laser light transmitted through the optical path selection unit 52 is used as stimulation light, and laser light reflected by the optical path selection unit 52 is used as excitation light. In this example, for example, if an acoustooptic filter is disposed between the optical path selection unit 52 and the scanning unit 53 and an acoustooptic filter is disposed between the optical path selection unit 52 and the scanning unit 54, The light amount adjustment of the stimulation light and the excitation light can be performed independently.

  As described above, in the observation system, the optical path selection unit is arranged on the optical path of the light so that the excitation light and the stimulation light pass through the optical path suitable for the observation purpose of the specimen 17, that is, enter the desired scanning unit. 52 and an optical member for optical path selection held in the optical path selector 55 are selectively arranged. Thereby, the freedom degree of observation of the sample 17 can be improved with a simple configuration.

[Description of operation of observation system 3]
Next, KAEDE, which is a fluorescent protein, is introduced into the cell as the specimen 17, and the specimen 17 is scanned with excitation light having a wavelength of 488 nm while stimulating the specimen 17 with stimulation light having a wavelength of 405 nm. The case of observation will be described. The observed fluorescence wavelength is 500 nm or more.

  In such a case, the controller 12 causes the optical path selection unit 52 to place the double-sided mirror 52A on the optical path of the stimulation light and the excitation light, and causes the optical path selection unit 55 to place the dichroic mirror 55B on the stimulation light and It arrange | positions on the optical path of excitation light. Then, the controller 12 causes the stimulation laser unit 15 to emit stimulation light having a wavelength of 405 nm, and causes the excitation laser unit 16 to emit excitation light having a wavelength of 488 nm.

  Then, the stimulation light emitted from the stimulation laser unit 15 is reflected by the double-sided mirror 52 </ b> A through the optical fiber 18 and the collimating lens 51, and enters the scanning unit 53. Then, the stimulation light from the double-sided mirror 52A is deflected by the scanning unit 53 and passes through the dichroic mirror 55B to the objective lens 57 and is irradiated onto the sample 17. At this time, the scanning unit 53 scans the observation surface of the specimen 17 with the stimulation light by deflecting the stimulation light.

  The excitation light emitted from the excitation laser unit 16 is reflected by the double-sided mirror 52A through the optical fiber 19 through the dichroic mirror 59 and deflected by the scanning unit 54. The excitation light deflected by the scanning unit 54 passes through the dichroic mirror 55B through the objective lens 57 and is applied to the sample 17. At this time, the scanning unit 54 scans the observation surface of the specimen 17 with the excitation light by deflecting the excitation light.

  Here, the dichroic mirror 55B has a characteristic of transmitting incident light having a wavelength of less than 410 nm and reflecting incident light having a wavelength of 450 nm or more.

  When such a dichroic mirror 55B is arranged on the optical path, the stimulation light having a wavelength of 405 nm is transmitted through the dichroic mirror 55B, and the excitation light having a wavelength of 488 nm and the fluorescence having a wavelength of 500 nm or more are reflected by the dichroic mirror 55B. Will do. Therefore, also in this case, it is possible to suppress a decrease in the amount of fluorescent light in the dichroic mirror 55B.

  In this way, when the sample 17 is irradiated with excitation light, fluorescence is generated from the sample 17, and this fluorescence passes through the objective lens 57 and the imaging lens 56 and is reflected by the dichroic mirror 55 </ b> B, and is further scanned. Is descanned. Then, the fluorescence emitted from the scanning unit 54 is reflected by the double-sided mirror 52A, passes through the dichroic mirror 59 through the pinhole 61, and is received by the photodetector 62.

  When fluorescence is photoelectrically converted by the photodetector 62 to obtain an electric signal, the controller 12 generates an observation image from the electric signal, and the computer 11 displays the observation image.

  Since the excitation laser unit 16 is provided with the laser light source 41 and the laser light source 42, the excitation light and the excitation light are emitted from the excitation laser unit 16 when the wavelengths of the stimulation light and the excitation light are different. You may do it.

  In such a case, for example, the optical path selection unit 52 arranges a dichroic mirror (not shown) on the optical path of the stimulation light and the excitation light, and the optical path selection unit 55 places the dichroic mirror 55B on the optical path of the stimulation light and the excitation light. Deploy.

  Accordingly, one of the stimulation light and the excitation light incident on the optical path selection unit 52 from the dichroic mirror 59 is incident on the scanning unit 53 by the dichroic mirror disposed on the optical path, and the other light is scanned. Can be made incident. Similarly, in the dichroic mirror 55 </ b> B, the stimulation light and the excitation light that have entered through different optical paths can be combined and incident on the imaging lens 56.

<Second Embodiment>
[Configuration of observation system]
In the above description, it has been described that the stimulation light and the excitation light are incident on the confocal head 14 from the external stimulation laser unit 15 and the excitation laser unit 16, but both the light sources of the stimulation light and the excitation light, or Either one of the light sources may be provided in the confocal head 14.

  For example, when a light source of stimulation light is provided in the confocal head 14, the observation system is configured as shown in FIG. In FIG. 3, parts corresponding to those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

  In the observation system of FIG. 3, a laser light source 91 is provided inside the confocal head 14 instead of the stimulation laser unit 15 of the observation system of FIG. 1, and other configurations are the same as those of the observation system of FIG. It is said that.

  The laser light source 91 is a point light source such as a laser diode, for example, and emits stimulation light used for optical stimulation of the specimen 17 based on the control of the controller 12. The stimulus light emitted from the laser light source 91 is collected by the collimator lens 51 and enters the optical path selection unit 52.

  The operation of the observation system of FIG. 3 is the same as that of the observation system of FIG. 1 except that the light source that emits stimulation light is the laser light source 91, and thus the description of the operation is omitted.

<Third Embodiment>
[Configuration of observation system]
Further, in the example of FIG. 1, it has been described that the stimulation light and the excitation light from different laser units are incident on the confocal head 14, but the stimulation light and the excitation light are incident on the confocal head 14 from one laser unit. You may do it.

  In such a case, the observation system is configured as shown in FIG. 4, for example. In FIG. 4, parts corresponding to those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

  The observation system of FIG. 4 is provided with a laser unit 121 that emits stimulation light and excitation light instead of the stimulation laser unit 15 and excitation laser unit 16 of the observation system of FIG. This is the same as the observation system of FIG.

  The laser unit 121 is provided with a laser light source 131 and a laser light source 132 that emit laser light of the same wavelength. The laser light emitted from the laser light source 131 is reflected by the mirror 133 and further by the half mirror 134. Part of it is reflected. On the other hand, part of the laser light emitted from the laser light source 132 passes through the half mirror 134.

  Thus, the laser beams emitted from the laser light source 131 and the laser light source 132 are combined in the same optical path by the half mirror 134 and enter the half mirror 135. Part of the laser light incident on the half mirror 135 from the half mirror 134 is reflected by the half mirror 135 and incident on the mirror 136, and the remaining laser light passes through the half mirror 135.

  The laser beam reflected by the half mirror 135 is reflected by the mirror 136, is appropriately wavelength-selected and intensity-modulated by the acousto-optic filter 137, passes through the shutter 138, and is guided to the optical fiber 18 by the collimator lens 139. The laser light incident on the optical fiber 18 enters the collimating lens 51 of the confocal head 14 as stimulation light.

  On the other hand, the laser light transmitted through the half mirror 135 is appropriately wavelength-selected and intensity-modulated by the acousto-optic filter 140, and is guided to the optical fiber 19 by the collimator lens 142 through the shutter 141. The laser light incident on the optical fiber 19 enters the collimating lens 58 of the confocal head 14 as excitation light.

  As described above, in the laser unit 121, the laser light having the same wavelength is separated by the half mirror 135, so that the separated laser light can be incident on the confocal head 14 as stimulation light and excitation light, respectively.

  In the laser unit 121, since the acoustooptic filter 137 and the acoustooptic filter 140 are provided in the optical path of the laser beam separated by the half mirror 135, the intensity of the stimulating light and the excitation light in one laser unit 121 is provided. The (light quantity) can be adjusted independently.

  The operation of the observation system of FIG. 4 is the same as that of the observation system of FIG. 1 except that the light source that emits stimulation light and excitation light is the laser unit 121, and thus the description of the operation is omitted. For example, when using a laser beam having a wavelength of 488 nm as stimulation light and excitation light and performing fluorescence observation while stimulating the specimen 17, the double-sided mirror 52A and the half mirror 55A are on the optical path of the stimulation light and excitation light. Placed in.

  In the observation system of FIG. 4, if laser beams having different wavelengths are emitted from the laser light source 131 and the laser light source 132, laser beams having different wavelengths can be used as stimulation light and excitation light. In such a case, the half mirror 134 combines laser beams having different wavelengths from the laser light source 131 and the laser light source 132.

  Then, the acousto-optic filter 137 emits only the laser light having the wavelength to be the stimulation light out of the laser light incident from the mirror 136 with the intensity specified by the controller 12, and passes through the shutter 138 and the collimating lens 139. The light is incident on the optical fiber 18.

  Similarly, the acousto-optic filter 140 emits only the laser light having the wavelength to be the excitation light out of the laser light incident from the half mirror 135 with the intensity specified by the controller 12, and the shutter 141 and the collimating lens 142. Through the optical fiber 19.

  Note that the scanning unit 54 may be disposed at the position of the scanning unit 53, and the scanning unit 53 may be disposed at the position of the scanning unit 54. Further, both the scanning unit 53 and the scanning unit 54 may be configured by two galvano scanners, or may be configured by two resonant scanners.

  Further, UV light having a wavelength of 400 nm or less, near-infrared light having a wavelength of 700 nm or more, or ultrashort pulse laser light other than visible light may be used as stimulation light or excitation light.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

  11 Computer, 12 Controller, 13 Microscope, 14 Confocal Head, 15 Stimulation Laser Unit, 16 Excitation Laser Unit, 17 Sample, 35 Acoustooptic Filter, 45 Acoustooptic Filter, 51 Collimator Lens, 52 Optical Path Selector, 53 Scanning Unit, 54 scanning unit, 55 optical path selection unit, 91 laser light source, 121 laser unit, 135 half mirror, 137 acoustooptic filter, 140 acoustooptic filter

Claims (7)

A first scanning unit that scans a specimen to be observed with incident light;
A second scanning unit that scans the sample with incident light;
The first light incident from the first point light source is reflected by the reflecting surface and incident on the first scanning unit, and the second light incident from the second point light source different from the first point light source. A reflecting portion that reflects the light of the light from another reflecting surface different from the reflecting surface and enters the second scanning portion;
By transmitting at least part of the first light incident from the first scanning unit and reflecting at least part of the second light incident from the second scanning unit, the first light is transmitted. And a combining unit that irradiates the specimen with the second light and the second light. While holding a plurality of first optical members including the reflecting portion, any one of the plurality of first optical members is arranged on an optical path of the first light and the second light. The scanning microscope according to claim 1, further comprising a first optical path selection unit. The scanning according to claim 2, wherein the first optical path selection unit arranges the reflection unit on the optical path when the wavelengths of the first light and the second light are the same. Type microscope. While holding a plurality of second optical members including the combining unit, any one of the plurality of second optical members is disposed on the optical path of the first light and the second light. The scanning microscope according to claim 3, further comprising a second optical path selection unit. A condensing unit that is disposed between the first point light source and the first scanning unit and changes a condensing position of the first light in the sample in an optical path direction of the first light; The scanning microscope according to claim 1, comprising: a scanning microscope. A separation unit that separates laser light emitted from a light source into the first light and the second light;
A first light amount adjustment unit that adjusts a light amount of the first light from the separation unit and makes the first light after the light amount adjustment enter the reflection unit;
The apparatus further comprises: a second light amount adjustment unit that adjusts a light amount of the second light from the separation unit and causes the second light after the light amount adjustment to enter the reflection unit. 2. A scanning microscope according to 1. The scanning microscope according to claim 1, wherein the other reflection surface of the reflection unit is a back surface of the reflection surface.

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