JP2010266813A - Observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a scanning observation device capable of suppressing an influence of condensed laser light on an optical element, and also, achieving satisfactory observation performance. <P>SOLUTION: The observation device 100 includes: a stage 2 for placing a sample 1; an objective lens 3 for condensing the laser light on the sample 1; an illumination means 10 for guiding the laser light to the objective lens 3; a condensing position-scanning means 20 for scanning the sample light condensation position being a condensing position in the sample 1 in the optical axis direction; and a control means 40 which controls the illumination means 10 and the condensing position scanning means 20. In this case, the control means 40 controls the illumination means 10 and the condensing position scanning means 20 based on the intermediate condensing position being the condensing position in the illumination optical path, and a condensing restricting position to restrict the condensation of the laser light in the illumination optical path. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an observation apparatus, and more particularly to a scanning observation apparatus using a laser light source.

  There is a laser scanning type observation apparatus having a high resolution in the optical axis direction by the confocal effect. A laser scanning type observation device is suitable for observation inside a sample and acquisition of a three-dimensional image of the sample, and is widely used as a biological or industrial observation device.

  Such an observation apparatus generally has a configuration in which the focal position is scanned in the optical axis direction by driving an objective lens or a stage on which a sample is arranged. However, in such a configuration, driving the objective lens and the stage may cause the specimen to vibrate and affect the observation result. In addition, since the objective lens and the stage cannot be driven at high speed, it may be difficult to scan the specimen at high speed.

  In order to solve the above problems, in Patent Documents 1 to 3, other optical elements (hereinafter referred to as z scanning control elements) arranged in the illumination optical path without driving the objective lens and the stage are described. ), And a technique for scanning the focal position in the optical axis direction has been proposed.

Patent Document 1 discloses a technique for scanning a focal position in the optical axis direction using a reference lens and a reference mirror.
Patent Document 2 discloses a technique for scanning a focal position in the optical axis direction using a liquid crystal spatial light modulator.
Patent Document 3 discloses a technique for scanning a focal position in the optical axis direction using a deformable mirror.
By controlling the z-scanning control element using these techniques, the focal position on the specimen can be scanned without moving the objective lens or the stage.
Note that the techniques disclosed in Patent Document 2 and Patent Document 3 can also correct spherical aberration caused by the movement of the focal position.

International Publication No. 2008/078083 Pamphlet JP-A-11-326860 JP 2004-341394 A

By the way, since the laser light emitted from the laser light source has strong directivity and convergence, a condensing position on the illumination optical path (hereinafter referred to as an intermediate condensing position), a focal position, that is, Very strong light energy is generated at the light collection position in the specimen (hereinafter referred to as the specimen collection position). When such strong light energy is applied to the optical element, the optical element may be deformed and the observation apparatus may not be able to obtain good observation performance.
In addition, when a condensing position on the observation optical path (hereinafter referred to as an intermediate image position) is located on the optical element, dust and scratches on the optical element affect the image and degrade the observation performance. There is.
For this reason, the normal observation apparatus is designed so that the optical element inside the observation apparatus is not located at the intermediate condensing position or the intermediate image position.

However, in the case of a configuration in which the specimen condensing position is scanned by controlling the z scanning control element as in the technique described above, the intermediate condensing position and the intermediate image position also change. More specifically, the intermediate condensing position and the intermediate image position move about the square of the magnification of the objective lens with respect to the scanning amount of the sample condensing position. For this reason, the intermediate condensing position and the intermediate image position change greatly even when scanning a slight specimen condensing position, and the optical element is positioned at the intermediate condensing position and the intermediate image position during scanning. Become.
In light of the above circumstances, an object of the present invention is to provide a scanning observation apparatus that realizes good observation performance.

  According to a first aspect of the present invention, a stage on which a specimen is arranged, an objective lens that condenses laser light on the specimen, illumination means that guides the laser light to the objective lens, and a specimen collection that is a condensing position in the specimen. Based on the first scanning means that scans the optical position in the optical axis direction, the intermediate condensing position that is the condensing position in the illumination optical path, and the condensing restriction position where the condensing of the laser light is restricted, There is provided an observation apparatus including a control means for controlling one scanning means.

  According to a second aspect of the present invention, in the observation apparatus according to the first aspect, the control unit includes a scanning control unit that controls driving of the first scanning unit, and an intermediate condensing position calculation that calculates an intermediate condensing position. There is provided an observation apparatus including means, a light collection restriction position calculation means for calculating a light collection restriction position, and an illumination control means for controlling the illumination means.

  According to a third aspect of the present invention, in the observation apparatus according to the second aspect, the illumination unit includes a light source, and the illumination control unit is emitted from the light source when the condensing restriction position and the intermediate condensing position overlap. An observation device that reduces the intensity of laser light is provided.

  According to a fourth aspect of the present invention, in the observation apparatus according to the second aspect, the illumination unit includes a light source, and a light amount modulation unit disposed between the light source and the condensing restriction position, and the illumination control unit Provides an observation device that reduces the intensity of the laser light passing through the light amount modulation means when the condensing restriction position and the intermediate condensing position overlap.

  According to a fifth aspect of the present invention, in the observation apparatus according to the third aspect or the fourth aspect, the intensity of the laser light changes according to a position where the condensing restriction position and the intermediate condensing position overlap. I will provide a.

  According to a sixth aspect of the present invention, in the observation apparatus according to the second aspect, the illumination unit includes a light source and a shutter device disposed between the light source and the condensing restriction position, and the illumination control unit includes An observation device is provided that causes the shutter device to block the laser beam when the condensing restriction position and the intermediate condensing position overlap.

  According to a seventh aspect of the present invention, in the observation apparatus according to the second aspect, the scanning control means restricts scanning by the first scanning means to a position where the condensing restriction position and the intermediate condensing position overlap. I will provide a.

  An eighth aspect of the present invention is the observation apparatus according to the second aspect, further comprising second scanning means for scanning the specimen condensing position in a direction perpendicular to the optical axis. An observation device is provided that restricts scanning by a second scanning unit to a position where a restriction position and an intermediate condensing position overlap.

According to a ninth aspect of the present invention, the observation apparatus according to the second aspect further includes third scanning means for changing a distance between the specimen and the objective lens, and the scanning control means includes the third control means. An observation device for controlling driving is provided.
According to a tenth aspect of the present invention, there is provided the observation apparatus according to the ninth aspect, wherein the third scanning unit drives the objective lens in the optical axis direction.
According to an eleventh aspect of the present invention, there is provided the observation apparatus according to the ninth aspect, wherein the third scanning unit drives the stage in the optical axis direction.

  According to a twelfth aspect of the present invention, in the observation apparatus according to the ninth aspect, the scanning control unit prevents the condensing limit position and the intermediate condensing position from overlapping by the first scanning unit and the third scanning unit. An observation apparatus that scans the sample collection position in the optical axis direction is provided.

  According to a thirteenth aspect of the present invention, in the observation apparatus according to the twelfth aspect, when the condensing restriction position and the intermediate condensing position do not overlap, the scanning control means emits the sample condensing position by the first scanning means. An observation device that scans in the axial direction and scans the sample condensing position in the optical axis direction by a third scanning unit when the condensing restriction position and the intermediate condensing position overlap is provided.

  According to a fourteenth aspect of the present invention, in the observation apparatus according to the twelfth aspect, the scanning control means is arranged so that the third condensing position and the intermediate condensing position are collected by the third scanning means before scanning the condensing limit position. An observation apparatus for moving a specimen to a position where light limiting positions do not overlap is provided.

  According to a fifteenth aspect of the present invention, the observation apparatus according to the second aspect further includes an imaging unit that images the specimen, and the imaging unit does not capture an image when the condensing restriction position and the intermediate condensing position overlap. An observation device is provided.

  According to a sixteenth aspect of the present invention, in the observation apparatus according to the second aspect, the intermediate condensing position calculation means is based on the arrangement of the optical elements constituting the objective lens, the illumination means, and the first scanning means. An observation apparatus for calculating a condensing position is provided.

  According to a seventeenth aspect of the present invention, there is provided the observation device according to the second aspect, wherein the intermediate condensing position calculation means calculates the intermediate condensing position based on the sample condensing position.

  An eighteenth aspect of the present invention is the observation apparatus according to the second aspect, further comprising wavefront measuring means for measuring the wavefront of the laser light, wherein the intermediate condensing position calculating means is based on the measurement result by the wavefront measuring means. Based on this, an observation apparatus for calculating an intermediate condensing position is provided.

  According to a nineteenth aspect of the present invention, in the observation apparatus according to the second aspect, the condensing restriction position calculating unit is configured to collect light based on the arrangement of the optical elements constituting the objective lens, the illuminating unit, and the first scanning unit. An observation device for calculating a light-restricted position is provided.

  According to a twentieth aspect of the present invention, a stage on which a specimen is arranged, an objective lens that condenses laser light on the specimen, and a specimen condensing position that is a condensing position in the specimen are scanned in the optical axis direction. Based on the scanning means, the observation means for observing the specimen condensing position, the intermediate image position which is the condensing position in the observation optical path, and the condensing restriction position where the condensing of the laser light is restricted, An observation device is provided that includes a control unit that controls the scanning unit.

  According to a twenty-first aspect of the present invention, in the observation apparatus according to the twentieth aspect, the control unit includes a scanning control unit that controls driving of the first scanning unit, and an intermediate condensing position calculation unit that calculates an intermediate image position. And an observation apparatus including a condensing restriction position calculating unit that calculates a condensing restriction position and an observation control unit that controls the observation unit.

  According to a twenty-second aspect of the present invention, in the observation apparatus according to the twenty-first aspect, the scanning control means limits the scanning by the first scanning means to a position where the condensing restriction position and the intermediate image position overlap. provide.

  A twenty-third aspect of the present invention is the observation apparatus according to the twenty-first aspect, further comprising second scanning means for scanning the specimen condensing position in a direction perpendicular to the optical axis. An observation device is provided that restricts scanning by a second scanning unit to a position where a restriction position and an intermediate image position overlap.

A twenty-fourth aspect of the present invention is the observation apparatus according to the twenty-first aspect, further comprising third scanning means for changing a distance between the sample and the objective lens, wherein the scanning control means is the third control means. An observation device for controlling driving is provided.
According to a twenty-fifth aspect of the present invention, in the observation apparatus according to the twenty-fourth aspect, the third scanning unit provides an observation apparatus that drives the objective lens in the optical axis direction.
According to a twenty-sixth aspect of the present invention, there is provided the observation device according to the twenty-fourth aspect, wherein the third scanning unit drives the stage in the optical axis direction.

  According to a twenty-seventh aspect of the present invention, in the observation apparatus according to the twenty-fourth aspect, the scanning control unit is configured so that the condensing restriction position and the intermediate image position are not overlapped by the first scanning unit and the third scanning unit. Provided is an observation device that scans a sample condensing position in an optical axis direction.

  According to a twenty-eighth aspect of the present invention, in the observation apparatus according to the twenty-seventh aspect, when the condensing restriction position and the intermediate image position do not overlap, the scanning control means sets the sample condensing position by the first scanning means on the optical axis. An observation device is provided that scans in the direction and scans the sample condensing position in the optical axis direction by a third scanning unit when the condensing restriction position and the intermediate image position overlap.

  According to a twenty-ninth aspect of the present invention, in the observation apparatus according to the twenty-seventh aspect, the scanning control means uses the third scanning means to collect the intermediate image position and the light condensing during the scanning before scanning the light condensing limit position. An observation apparatus for moving a specimen to a position where the restriction positions do not overlap is provided.

  According to a thirtieth aspect of the present invention, in the observation apparatus according to the twenty-first aspect, the observation means includes an imaging means, and the imaging means is an observation apparatus that does not image when the condensing restriction position and the intermediate image position overlap. provide.

  According to a thirty-first aspect of the present invention, in the observation device according to the twenty-first aspect, the intermediate condensing position calculation means is based on the arrangement of the optical elements constituting the objective lens, the observation means, and the first scanning means. An observation apparatus for calculating an image position is provided.

  A thirty-second aspect of the present invention provides the observation apparatus according to the twenty-first aspect, wherein the intermediate condensing position calculation means calculates the intermediate image position based on the specimen condensing position.

  According to a thirty-third aspect of the present invention, in the observation device according to the twenty-first aspect, the condensing restriction position calculating means is a collecting unit based on the arrangement of the optical elements constituting the objective lens, the illuminating means, and the first scanning means. An observation device for calculating a light-restricted position is provided.

  A thirty-fourth aspect of the present invention is a microscope illumination method, wherein a condensing restriction position in an illumination optical path where condensing of laser light is restricted and an intermediate condensing position that is a condensing position in the illumination optical path overlap. A first step of displaying a range; a second step of setting a range in which the in-sample condensing position which is a condensing position in the sample is scanned in the optical axis direction; and a third step of illuminating the sample with laser light In a third step, there is provided a microscope illumination method for modulating the intensity of laser light when the condensing limit position and the intermediate condensing position overlap.

  A thirty-fifth aspect of the present invention is a microscope observation method in which a condensing restriction position in an observation optical path where condensing of laser light is restricted overlaps an intermediate image position that is a condensing position in the observation optical path. A second step of setting a range for scanning the in-sample condensing position, which is a condensing position in the sample, in the optical axis direction, and a third step of observing the sample. In a third step, a microscope observation method is provided in which the specimen is not imaged when the condensing restriction position and the intermediate image position overlap.

  According to the present invention, it is possible to provide a scanning observation apparatus that realizes good observation performance.

3 is a diagram for explaining a configuration of an observation apparatus according to Example 1. FIG. FIG. 3 is a diagram for explaining a configuration of a control unit according to the first embodiment. It is a conceptual diagram which shows an example of the calculation result of the condensing restriction position by the control means which concerns on Example 1. FIG. It is a conceptual diagram which shows an example of the calculation result of the condensing position by a control means. 6 is a flowchart illustrating scanning control of the observation apparatus according to the first embodiment. 10 is a flowchart illustrating a modification of scanning control of the observation apparatus according to the first embodiment. FIG. 6 is a diagram for explaining a configuration of an observation apparatus according to Example 2. 6 is a diagram for explaining a configuration of an observation apparatus according to Example 3. FIG. FIG. 6 is a schematic diagram illustrating a configuration of a wavefront measuring unit according to a third embodiment. 10 is a flowchart showing scanning control of the observation apparatus according to Example 3. FIG. 10 is a diagram for explaining the operation of the observation apparatus according to Example 4. FIG. 10 is a diagram for explaining a configuration of an observation apparatus according to Example 5. 10 is a flowchart showing scanning control of the observation apparatus according to Example 5. FIG. 10 is a diagram for explaining a configuration of an observation apparatus according to Example 6. FIG. 10 is a diagram for explaining a configuration of an observation apparatus according to Example 7. 10 is a flowchart showing scanning control of the observation apparatus according to Example 7.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 is a diagram for explaining the configuration of the observation apparatus according to the present embodiment. The observation apparatus 100 illustrated in FIG. 1 is a two-photon excitation fluorescence microscope. The observation apparatus 100 includes a stage 2 on which the sample 1 is arranged, an objective lens 3 that condenses the laser light on the sample, an illuminating unit 10 that guides the laser light to the objective lens 3, and a sample condensing position in the optical axis direction. Condensing position scanning means 20 for scanning, observation means 30 for observing fluorescence generated from the specimen, control means 40 for controlling the entire observation apparatus 100, input means 50, and display means 60 are configured. ing.

  The illumination means 10 includes an ultra-short pulse laser light source 11, an AOM (Acoustic Optics Modulator) shutter device 12, a dispersion compensator 13, a beam expander 14, a zoom beam expander 15 having a magnification changing function, A galvanometer mirror 16 that scans the specimen condensing position in a direction perpendicular to the optical axis, a relay lens 17, and an imaging lens 18 are configured.

  The condensing position scanning unit 20 is disposed between the beam expander 14 and the beam expander 15, and includes a polarization beam splitter 21, a half-wave plate 22, a z scan lens 23, and a piezo mirror 24. It is configured. The condensing position scanning unit 20 is a first scanning unit of the observation apparatus 100.

  The ultrashort pulse laser beam emitted intermittently from the ultrashort pulse laser light source 11 enters an AOM shutter device 12 configured using an acoustooptic device. In the state where the shutter of the AOM shutter device 12 is opened, the laser light is incident on the dispersion compensator 13 and the aberration caused by the chromatic dispersion of the laser light is compensated. Then, the beam diameter and the divergence angle are changed by the beam expander 14 and the laser light is incident on the condensing position scanning unit 20. The laser light incident on the condensing position scanning unit 20 is reflected by the polarization beam splitter 21. Then, the light travels back and forth along the optical path between the polarizing beam splitter 21 and the piezo mirror 24 and enters the polarizing beam splitter 21 again. During this reciprocation, the laser beam passes through the half-wave plate 22 twice. As a result, the polarization direction of the laser light is rotated by 90 degrees, so that the laser light passes through the polarization beam splitter 21 and enters the beam expander 15.

The z scan lens 23 and the piezo mirror 24 are optical elements corresponding to the reference lens and the reference mirror in Patent Document 1. The specimen condensing position can be scanned in the optical axis direction by driving the piezo mirror 24 in the optical axis direction and changing the relative distance between the piezo mirror 24 and the z scan lens 23. The optical system (first optical system) including the objective lens 3 and the optical system (second optical system) including the z scan lens 23 and the piezo mirror 24 are configured to satisfy the following conditional expression (1). Here, f ₁ is a focal length of the objective lens 3, and n ₁ is a refractive index between the sample 1 and the objective lens 3. f ₂ is a focal length of the z scan lens 23, and n ₂ is a refractive index between the z scan lens 23 and the piezo mirror 24. Further, M ₁₂ is the projection magnification between the first optical system and second optical system.
n ₁ f ₁ = M ₁₂ n ₂ f ₂ (1)
Thereby, since the Herschel's condition is satisfied, the observation apparatus 100 can correct the spherical aberration associated with the scanning of the sample condensing position in the optical axis direction.

The laser light incident on the beam expander 15, the scaling function of the beam expander 15, as the conditional expression (1) is satisfied, projection magnification M ₁₂ is adjusted. Adjustment of the projection magnification M ₁₂ by the beam expander 15, for example, when the focal length f ₁ by replacement of the objective lens 3 is changed, is performed in a case where the refractive index n ₁ by scanning the optical axis direction is changed.

  Thereafter, the laser light is incident on the objective lens 3 via the galvanometer mirror 16, the relay lens 17, the imaging lens 18, and the dichroic mirror 31. The objective lens 3 irradiates the sample condensing position on the sample 1 with laser light. Note that the galvanometer mirror 16 is a second scanning unit of the observation apparatus 100.

  In the specimen irradiated with laser light, fluorescence is generated by two-photon excitation. This fluorescence enters the observation means 30 via the objective lens 3. The fluorescence reflects from the dichroic mirror 31 and passes through the barrier filter 32. The barrier filter 32 transmits only the fluorescence generated by the two-photon excitation. Thereby, only the fluorescence generated from the specimen condensing position is condensed by the condenser lens 33 and enters the photomultiplier tube 34. The photomultiplier tube 34 functions as an image sensor and converts the fluorescence into an electrical signal to generate an image.

  FIG. 2 is a diagram for explaining the configuration of the control means according to the present embodiment. The control unit 40 includes a scanning control unit 41, an illumination control unit 42, an intermediate condensing position calculation unit 43, a condensing restriction position calculation unit 44, and an observation control unit 45.

  The scanning control means 41 controls the scanning of the sample condensing position. More specifically, the scanning control unit 41 controls scanning of the sample condensing position in the optical axis direction by controlling the piezo mirror 24. Further, the scanning control unit 41 controls scanning in the direction perpendicular to the optical axis of the specimen condensing position by controlling the galvanometer mirror 16. The scanning control means 41 desirably controls the zoom beam expander 15 together with the piezo mirror 24 so as to satisfy the conditional expression (1).

  The illumination control unit 42 controls the illumination unit 10. More specifically, the illumination control means 42 controls the intensity of laser light emitted from the ultrashort pulse laser light source 11 and ON / OFF of the ultrashort pulse laser light source 11. The illumination control means 42 also controls the opening / closing of the shutter of the AOM shutter device 12.

  The intermediate condensing position calculation means 43 calculates an intermediate condensing position that is a condensing position on the illumination optical path. The intermediate condensing position is calculated based on the physical arrangement of the optical elements, for example. Specifically, it is calculated based on the arrangement of the objective lens 3 and the optical elements constituting the illumination unit 10 and the condensing position scanning unit 20. Further, the intermediate condensing position may be calculated based on the sample condensing position.

Further, the intermediate condensing position calculation unit 43 may calculate the intermediate image position on the observation optical path when the intermediate image is on the observation optical path. Further, the intermediate image position on the illumination optical path may be calculated. Here, the intermediate image position on the illumination optical path refers to a position on the illumination optical path that is conjugate to the object point position of the observation means 30. In this embodiment, since there is no intermediate image on the observation optical path, the position is conjugate to the object point of the observation means 30 on the illumination optical path.
The intermediate image position is calculated based on, for example, the physical arrangement of the optical elements. Specifically, it is calculated based on the arrangement of the optical elements constituting the objective lens 3, the illumination unit 10, the condensing position scanning unit 20, and the observation unit 30. Further, the intermediate image position may be calculated based on the sample condensing position.

The condensing restriction position calculation unit 44 calculates a condensing restriction position in the optical path where the condensing of the laser light is restricted. FIG. 3A is a conceptual diagram illustrating an example of a calculation result of a condensing restriction position by the control unit according to the present embodiment. Here, only the condensing restriction position on the illumination optical path is shown. In FIG. 3A, the range of the condensing restriction position is indicated by hatching. As illustrated in FIG. 3A, the condensing restriction position is, for example, on the optical element or around the optical element. The reason why the light condensing limit position is set on the optical element is as described above. The reason why the periphery of the optical element is set as the condensing limit position is that dust and scratches on the optical element are reflected in the image if there is a condensing position around the optical element. Note that the control unit 40 may include a condensable position calculation unit that calculates a condensable position in an optical path where the condensing of the laser light is not limited, instead of the condensing limitation position calculation unit 44. . FIG. 3B is a conceptual diagram illustrating an example of a calculation result of a condensable position by the control unit. In FIG. 3B, the range of the condensable position is indicated by hatching.
The observation control means 45 controls the observation means. More specifically, the observation control unit 45 controls the imaging of the sample 1 by the photomultiplier tube 34.

  Based on the information obtained from the intermediate condensing position calculation means 43 and the condensing restriction position calculation means 44, the control means 40 condenses light so that the intermediate condensing position or the intermediate image position does not overlap with the condensing restriction position. The position scanning means 20 is controlled. For example, the scanning control unit 41 does not stop the condensing position scanning unit 20 in a state where the intermediate condensing position or the intermediate image position overlaps with the condensing restriction position, and the intermediate condensing position or the intermediate image position and the condensing restriction. The condensing position scanning means 20 is driven until the positions do not overlap.

  Alternatively, the control unit 40 controls the illumination unit 10 to limit the condensing state of the laser light when the intermediate condensing position and the condensing restriction position overlap. For example, the illumination control unit 42 causes the ultrashort pulse laser light source 11 to stop emitting laser light or reduce the intensity of the laser light when the intermediate condensing position and the condensing limit position overlap. In addition, the illumination control unit 42 may cause the AOM shutter device 12 to maintain a closed state. When the intensity of the laser beam is reduced, the intensity of the laser beam may be changed according to the position where the intermediate condensing position and the condensing limit position overlap. The control means 40 may change the intensity of the laser light in consideration of, for example, the material of the optical element at the overlapping position.

Further, the control unit 40, when the intermediate condensing position or the intermediate image position and the condensing limit position overlap only in a part of the plane including the position (z position) in the optical axis direction controlled by the scanning control unit 41, for example, In a case where a part of an optical element such as a mirror disposed at an oblique angle in the optical path is placed on the condensing limit position, the galvano mirror 16 is controlled so as to be in a direction perpendicular to the optical axis of the sample condensing position. The scanning range may be limited.
The input unit 50 and the display unit 60 are used, for example, for inputting or changing a scanning range by an observer.

  FIG. 4 is a flowchart illustrating the scanning control of the observation apparatus according to the present embodiment. Hereinafter, an example of scanning control by the control means 40 of the observation apparatus 100 will be described with reference to FIG.

  First, when scanning control of the observation apparatus 100 is started, the shutter of the AOM shutter apparatus 12 is closed (step S1-1). Next, the condensing restriction position is calculated, and the range of the z position of the sample condensing position corresponding to the condensing restriction position is displayed on the display means 60 (step S1-2). The observer uses the input unit 50 while referring to the display unit 60 to input a range for scanning the sample 1 (hereinafter referred to as a scanning range) (step S1-3).

  Next, an intermediate condensing position and a condensing restriction position corresponding to each position within the input scanning range are calculated. The intermediate condensing position and the condensing restriction position are compared, and it is determined whether or not the intermediate condensing position and the condensing restriction position may overlap within the scanning range (step S1-4). If there is no overlap within the scanning range, the control transitions to step S1-7.

  If the intermediate condensing position and the condensing restriction position may overlap within the scanning range, the display means 60 may overlap the intermediate condensing position and the condensing restriction position, or the intermediate condensing position and the condensing restriction position may overlap. The sample collection position at the time is displayed (step S1-5).

The observer selects whether to change the scanning range based on the information displayed on the display unit 60 (step S1-6). When the change of the scanning range is selected, the control proceeds to step S1-3. If it is determined by the observer that the scanning range does not need to be changed and the scanning range is not changed, the control proceeds to step S1-7.
In step S1-7, the condensing position operation means 20 moves the sample condensing position to the scanning start position.

  In step S1-8, it is determined whether or not the current intermediate condensing position and the condensing restriction position overlap. If the intermediate condensing position and the condensing restriction position overlap, the control proceeds to step S1-12.

  When the intermediate condensing position and the condensing restriction position do not overlap, the shutter of the AOM shutter device 12 is opened (step S1-9), and the specimen 1 is imaged (step S1-10). After completion of imaging, the shutter of the AOM shutter device 12 is closed (step S1-11).

In step S1-12, it is determined whether or not the current sample collection position is the scanning end position. If the sample condensing position is not the scanning end position, the condensing position operating means 20 moves the sample condensing position to the next z position (step S1-13). Thereafter, the control proceeds to step S1-8. When the sample condensing position is the scanning end position, the scanning control ends.
In the above control, it is desirable to control in consideration of the overlap between the intermediate image position and the condensing restriction position in addition to the overlap between the intermediate condensing position and the condensing restriction position.

  As described above, by performing the control illustrated in FIG. 4, it is possible to prevent the laser light from being focused on the optical element or the like. In addition, it is possible to suppress specimen vibration and spherical aberration associated with scanning in the optical axis direction. As a result, good observation performance of the observation apparatus is realized.

FIG. 5 is a flowchart illustrating a modification of the scanning control of the observation apparatus according to the present embodiment. Hereinafter, a modified example of the scanning control by the control means 40 of the observation apparatus 100 will be described with reference to FIG.
Steps S1-1 to S1-7 of the scanning control illustrated in FIG. 5 are the same as the scanning control illustrated in FIG. Therefore, the description is omitted.

  In step S1-8, it is determined whether or not the intermediate condensing position and the condensing restriction position overlap with the sample condensing position at the scanning start position. When the intermediate condensing position and the condensing restriction position do not overlap, the same control as the scanning control illustrated in FIG. 4 is performed. Specifically, the shutter of the AOM shutter device 12 is opened (step S1-9), and the specimen 1 is imaged (step S1-10). After completion of imaging, the shutter of the AOM shutter device 12 is closed (step S1-11). Thereafter, the control proceeds to step S1-12.

  On the other hand, when the intermediate condensing position and the condensing restriction position overlap, control different from the scanning control illustrated in FIG. 4 is performed. First, the intensity of the laser light emitted from the ultrashort pulse laser light source 11 is reduced (step S1-14). As described above, the intensity of the laser beam may be changed according to the position where the intermediate condensing position and the condensing restriction position overlap. Thereafter, the shutter of the AOM shutter device 12 is opened (step S1-15), and the specimen 1 is imaged (step S1-16). After the imaging is finished, the shutter of the AOM shutter device 12 is closed (step S1-17), and the intensity of the laser light is also returned to the intensity before the intensity reduction (step S1-18). Thereafter, the control proceeds to step S1-12.

  In step S1-12, it is determined whether or not the current sample collection position is the scanning end position. If the sample condensing position is not the scanning end position, the sample condensing position is moved to the next z position (step S1-13). Thereafter, the control proceeds to step S1-8. When the sample condensing position is the scanning end position, the scanning control ends.

  As described above, by performing the control illustrated in FIG. 5, the sample 1 can be imaged at all the z positions. In this control, even when the laser beam is condensed on the optical element at the time of imaging, the influence of the laser beam on the optical element is suppressed because the intensity of the laser beam is reduced. In addition, it is possible to suppress specimen vibration and spherical aberration associated with scanning in the optical axis direction, and as a result, good observation performance is realized.

  FIG. 6 is a diagram for explaining the configuration of the observation apparatus according to the present embodiment. The observation apparatus 101 illustrated in FIG. 6 is a two-photon excitation fluorescence microscope, similarly to the observation apparatus 100 illustrated in FIG. Note that among the constituent elements of the observation apparatus 101, the same constituent elements as those of the observation apparatus 100 are denoted by the same reference numerals, and description thereof is omitted.

  Similar to the observation apparatus 100, the observation apparatus 101 includes a stage 2 on which the specimen 1 is arranged, an objective lens 3 that focuses the laser light on the specimen, an illumination unit 10 that guides the laser light to the objective lens 3, and a specimen collection. Condensing position scanning means 20 for scanning the optical position in the optical axis direction, observation means 30 for observing fluorescence generated from the specimen, control means 40 for controlling the entire observation apparatus 101, input means 50, and display means 60 And.

  The observation apparatus 101 is different from the observation apparatus 100 in that the illumination unit 10 includes a light amount modulation unit 19 between the polarization beam splitter 21 and the beam expander 15. Other configurations are the same as those of the observation apparatus 100. Further, the configuration of the control means 40 is the configuration illustrated in FIG.

  The light amount modulation unit 19 is controlled by the illumination control unit 42 and adjusts the intensity of the laser light passing through the light amount modulation unit 19 in accordance with the intermediate condensing position changed by the condensing position scanning unit 20. The light quantity modulation means 19 is constituted by, for example, a neutral density filter (ND filter).

  In the observation apparatus 101, scanning control illustrated in FIG. 5 is performed. However, the observation apparatus 101 controls the intensity of the laser light transmitted through the light amount modulation means 19 instead of controlling the intensity of the laser light emitted from the ultrashort pulse laser light source 11. Thereby, the same effect as the first embodiment can be obtained without changing the intensity of the laser light emitted from the ultrashort pulse laser light source 11.

  FIG. 7 is a diagram for explaining the configuration of the observation apparatus according to the present embodiment. The observation apparatus 102 illustrated in FIG. 7 is a two-photon excitation fluorescence microscope, similarly to the observation apparatus 101 illustrated in FIG. Of the constituent elements of the observation apparatus 102, the same constituent elements as those of the observation apparatus 101 are denoted by the same reference numerals, and description thereof is omitted.

  Similar to the observation apparatus 101, the observation apparatus 102 includes a stage 2 on which the specimen 1 is arranged, an objective lens 3 that focuses the laser light on the specimen, an illumination unit 10 that guides the laser light to the objective lens 3, and a specimen collection. Condensing position scanning means 20 that scans the light position in the optical axis direction, observation means 30 that observes fluorescence generated from the specimen, control means 40 that controls the entire observation apparatus 102, input means 50, and display means 60 And.

  The observation apparatus 102 is further different from the observation apparatus 101 in that the observation apparatus 102 includes a wavefront measuring unit 70 that measures the wavefront of the light beam in the illumination optical path. Other configurations are the same as those of the observation apparatus 101. Further, the configuration of the control means 40 is the configuration illustrated in FIG.

  FIG. 8 is a schematic diagram showing the configuration of the wavefront measuring means according to the present embodiment. As illustrated in FIG. 8, the wavefront measuring unit 70 is a so-called Shack-Hartmann wavefront measuring device, and includes a microlens array 71 and a charge coupled device (CCD) 72.

  FIG. 9 is a flowchart illustrating the scanning control of the observation apparatus according to the present embodiment. Hereinafter, an example of scanning control by the control unit 40 of the observation apparatus 102 will be described with reference to FIG.

  First, when scanning control of the observation apparatus 102 is started, the shutter of the AOM shutter apparatus 12 is closed (step S3-1). Further, the light quantity modulation means 19 is controlled to reduce the intensity of the laser light passing through the light quantity modulation means 19 (step S3-2). The intensity of the laser light that passes through the light amount modulation unit 19 is determined in accordance with, for example, the optical element that is most susceptible to the laser light among the optical elements in the illumination optical path.

Next, the condensing restriction position is calculated, and the z position of the sample condensing position corresponding to the condensing restriction position is displayed on the display unit 60 (step S3-3). The observer inputs the scanning range by using the input unit 50 while referring to the display unit 60 (step S3-4).
Thereafter, the sample collection position is moved to the scanning start position by the collection position operating means 20 (step S3-5), and the shutter of the AOM shutter device 12 is opened (step S3-6).

  In step S3-7, the wavefront measuring means 70 is used to measure the wavefront of the light beam in the illumination optical path. Then, based on the wavefront measurement result by the wavefront measuring means 70, the intermediate condensing position calculating means 43 calculates the current intermediate condensing position (step S3-8).

  It is determined whether or not the current intermediate condensing position and the condensing restriction position overlap (step S3-9). If the intermediate condensing position and the condensing restriction position overlap, the control proceeds to step S3-13.

  If the intermediate condensing position and the condensing restriction position do not overlap, the light quantity modulation means 19 is controlled to return the intensity of the laser light passing through the light quantity modulation means 19 (step S3-10), and the sample 1 is An image is taken (step S3-11). After the imaging is completed, the light quantity modulation means 19 is controlled to reduce the intensity of the laser light passing through the light quantity modulation means 19 again (step S3-12).

In step S3-13, the shutter of the AOM shutter device 12 is closed. Then, it is determined whether or not the current sample collection position is the scanning end position (step S3-14). If the sample condensing position is not the scanning end position, the sample condensing position is moved to the next z position (step S3-15). Thereafter, the control proceeds to step S3-6. When the sample condensing position is the scanning end position, the scanning control ends.
In the above control, it is desirable to control in consideration of the overlap between the intermediate image position and the condensing restriction position in addition to the overlap between the intermediate condensing position and the condensing restriction position.

  As described above, by performing the control illustrated in FIG. 9, the intensity of the laser light at the condensing position is reduced even when the laser light is condensed on the optical element during scanning. Further, imaging is not performed in a state where the laser light is focused on the optical element. For this reason, the time for which the laser beam is focused on the optical element is only the time required for wavefront measurement. As described above, since the laser light is focused on the optical element with a low intensity for a short time, the influence of the laser light on the optical element is suppressed. In addition, it is possible to suppress specimen vibration and spherical aberration associated with scanning in the optical axis direction. As a result, good observation performance of the observation apparatus is realized.

  FIG. 10 is a diagram for explaining the operation of the observation apparatus according to the present embodiment. FIG. 10 shows only a part of the configuration of the observation apparatus 103 according to the present embodiment. Note that the overall configuration of the observation apparatus 103 is the same as that of the observation apparatus 100 illustrated in FIG. The observation apparatus 103 is different from the observation apparatus 100 in that the beam expander 14 is controlled by the scanning control means 41.

  When the beam expander 14 emits laser light as parallel light, the laser light enters the z-scan lens 23 as parallel light through the polarization beam splitter 21 and the half-wave plate 22. For this reason, when the piezo mirror 24 is located away from the z scan lens 23 by the focal length of the z scan lens 23, the laser light is condensed on the piezo mirror 24.

  In the observation apparatus 103, the scanning control unit 41 controls the converging position scanning unit 20 and the beam expander 14 in order to prevent the laser beam from condensing on the piezo mirror 24. This makes it possible to control the spread angle of the laser light emitted from the beam expander 14 in accordance with the position of the piezo mirror 24, and avoid a state where the laser light is condensed on the piezo mirror 24. FIG. 10 illustrates a state in which the laser light emitted from the beam expander 14 is converged light, thereby avoiding condensing the laser light on the piezo mirror 24.

  As described above, also in the observation apparatus 103 according to the present embodiment, the same effects as those of the first embodiment can be obtained. In addition, it is possible to prevent laser light from being focused on the piezo mirror 24.

  FIG. 11 is a diagram for explaining the configuration of the observation apparatus according to the present embodiment. The observation apparatus 104 illustrated in FIG. 11 is a two-photon excitation fluorescence microscope, similarly to the observation apparatus 100 illustrated in FIG. Note that among the constituent elements of the observation apparatus 104, the same constituent elements as those of the observation apparatus 100 are denoted by the same reference numerals, and description thereof is omitted.

  Similar to the observation apparatus 100, the observation apparatus 104 includes a stage 2 on which the specimen 1 is arranged, an objective lens 3 that focuses the laser light on the specimen, an illumination unit 10 that guides the laser light to the objective lens 3, and a specimen collection. Condensing position scanning means 20 that scans the light position in the optical axis direction, observation means 30 that observes fluorescence generated from the specimen, control means 40 that controls the entire observation apparatus 104, input means 50, and display means 60 And.

  The observation apparatus 104 further includes a piezo element 25 used for driving the objective lens 3 in the optical axis direction and a piezo element 26 used for driving the stage 2 in the optical axis direction. Is different. Other configurations are the same as those of the observation apparatus 100. Further, the configuration of the control means 40 is the configuration illustrated in FIG.

  In the observation device 104, the objective lens 3 is controlled by the scanning control means 41 via the piezo element 25. The stage 2 is also controlled by the scanning control means 41 via the piezo element 26. That is, the piezo element 25 and the piezo element 26 function as a third scanning unit that changes the relative distance between the objective lens 3 and the stage 2 by being controlled by the scanning control unit 41.

  FIG. 12 is a flowchart illustrating scanning control of the observation apparatus according to the present embodiment. Hereinafter, scanning control by the control means 40 of the observation apparatus 104 will be described with reference to FIG.

  First, when scanning control of the observation device 104 is started, the shutter of the AOM shutter device 12 is closed (step S5-1). Then, the z position of the sample condensing position corresponding to the condensing limit position is displayed on the display means 60 (step S5-2). The observer uses the input unit 50 while referring to the display unit 60 to input the scanning range (step S5-3). Thereafter, the specimen collection position is moved to the scanning start position (step S5-4).

  In step S5-5, it is determined whether or not the current intermediate condensing position and the condensing restriction position overlap. If the intermediate condensing position and the condensing restriction position do not overlap, control proceeds to step S5-8.

  If the intermediate condensing position and the condensing limit position overlap, the z position is returned to the previous position by the condensing position scanning unit 20 (first scanning unit) (step 5-6). When the sample condensing position is at the scanning start position, the specimen is returned to a position where the intermediate condensing position and the condensing restriction position do not overlap. Thereafter, the z position is moved to the next position by the third scanning means (piezo element 25 and piezo element 26) (step S5-7).

  In step S5-8, the shutter of the AOM shutter device 12 is opened. Then, the sample 1 is imaged (step S5-9). After completion of imaging, the shutter of the AOM shutter device 12 is closed (step S5-10).

In step S5-11, it is determined whether the current sample collection position is the scan end position. If the sample condensing position is not the scanning end position, the first condensing position is moved to the next z position by the first scanning means (step S5-12). Thereafter, the control proceeds to step S5-6. When the sample condensing position is the scanning end position, the scanning control ends.
In the above control, it is desirable to control in consideration of the overlap between the intermediate image position and the condensing restriction position in addition to the overlap between the intermediate condensing position and the condensing restriction position.

  As described above, by performing the control illustrated in FIG. 12, it is possible to take an image while illuminating the specimen 1 with a necessary laser intensity at all z positions. In this control, the third scanning unit is used for scanning only when the laser beam is condensed on the optical element by the scanning using the first scanning unit. Compared to the case of scanning only by the first scanning unit, the third scanning unit can largely scan the condensing position. For this reason, when the intermediate condensing position or the intermediate image position overlaps the condensing limit position when scanning in the optical axis direction by the first scanning means, the third scanning means is used as the intermediate condensing position or the intermediate condensing position. The intermediate condensing position or the intermediate image position can be moved so that the image position quickly passes the condensing restriction position. As a result, good observation performance of the observation apparatus is realized.

  In the above control, it may be calculated in advance whether the intermediate condensing position or the intermediate image position overlaps with the condensing restriction position at the z position after scanning, and the scanning means used for scanning may be selected. In this case, when the intermediate condensing position or the intermediate image position and the condensing restriction position overlap, control is performed so that scanning is performed by the third scanning unit, and when they do not overlap, scanning is performed by the first scanning unit.

  In addition to the above-described control, after the scanning range is input, control may be performed to determine whether the intermediate condensing position or the intermediate image position may overlap with the condensing restriction position during scanning. . In this case, if there is an overlap, the scanning start position may be moved by the third scanning means to a position where the intermediate condensing position or the intermediate image position and the condensing restriction position do not overlap during scanning.

  FIG. 13 is a diagram for explaining the configuration of the observation apparatus according to the present embodiment. The observation apparatus 105 illustrated in FIG. 13 is a two-photon excitation fluorescence microscope, similarly to the observation apparatus 102 illustrated in FIG. Of the constituent elements of the observation apparatus 105, the same constituent elements as those of the observation apparatus 102 are denoted by the same reference numerals, and description thereof is omitted.

  Similar to the observation apparatus 102, the observation apparatus 105 includes a stage 2 on which the specimen 1 is arranged, an objective lens 3 that focuses the laser light on the specimen, an illumination unit 10 that guides the laser light to the objective lens 3, and a specimen collection. Condensing position scanning means 20 for scanning the optical position in the optical axis direction, observation means 30 for observing fluorescence generated from the specimen, control means 40 for controlling the entire observation apparatus 105, input means 50, and display means 60 And.

  The observation apparatus 105 is different from the observation apparatus 102 in the configuration of the condensing position scanning unit 20. More specifically, a deformable mirror 27 is included instead of the z scan lens 23 and the piezo mirror 24 of the observation apparatus 102. In the observation device 105, the scanning control unit 41 controls the deformable mirror 27, whereby the sample condensing position can be scanned in the optical axis direction. Instead of the deformable mirror 27, a reflective liquid crystal device 28 such as a liquid crystal spatial light modulator described in Patent Document 2 may be used.

  Further, in the deformable mirror 27 and the reflective liquid crystal device 28, spherical aberration accompanying scanning in the optical axis direction can be corrected by itself. For this reason, the beam expander 15 included in the illumination unit 10 does not have to be a zoom type having a zoom function.

  In the observation apparatus 105, similarly to the observation apparatus 102, scanning control illustrated in FIG. 9 is performed. Thereby, the same effect as Example 3 can be acquired.

  FIG. 14 is a diagram for explaining the configuration of the observation apparatus according to the present embodiment. An observation apparatus 106 illustrated in FIG. 14 is a fluorescence microscope having a light source for light stimulation and a light source for observation.

The observation device 106 includes a stage 2 on which the sample 1 is arranged, an objective lens 3 that focuses laser light on the sample, laser light for light stimulation (hereinafter referred to as stimulation light), and laser for observation. Illumination means 80 for guiding light (hereinafter referred to as illumination light) to the objective lens 3, condensing position scanning means 20, observation means 30 for observing fluorescence generated from the specimen, and control for controlling the entire observation apparatus 106 The unit 40 includes a piezo element 25 used for driving the objective lens 3, a piezo element 26 used for driving the stage 2, an input unit 50, and a display unit 60. Note that the configuration of the control means 40 is the configuration illustrated in FIG.
The illumination means 80 includes a first illumination means, a second illumination means, a dichroic mirror 92, and an imaging lens 93.

  The first illumination means includes an ultrashort pulse laser light source 81 that intermittently emits an ultrashort pulse laser beam, an AOM shutter device 82, a dispersion compensator 83, a beam expander 84, and a zoom type having a magnification changing function. Beam expander 85, galvanometer mirror 86, and relay lens 87.

  On the other hand, the second illumination means includes a CW laser light source 88 that continuously emits laser light, an AOM shutter device 89, a galvano mirror 90, and a relay lens 91.

  The condensing position scanning unit 20 is disposed between the beam expander 84 and the beam expander 85, and includes a polarization beam splitter 21, a half-wave plate 22, a z scan lens 23, and a piezo mirror 24. It is configured. Instead of the z scan lens 23 and the piezo mirror 24 of the condensing position scanning unit 20, a deformable mirror or a reflective liquid crystal device can be used. In that case, the beam expander 15 does not have to be a zoom beam expander.

  In the observation device 106, the condensing position scanning unit 20 is a first scanning unit that scans the sample condensing position of the stimulation light in the optical axis direction. The galvanometer mirror 86 is a second scanning unit that scans the sample collection position of the stimulation light in a direction perpendicular to the optical axis. The piezo element 25 and the piezo element 26 are third scanning means for scanning the sample collection positions of the stimulation light and the illumination light in the optical axis direction. The galvanometer mirror 90 is a fourth scanning unit that scans the specimen collection position of the illumination light in a direction perpendicular to the optical axis.

  The action of the stimulation light emitted from the ultrashort pulse laser light source 81 is the same as the action of the laser light emitted from the ultrashort pulse laser light source 11 of the observation apparatus 100. Therefore, the description is omitted.

  Laser light (illumination light) emitted from the CW laser light source 88 enters an AOM shutter device 89 configured using an acoustooptic device. When the shutter of the AOM shutter device 89 is opened, the illumination light is reflected by the dichroic mirror 31 and enters the dichroic mirror 92 via the galvano mirror 90 and the relay lens 91. Further, the illumination light passes through the dichroic mirror 92 and enters the objective lens 3 through the imaging lens 93. The objective lens 3 irradiates illumination light to the specimen condensing position on the specimen 1. Here, usually, the sample collection position of the stimulation light and the sample collection position of the illumination light are different.

  In a specimen irradiated with stimulation light and illumination light, fluorescence is generated by each light. At this time, the stimulation light excites the specimen through a two-photon process to generate fluorescence, whereas the illumination light excites the specimen through a one-photon process to generate fluorescence. When the sample is excited through a two-photon process using the ultrashort pulse laser light source 81, it is necessary to irradiate the sample with a stronger laser density than when the sample is excited through a one-photon process using the CW laser light source 88. There is. As a result, the laser intensity at the intermediate condensing position of the stimulation light is stronger than the laser intensity at the intermediate condensing position of the illumination light. For this reason, in the observation apparatus 106, the condensing position scanning unit 20 is provided only in the illumination optical path of the first illumination unit including the ultrashort pulse laser light source 81. A condensing position scanning unit may be further provided in the illumination optical path of the second illumination unit including the CW laser light source 88.

  Fluorescence generated from the specimen enters the observation means 30 via the objective lens 3, the imaging lens 93, the dichroic mirror 92, the relay lens 91, and the galvanometer mirror 90. The fluorescence passes through the dichroic mirror 31 and is condensed on the confocal pinhole 35 by the condenser lens 33. Only the fluorescence generated from the specimen collection position of the illumination light passes through the confocal pinhole 35 and enters the photomultiplier tube 34. The photomultiplier tube 34 functions as an image sensor and converts the fluorescence into an electrical signal to generate an image.

  The observation device 106 can separately scan the sample collection position of stimulus light (hereinafter referred to as stimulus coordinates) and the sample collection position of illumination light. For this reason, it is possible to observe the change of the sample when the stimulus coordinates for applying the light stimulus are changed.

  FIG. 15 is a flowchart illustrating the scanning control of the observation apparatus according to the present embodiment. Hereinafter, an example of scanning control by the control unit 40 of the observation apparatus 106 will be described with reference to FIG. Note that FIG. 15 focuses only on scanning of stimulus coordinates.

  First, when scanning control of the observation device 106 is started, the shutter of the AOM shutter device 82 is closed (step S7-1). Next, the condensing restriction position of the stimulation light is calculated, and the range of the z position of the stimulation coordinates corresponding to the condensing restriction position is displayed on the display means 60 (step S7-2). The observer inputs stimulus coordinates using the input unit 50 while referring to the display unit 60 (step S7-3).

  Next, an intermediate condensing position and a condensing restriction position corresponding to each coordinate of the input plurality of stimulus coordinates are calculated. The intermediate condensing position and the condensing restriction position are compared, and it is determined whether or not the intermediate condensing position and the condensing restriction position may overlap in the stimulus coordinates (step S7-4). If there is no overlap in the stimulus coordinates, control proceeds to step S7-9.

  When the intermediate condensing position and the condensing restriction position may overlap with each other in the stimulus coordinates, the display means 60 may overlap the intermediate condensing position and the condensing restriction position, or the intermediate condensing position and the condensing restriction position may overlap. Are displayed (step S7-5).

The observer selects whether to change the stimulus coordinates based on the information displayed on the display means 60 (step S7-6). If the change of the stimulus coordinates is selected, the control proceeds to step S7-3. When the change of the stimulus coordinates is not selected, the stimulus coordinates where the intermediate condensing position and the condensing restriction position overlap are deleted (step S7-7).
In step S7-8, the stimulus coordinates are moved to the scan start position.

  Then, the shutter of the AOM shutter device 82 is opened, and the specimen 1 is stimulated by the stimulation light. At this time, the specimen 1 is imaged by the observation means 30. After completion of imaging, the shutter of the AOM shutter device 82 is closed (step S7-9).

In step S7-10, it is determined whether the current stimulus coordinate is the last input stimulus coordinate. If it is not the last stimulus coordinate, the stimulus coordinate is moved to the next z position (step S7-11). Thereafter, the control proceeds to step S7-9. If the current stimulus coordinate is the last stimulus coordinate, the scanning control is terminated.
In the above control, it is desirable to control in consideration of the overlap between the intermediate image position and the condensing restriction position in addition to the overlap between the intermediate condensing position and the condensing restriction position.

  As described above, by performing the control exemplified in FIG. 15, it is possible to prevent the stimulation light from being collected on the optical element or the like. In addition, it is possible to suppress specimen vibration and spherical aberration associated with scanning of stimulus coordinates in the optical axis direction. As a result, good observation performance of the observation apparatus is realized.

DESCRIPTION OF SYMBOLS 1 ... Specimen 2 ... Stage 3 ... Objective lens 4 ... Condensing restriction position 5 ... Condensing possible position 10, 80 ... Illuminating means 11, 81 ... Ultrashort pulse laser Light source 12, 82, 89 ... AOM shutter device 13, 83 ... Dispersion compensator 14, 15, 84, 85 ... Beam expander 16, 86, 90 ... Galvano mirror 17, 87, 91 .. Relay lenses 18, 93... Imaging lens 19. Light amount modulation means 20... Condensing position scanning means 21... Polarization beam splitter 22. Scan lens 24 ... Piezo mirror 25, 26 ... Piezo element 27 ... Deformable mirror 28 ... Reflective liquid crystal device 30 ... Observation means 31, 92 ... Dichroic mirror 32 ... Barrier filter 33 ... Condensing lens 34 ... Photomultiplier tube 35 ... Confocal pinhole 40 ... Control means 41 ... Scanning control means 42 ... Illumination control means 43 ... Intermediate collection Light position calculating means 44 ... Condensation restriction position calculating means 45 ... Observation control means 50 ... Input means 60 ... Display means 70 ... Wavefront measuring means 71 ... Micro lens array 72 ...・ Charge coupled device (CCD)
88 ... CW laser light source 100, 101, 102, 103, 104, 105, 106 ... Observation device

Claims (35)

A stage to place the specimen;
An objective lens for focusing the laser beam on the specimen;
Illumination means for guiding the laser light to the objective lens;
First scanning means for scanning a specimen condensing position, which is a condensing position in the specimen, in an optical axis direction;
Control means for controlling the illuminating means and the first scanning means based on an intermediate condensing position which is a condensing position in the illumination optical path and a condensing restriction position where the condensing of the laser light is restricted. An observation apparatus comprising: The observation apparatus according to claim 1,
The control means includes
Scanning control means for controlling driving of the first scanning means;
Intermediate condensing position calculating means for calculating the intermediate condensing position;
A condensing restriction position calculating means for calculating the condensing restriction position;
And an illumination control means for controlling the illumination means. The observation apparatus according to claim 2,
The illumination means includes a light source,
The observation device, wherein the illumination control means reduces the intensity of laser light emitted from the light source when the condensing restriction position and the intermediate condensing position overlap. The observation apparatus according to claim 2,
The illumination means includes
A light source;
A light amount modulation means disposed between the light source and the light collection restriction position,
The observation apparatus characterized in that the illumination control means reduces the intensity of the laser light passing through the light amount modulation means when the condensing restriction position and the intermediate condensing position overlap. In the observation apparatus according to claim 3 or 4,
The intensity of the laser beam changes in accordance with a position where the condensing restriction position and the intermediate condensing position overlap. The observation apparatus according to claim 2,
The illumination means includes
A light source;
A shutter device disposed between the light source and the light collection restriction position,
The observation device, wherein the illumination control unit causes the shutter device to block laser light when the condensing restriction position and the intermediate condensing position overlap. The observation apparatus according to claim 2,
The scanning control means restricts scanning by the first scanning means to a position where the condensing restriction position and the intermediate condensing position overlap. The observation apparatus according to claim 2,
And a second scanning means for scanning the specimen condensing position in a direction perpendicular to the optical axis,
The scanning control means restricts scanning by the second scanning means to a position where the condensing restriction position and the intermediate condensing position overlap each other. The observation apparatus according to claim 2,
Furthermore, it includes a third scanning means for changing a distance between the sample and the objective lens,
The observation apparatus characterized in that the scanning control means controls driving of the third control means. The observation apparatus according to claim 9, wherein
The third scanning unit drives the objective lens in the optical axis direction. The observation apparatus according to claim 9, wherein
The third scanning unit drives the stage in the optical axis direction. The observation apparatus according to claim 9, wherein
The scanning control means scans the sample condensing position in the optical axis direction by the first scanning means and the third scanning means so that the condensing restriction position and the intermediate condensing position do not overlap. A characteristic observation device. The observation device according to claim 12,
The scanning control means includes
When the condensing restriction position and the intermediate condensing position do not overlap, the first scanning means scans the sample condensing position in the optical axis direction,
An observation apparatus, wherein when the condensing restriction position and the intermediate condensing position overlap, the third condensing position scans the sample condensing position in the optical axis direction. The observation device according to claim 12,
The scanning control means moves the sample to a position where the intermediate condensing position and the condensing restriction position do not overlap during scanning by the third scanning means before scanning the condensing restriction position. A characteristic observation device. The observation apparatus according to claim 2,
Furthermore, the imaging means for imaging the sample,
The observation apparatus according to claim 1, wherein the imaging unit does not capture an image when the condensing restriction position and the intermediate condensing position overlap. The observation apparatus according to claim 2,
The observation apparatus according to claim 1, wherein the intermediate condensing position calculating unit calculates the intermediate condensing position based on an arrangement of optical elements constituting the objective lens, the illuminating unit, and the first scanning unit. The observation apparatus according to claim 2,
The intermediate condensing position calculation means calculates the intermediate condensing position based on the sample condensing position. The observation apparatus according to claim 2,
Furthermore, it includes a wavefront measuring means for measuring the wavefront of the laser beam,
The said intermediate condensing position calculation means calculates the said intermediate condensing position based on the measurement result by the said wavefront measurement means, The observation apparatus characterized by the above-mentioned. The observation apparatus according to claim 2,
The observation apparatus characterized in that the condensing restriction position calculating means calculates the condensing restriction position based on an arrangement of optical elements constituting the objective lens, the illuminating means, and the first scanning means. A stage to place the specimen;
An objective lens for focusing the laser beam on the specimen;
First scanning means for scanning a specimen condensing position, which is a condensing position in the specimen, in an optical axis direction;
Observation means for observing the specimen collection position;
Control means for controlling the observation means and the first scanning means based on an intermediate image position that is a condensing position in the observation optical path and a condensing restriction position where condensing of the laser light is restricted. An observation apparatus characterized by that. The observation apparatus according to claim 20,
The control means includes
Scanning control means for controlling driving of the first scanning means;
Intermediate condensing position calculating means for calculating the intermediate image position;
A condensing restriction position calculating means for calculating the condensing restriction position;
An observation control means for controlling the observation means. The observation apparatus according to claim 21,
The scanning control means restricts scanning by the first scanning means to a position where the condensing restriction position and the intermediate image position overlap each other. The observation apparatus according to claim 21,
And a second scanning means for scanning the specimen condensing position in a direction perpendicular to the optical axis,
The scanning control means restricts scanning by the second scanning means to a position where the condensing restriction position and the intermediate image position overlap. The observation apparatus according to claim 21,
Furthermore, it includes a third scanning means for changing a distance between the sample and the objective lens,
The observation apparatus characterized in that the scanning control means controls driving of the third control means. The observation apparatus according to claim 24,
The third scanning unit drives the objective lens in the optical axis direction. The observation apparatus according to claim 24,
The third scanning unit drives the stage in the optical axis direction. The observation apparatus according to claim 24,
The scanning control unit scans the sample condensing position in the optical axis direction by the first scanning unit and the third scanning unit so that the condensing restriction position and the intermediate image position do not overlap. An observation device. The observation device according to claim 27,
The scanning control means includes
When the condensing restriction position and the intermediate image position do not overlap, the first scanning means scans the sample condensing position in the optical axis direction,
An observation apparatus, wherein when the condensing limit position and the intermediate image position overlap, the third condensing unit scans the sample condensing position in the optical axis direction. The observation device according to claim 27,
The scanning control means moves the sample to a position where the intermediate image position and the light collection restriction position do not overlap during the scanning by the third scanning means before scanning the light collection restriction position. An observation device. The observation apparatus according to claim 21,
The observation means includes imaging means,
The observation apparatus according to claim 1, wherein the imaging unit does not capture an image when the condensing restriction position and the intermediate image position overlap. The observation apparatus according to claim 21,
The intermediate condensing position calculation means calculates the intermediate image position based on an arrangement of optical elements constituting the objective lens, the observation means, and the first scanning means. The observation apparatus according to claim 21,
The observation apparatus characterized in that the intermediate condensing position calculation means calculates the intermediate image position based on the sample condensing position. The observation apparatus according to claim 21,
The observation apparatus characterized in that the condensing restriction position calculating means calculates the condensing restriction position based on an arrangement of optical elements constituting the objective lens, the illuminating means, and the first scanning means. A microscope illumination method,
A first step of displaying a range in which a condensing restriction position in an illumination optical path where condensing of laser light is restricted and an intermediate condensing position that is a condensing position in the illumination optical path overlap;
A second step of setting a range in which the in-sample condensing position that is the condensing position in the sample is scanned in the optical axis direction;
Illuminating the specimen with the laser light, and
In the third step, the intensity of laser light is modulated when the condensing restriction position and the intermediate condensing position overlap with each other. A microscope observation method,
A first step of displaying a range in which a condensing restriction position in an observation optical path where condensing of laser light is restricted and an intermediate image position that is a condensing position in the observation optical path overlap;
A second step of setting a range in which the in-sample condensing position that is the condensing position in the sample is scanned in the optical axis direction;
A third step of observing the specimen,
In the third step, the specimen is not imaged when the condensing restriction position and the intermediate image position overlap with each other.

Images