JP2006003805A - Confocal observation system, photoirradiation method and photoirradiation program - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately perform luminous stimulus and photobleach in the corresponding area of a sample by specifying an optional three-dimensional area in a stereoscopic sample image, and to accurately perform the analysis of the behavior of the sample having a stereostructure after the luminous stimulus is performed. <P>SOLUTION: The confocal laser microscope as the confocal observation system is provided with a scanning optical system A and a detection optical system B for obtaining the optical cross section image of the stereoscopic sample 9, and a control unit 14 or the like for performing processing of forming the three-dimensional image from the obtained optical cross section image, processing of specifying a desired three-dimensional area in the formed three-dimensional image, processing of obtaining a cross section area to be irradiated with exciting light or stimulating light based on the specified three-dimensional area and processing of irradiating the area of the stereoscopic sample 9 corresponding to the obtained cross section area with the exciting light or the stimulating light. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a confocal observation system, and more particularly to a confocal observation system represented by a microscope or an endoscope having a scanning optical system.

  A confocal microscope is a microscope that can detect only information on a focal plane in a sample by providing a pinhole at a position conjugate with the sample in a detection optical system. The information on the focal plane of the sample becomes an optical cross-sectional image, and it is possible to construct a three-dimensional structure of the sample by continuously acquiring the cross-sectional image.

  In such a confocal microscope, a scanning confocal laser microscope irradiates a laser beam from a light source while scanning in the X-axis and Y-axis directions of the sample via a scanning optical system and an objective lens. This is a microscope that obtains two-dimensional luminance information of a specimen by capturing fluorescence or reflected light again into a detector via an objective lens and a scanning optical system. It is also possible to observe the fluorescence image or reflection image of the specimen by associating the two-dimensional luminance information with the scanning position information of the scanning optical system and displaying the two-dimensional distribution of the bright spots.

  If multi-photon absorption is generated using a laser light source that emits pulsed laser light, etc., only information on the focal plane of the specimen can be obtained by exciting only the focal plane of the specimen without providing a pinhole. It is possible to obtain.

Further, if a scanning optical system is provided in addition to the optical system for acquiring an image, it is possible to independently control the irradiation of the laser beam for light stimulation and the image acquisition.
With respect to these confocal laser microscopes, Patent Document 1 proposes a method for efficiently acquiring an image by scanning a specimen at high speed in order to perform stereoscopic observation of a living specimen in the confocal laser microscope. ing. Patent Document 2 proposes a multi-photon excitation laser microscope that requires adjustment for adjusting the optimum observation conditions, and allows easy setting of conditions by arrangement of optical members. Patent Document 3 proposes a method in which a scanning optical system is arranged in addition to the detection optical system, and a light stimulus is expressed independently of the observation position.

By using these conventional techniques, it is possible to construct a three-dimensional image (three-dimensional image) of a living specimen at high speed. In addition, by using pulsed laser light or the like for specimen observation or optical stimulation, it is possible to excite only the focal plane of the specimen by multiphoton excitation.
JP 2003-195714 A JP-A-11-326755 Japanese Patent Laid-Open No. 10-206742

  However, conventionally, an arbitrary three-dimensional region in the three-dimensional image of the constructed specimen is designated, and the region in the specimen corresponding to the three-dimensional region is irradiated with excitation light or stimulation light, so that light stimulation or photo No proposal has been made regarding the method of bleaching.

  In view of the above circumstances, the present invention can accurately perform light stimulation and photo bleaching on a corresponding region in a specimen by designating an arbitrary three-dimensional region in the three-dimensional image of the specimen. It is an object of the present invention to provide a confocal observation system, a light irradiation method, and a light irradiation program capable of accurately performing behavioral analysis of a specimen having a light stimulus after light stimulation.

  To achieve the above object, the confocal observation system according to the first aspect of the present invention includes an image acquisition unit that acquires an optical cross-sectional image of a three-dimensional specimen, and an optical cross-sectional image acquired by the image acquisition unit. Three-dimensional image construction means for constructing a three-dimensional image, designation means for designating a desired three-dimensional area in the three-dimensional image constructed by the three-dimensional image construction means, and the three-dimensional area designated by the designation means Area acquisition means for acquiring a cross-sectional area for irradiating excitation light or stimulation light based on the excitation light or stimulation to the area of the three-dimensional specimen corresponding to the cross-sectional area acquired by the area acquisition means It is the structure which irradiates light.

  According to this configuration, an optical cross-sectional image of the three-dimensional specimen is acquired by the image acquisition unit, a three-dimensional image is constructed from the optical cross-sectional image acquired by the image acquisition unit by the three-dimensional image construction unit, A desired three-dimensional region in the three-dimensional image constructed by the original image construction unit is designated, and a cross-sectional region for irradiating excitation light or stimulation light based on the three-dimensional region designated by the designation unit by the region acquisition unit is provided. The excitation light or the stimulation light is irradiated to the area of the three-dimensional specimen corresponding to the cross-sectional area acquired and acquired by the area acquisition means.

  The confocal observation system according to a second aspect of the present invention is the confocal observation system according to the first aspect described above, wherein the region acquisition unit is obtained from the optical cross-sectional image acquired by the image acquisition unit based on the three-dimensional region. Or a cross-sectional area obtained by interpolation from the optical cross-sectional image acquired by the image acquisition means as a cross-sectional area for irradiating the excitation light or stimulation light.

  According to this configuration, from the cross-sectional area obtained from the optical cross-sectional image obtained by the image obtaining unit based on the three-dimensional area by the region obtaining unit, or from the optical cross-sectional image obtained by the image obtaining unit. A cross-sectional area obtained by interpolation is acquired as a cross-sectional area for irradiating excitation light or stimulation light.

  The confocal observation system according to a third aspect of the present invention is the confocal observation system according to the first or second aspect, wherein the excitation light or stimulation light is pulsed laser light for exciting the three-dimensional specimen by a multiphoton excitation phenomenon. There is a configuration.

  The confocal observation system according to a fourth aspect of the present invention is the confocal observation system according to any one of the first to third aspects described above, wherein the system is a first optical unit for acquiring an optical cross-sectional image of the three-dimensional specimen. It is the structure which has a system and the 2nd optical system for irradiating the said excitation light or stimulation light independently.

  In addition, the present invention can also be configured as a light irradiation method and a light irradiation program.

  According to the present invention, by designating an arbitrary three-dimensional region in a three-dimensional image of a specimen, light stimulation and photo bleaching can be accurately performed on the corresponding region in the specimen. In addition, it is possible to accurately analyze the behavior of a specimen having a three-dimensional structure after light stimulation.

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

FIG. 1 is a configuration diagram of a confocal laser microscope apparatus that is a confocal observation system according to Embodiment 1 of the present invention.
As shown in the figure, this apparatus includes a laser light source 1 that emits laser light, a laser control unit 2 that adjusts the wavelength and intensity of the laser light emitted from the laser light source 1, a dichroic mirror 3, and laser light. The optical scanner unit 4 includes two optical scanners 4a and 4b, a relay lens 5, a reflection mirror 6, an imaging lens 7, and an objective lens 8. A stage 10 that is movable in the optical axis direction (Z direction) and on which the three-dimensional specimen 9 is placed, a lens 11, and a pinhole 12 provided at a position conjugate with the focal position of the objective lens 8, A photoelectric conversion unit 13 that converts light that has passed through the pinhole 12 into an electrical signal, a control unit 14 that controls the operation of the entire apparatus, and a display unit 15 are provided.

  In this apparatus, an optical system composed of the laser light source 1, the laser control unit 2, the dichroic mirror 3, the optical scanner unit 4, the relay lens 5, and the reflection mirror 6 is called a scanning optical system A, and the lens 11 and the pinhole 12 The optical system consisting of is called a detection optical system B.

  In this apparatus, the laser light emitted from the laser light source 1 is adjusted to an arbitrary wavelength and laser intensity by the laser control unit 2 controlled by the control unit 14, then reflected by the dichroic mirror 3, and the control unit 14. Are guided to the optical scanner unit 4 controlled by, and are deflected and scanned in an arbitrary direction. The laser beam that has been deflected and scanned passes through the relay lens 5, is reflected by the reflecting mirror 6, and is movable through the imaging lens 7 and the objective lens 8 in the optical axis direction under the control of the control unit 14. The three-dimensional specimen 9 placed thereon is reached. Accordingly, the optical scanner unit 2 deflects and scans the laser light in two dimensions, so that the laser light is two-dimensionally scanned on the focal plane 22 of the three-dimensional specimen 9. The focal plane 22 is a plane including the focal position of the objective lens 8 and is a plane perpendicular to the optical axis direction.

  On the other hand, the fluorescence or reflected light from the focal plane 22 generated by the laser light that has reached the three-dimensional specimen 9 in this way has the same optical path as the above-mentioned laser light in the opposite direction, and the objective lens 8, the imaging lens 7, It passes through the reflection mirror 6, the relay lens 5, and the optical scanner unit 4. Then, of the light that has passed through the optical scanner unit 4 by the dichroic mirror 3, only the light whose wavelength is selected reaches the optical detection system B. In the detection optical system B, the light whose wavelength is selected is transmitted through the lens 11, and only the light on the focal plane 22 of the three-dimensional specimen 9 is selected from the light transmitted through the lens 11 by the pinhole 12. It reaches the photoelectric conversion unit 13.

FIG. 2 is a block diagram illustrating a configuration of the control unit 14 that performs control such as driving the scanning optical system A and displaying a signal from the detection optical system B as an image.
In this figure, this unit 14 controls the movement of a first scanning optical system drive unit 14a for driving the optical scanners 4a and 4b, a first laser output control unit 14b for controlling the laser controller 2, and the stage 10. A Z-axis control unit 14c, a man-machine interface 14d for receiving various inputs from a user, a program memory 14j in which a control program is stored, and a control program is read from the program memory 14j and executed to thereby generate a photoelectric conversion unit A CPU 14e for controlling the operation of the entire apparatus including A / D conversion of the electrical signal obtained by 13; a memory 14f for storing the electrical signal A / D converted by the CPU 14e as an optical cross-sectional image; 15 is an optical cross-sectional image stored in the memory 14f when displaying an image. To an image processing circuit 14g for performing predetermined image processing.

Next, as an operation of this confocal laser microscope apparatus, an operation performed when irradiating an arbitrary three-dimensional region in the three-dimensional specimen 9 with excitation light will be described.
In this apparatus, it is possible to construct a three-dimensional image of the three-dimensional specimen 9 by continuously capturing the optical cross-sectional image of the three-dimensional specimen 9 while moving the stage 10 in the optical axis direction. The construction of the three-dimensional image at this time is performed as follows.

  First, an optical cross-sectional image is acquired at a reference position where Z = 0, and then the stage 10 is driven to continuously change the position of the three-dimensional specimen 9 while changing the position in the Z direction (hereinafter referred to as “Z position”). A cross-sectional image is captured, and the captured optical cross-sectional image is stored in the memory 14d together with information on the Z position. Then, the optical cross-sectional images thus stored in the memory 14d are superimposed on the display unit 15 along the z-axis direction based on the corresponding Z position information, and the respective optical cross-sectional images are superimposed on the z-axis direction. A three-dimensional image can be formed by providing a thickness according to predetermined conditions.

FIG. 3 is a diagram showing a processing flow relating to the operation of the confocal laser microscope apparatus. In the figure, S1 to S7 indicate processing related to the construction of the above-described three-dimensional image.
First, k indicating a value corresponding to the Z position (cross-sectional position) is set to 0 (S1), and the stage 10 is moved to the set image acquisition start position (S2). Thereby, the focal position is set to the origin position.

  Subsequently, the optical scanner unit 4 performs two-dimensional scanning, acquires a confocal image (k) that is an optical cross-sectional image, and stores it in the memory 14d together with information on the Z position at this time (S3). The confocal image (k) represents an optical cross-sectional image at the Z position corresponding to k.

  Subsequently, k = k + 1 is set (S4), the stage 10 is moved by a predetermined amount ΔZ (S5), and then it is determined whether or not the Z position of the stage 10 is the set image acquisition end position. (S6). If the determination result is No, the process returns to S3. As a result, the processes of S3 to S5 described above are repeated until the Z position of the stage 10 reaches the image acquisition end position.

  On the other hand, if the determination result in S6 is Yes, the optical cross-sectional image stored in the memory 14d is subsequently superimposed along the z-axis direction on the display unit 10 based on the corresponding Z position information. Then, a three-dimensional image of the three-dimensional specimen 9 is constructed (S7), and the three-dimensional image is displayed on the display unit 10.

Subsequently, an arbitrary three-dimensional region in the three-dimensional image displayed on the display unit 10 is designated in accordance with a user instruction made via the man-machine interface 14d (S8).
Subsequently, in accordance with a user instruction performed via the man-machine interface 14d, an interval (Z interval) between which the excitation light is irradiated is specified, and the interval between the cross sections and the three-dimensional region specified in S8 are determined. Based on this, a plurality of cross-sectional positions of the three-dimensional specimen 9 to be irradiated with the excitation light are determined (S9). In this embodiment, it is assumed that a plurality of equidistant cross-sectional positions are determined in S9.

  Subsequently, if any of the determined plurality of cross-sectional positions coincides with the cross-sectional position of the optical cross-sectional image acquired in S3, the optical cross-sectional image of the corresponding cross-sectional position is designated in S8. A cross-sectional area corresponding to the matching cross-sectional position of the three-dimensional area determined is obtained, and the cross-sectional position of the optical cross-sectional image acquired in S3 is determined among the determined cross-sectional positions. If there is not, the cross-sectional area of the three-dimensional area specified in S8 and corresponding to the non-coincident cross-sectional position is calculated by interpolation from the optical cross-sectional image acquired in S3 (S10). ). The cross-sectional area calculated by this interpolation is called a virtual cross-sectional area.

  Subsequently, the stage 10 is moved so that the focal plane 22 is aligned with one of the plurality of cross-sectional positions determined in S9 (S11), and corresponds to the cross-sectional area obtained in S10 related to the cross-sectional position. The laser beam is irradiated as excitation light for performing light stimulation or photobleaching on the region to be processed or the region corresponding to the virtual cross-sectional region calculated by interpolation in S10 (S12).

  Subsequently, it is determined whether or not the laser beam is irradiated at all of the cross-sectional positions determined in S9 (S13). If the determination result is No, the process returns to S11. The processes of S11 to S12 are repeated again for one of the cross-sectional positions not irradiated with the laser beam. On the other hand, if the determination result in S13 is Yes, the laser light irradiation has been completed at all the cross-sectional positions, and this flow ends.

4, 5 and 6 are diagrams showing specific examples when this flow is executed.
FIG. 4 shows an example of an optical cross-sectional image obtained by the processes of S1 to S7 and a three-dimensional image of the three-dimensional specimen 9 constructed from the optical cross-sectional image.

  In the example of the figure, four optical cross-sectional images 16a, 16b, 16c, and 16d are obtained, and the three-dimensional image 17 of the three-dimensional specimen 9 is constructed from the four optical cross-sectional images 16. .

FIG. 5 shows an example of the three-dimensional area designated by the process of S8 in the three-dimensional image 17 shown in FIG.
In the example of FIG. 5, an example is shown in which the three-dimensional region 18 is designated in accordance with a user instruction.

FIG. 6 shows the cross-sectional positions determined by the process of S9 and the cross-sectional areas and the virtual cross-sectional areas obtained by the process of S10 in the three-dimensional area 18 shown in FIG.
In the example of the figure, the interval of the cross section is designated according to the user's instruction, and five cross-sectional positions A, B, C, D, and E (which are shown as the cross-sectional positions on the display unit 10 here) are equally spaced. In this example, the cross-sectional areas 19a, 19b, 19c, 19d, and 19e of the three-dimensional area 18 are obtained at the respective cross-sectional positions.

  The cross-sectional position A matches the cross-sectional position of the optical cross-sectional image 16b, and the cross-sectional position E matches the cross-sectional position of the optical cross-sectional image 16c. Accordingly, the cross-sectional area 19a is obtained from the optical cross-sectional image 16b, and the cross-sectional area 19e is obtained from the optical cross-sectional image 16c. On the other hand, since the cross-sectional positions B, C, and D do not coincide with the cross-sectional position of the optical cross-sectional image 16, the cross-sectional areas 19b, 19c, and 19d are optical cross-sectional images 16b, 16c that sandwich the cross-sectional area. Is calculated by interpolation.

  When the cross-sectional areas 19a, 19b, 19c, 19d, and 19e at the cross-sectional positions A, B, C, D, and E are obtained in this way, the processes of S11 to S13 are performed, and areas corresponding to the cross-sectional areas 19 are obtained. All of these are irradiated with laser light.

  As described above, according to the present embodiment, by designating an arbitrary three-dimensional region in the three-dimensional image of the three-dimensional specimen 9 in accordance with a user instruction, light stimulation and photo bleaching can be performed in the corresponding region in the three-dimensional specimen 9. Therefore, it is possible to accurately analyze the behavior of the three-dimensional specimen 9 after light stimulation. In addition, if the total number of optical cross-sectional images acquired when constructing a three-dimensional image of the three-dimensional specimen 9 is reduced, the total amount of laser light irradiated on the three-dimensional specimen is reduced at the time of acquisition. Therefore, unnecessary light stimulation and photo bleaching can be avoided as much as possible.

  In this embodiment, the confocal laser microscope apparatus is configured to move the focal plane 22 by moving the stage 10 in the optical axis direction. However, the focal plane is moved by moving the objective lens 8 in the optical axis direction. It is also possible to move 22.

  Further, in this embodiment, in S9 of FIG. 3, the interval of the cross section for irradiating the excitation light is designated according to the user's instruction, but this apparatus automatically designates this. Is also possible.

  Further, in S9, the interval of the cross section for irradiating the excitation light is specified and the cross section position is determined according to the user's instruction. Instead, the user specifies an arbitrary point in the three-dimensional area. By doing so, it is possible to determine the cross-sectional position.

FIG. 7 is a diagram illustrating an example when the cross-sectional position is determined by the user instructing an arbitrary point in the three-dimensional region in S9.
In the figure, a three-dimensional region 20 indicates a part of the three-dimensional region specified in S8, and the cross-sections 21a and 21b indicated by solid lines both coincide with the cross-sectional position of the optical cross-sectional image acquired in S3. A cross section is shown.

  At this time, when a desired point in the three-dimensional region 20 (a point indicated by x in the figure) is designated by the user, and the designated point does not coincide with the cross-sectional position of the optical cross-sectional image acquired in S3. The cross-sectional position corresponding to the cross-section including the indicated point is obtained as follows.

  However, here, the z coordinate on the display unit 10 of the cross section 21a is z1, and the Z position of the stage 10 corresponding to z1 is Z1. Further, the z coordinate of the cross section 21b on the display unit 10 is set to z2, and the Z position of the stage 10 corresponding to z2 is set to Z2. Further, the z coordinate on the display unit 10 of the cross section including the point designated by the user (the cross section indicated by the dotted line) is z3, and the Z position of the stage 10 corresponding to z3 is Z3.

At this time, Z3 which is the cross-sectional position to be obtained is obtained by the following equation.
Z3 = Z1 + (Z2-Z1) / (z2-z1) * (z3-z1)
Thereby, the Z position of the stage 10 corresponding to the point instruct | indicated by the user can be calculated | required, and a cross-sectional position can be determined.

  In the present embodiment, the light source 1 that emits pulsed laser light for exciting the three-dimensional specimen 9 so as to emit fluorescence by a multiphoton excitation phenomenon may be used. Since the multi-photon excitation phenomenon is a phenomenon that occurs only on the focal plane where the laser light is concentrated, it is possible to irradiate the focal plane only with the excitation light for light stimulation or photo bleaching. In addition, when performing excitation with pulsed laser light, laser light having a longer wavelength can be used than in the case of using continuous wave laser light, so at a deeper position that cannot be reached with normal laser light, It is possible to generate an excitation phenomenon, which is effective for observation, light stimulation, and photobleaching of a specimen having a large focal direction such as a nerve cell in which a tissue or an axon is developed. In addition, since the excitation phenomenon due to multiphoton excitation occurs only at the focal plane where the energy density becomes very large, it is possible to cause optical stimulation or photo bleaching only at a desired site.

FIG. 8 is a configuration diagram of a confocal laser microscope apparatus that is a confocal observation system according to Embodiment 2 of the present invention.
In the figure, the difference from the configuration shown in FIG. 1 is that a laser light source 26 for irradiating laser light, a laser control unit 27 for adjusting the wavelength and intensity of the laser light emitted from the laser light source 26, and a laser The laser beam from the light source 26 is deflected and scanned two-dimensionally (XY direction), and includes an optical scanner unit 28 having two optical scanners 28a and 28b, a relay lens 29, and a dichroic mirror 30. Accordingly, the internal configuration of the control unit 14 is slightly different. Other configurations are the same as those shown in FIG.

In this apparatus, an optical system including the laser light source 26, the laser control unit 27, the optical scanner unit 28, and the relay lens 29 is referred to as a scanning optical system C.
In the apparatus according to the present embodiment, the laser light emitted from the laser light source 26 is adjusted to a desired wavelength and laser intensity by the laser control unit 27 controlled by the control unit 14 and then controlled by the control unit 14. Is guided to the optical scanner unit 28 and deflected and scanned in an arbitrary direction. The laser beam that has been deflected and scanned passes through the relay lens 29 and is then combined with the light from the scanning optical system A by the dichroic mirror 30, passes through the imaging lens 7 and the objective lens 8, and passes through the three-dimensional specimen 9. The upper focal plane 22 is irradiated.

FIG. 9 is a block diagram illustrating a configuration of the control unit 14 according to the present embodiment.
In the figure, the difference from the configuration shown in FIG. 2 is that a second scanning optical system drive unit 14 h that drives the optical scanners 28 a and 28 b and a second laser output control unit that controls the laser control unit 27. 14i. As a result, the optical scanner unit 28 and the laser control unit 26 are driven and controlled so that the laser light from the laser light source 26 can be irradiated to a desired position. Further, as necessary, the first scanning optical system A and the second scanning optical system C can be controlled independently or in synchronization. Other configurations are the same as those shown in FIG.

  The operation of the confocal laser microscope apparatus according to the present embodiment is related to the first embodiment except that the three-dimensional specimen 9 is irradiated with the excitation light using the scanning optical system C instead of the scanning optical system A. It is the same as the operation of the device. That is, in the present apparatus, the scanning optical system A is used when acquiring an optical cross-sectional image, and the scanning optical system C is used when irradiating the three-dimensional specimen 9 with excitation light. However, in this apparatus, the irradiation position of the laser beam by the scanning optical system C is associated with the irradiation position of the laser beam by the scanning optical system A in advance.

  As described above, according to the present embodiment, since it is possible to perform the irradiation of the excitation light to the three-dimensional specimen 9 and the acquisition of the optical cross-sectional image independently, after performing the light stimulation and the photo bleaching. It is possible to accurately measure a phenomenon appearing in a three-dimensional specimen in real time.

  Although the present invention has been described in detail above, the present invention is not limited to the above-described embodiment, and it is needless to say that various improvements and changes may be made without departing from the gist of the present invention.

  For example, the first and second embodiments have been described using a scanning confocal laser microscope apparatus having a scanning optical system as a means for acquiring an image. However, the confocal of the disk scanning method is limited to the means for acquiring an image. It is also possible to use a microscope.

  In the first and second embodiments, the confocal observation system according to the present invention is applied to the confocal laser microscope apparatus. However, it can also be applied to an endoscope system that requires a scanner unit for acquiring an image. It is.

1 is a configuration diagram of a confocal laser microscope apparatus that is a confocal observation system according to Embodiment 1. FIG. FIG. 3 is a block diagram illustrating a configuration of a control unit according to the first embodiment. FIG. 6 is a diagram illustrating a processing flow relating to the operation of the confocal laser microscope apparatus according to the first embodiment. It is a figure which shows the specific example when the processing flow which concerns on Example 1 is performed. It is a figure which shows the specific example when the processing flow which concerns on Example 1 is performed. It is a figure which shows the specific example when the processing flow which concerns on Example 1 is performed. It is a figure which shows an example at the time of making it determine a cross-sectional position by instruct | indicating the arbitrary points in a three-dimensional area | region. 6 is a configuration diagram of a confocal laser microscope apparatus which is a confocal observation system according to Embodiment 2. FIG. FIG. 6 is a block diagram illustrating a configuration of a control unit according to a second embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Laser light source 2 Laser control part 3 Dichroic mirror 4 Optical scanner part 5 Relay lens 6 Reflection mirror 7 Imaging lens 8 Objective lens 9 Three-dimensional sample 10 Stage 11 Lens 12 Pinhole 13 Photoelectric conversion part 14 Control unit 15 Display part 16 Optical Cross-sectional image 17 Three-dimensional image 18 Three-dimensional region 19 Cross-sectional region 20 Three-dimensional region 21 Cross-section 22 Focal plane 26 Laser light source 27 Laser control unit 28 Optical scanner unit 29 Relay lens 30 Dichroic mirror

Claims (6)

Image acquisition means for acquiring an optical cross-sectional image of a three-dimensional specimen;
Three-dimensional image construction means for constructing a three-dimensional image from the optical cross-sectional image acquired by the image acquisition means;
Designation means for designating a desired three-dimensional region in the three-dimensional image constructed by the three-dimensional image construction means;
Area acquiring means for acquiring a cross-sectional area for irradiating excitation light or stimulation light based on the three-dimensional area specified by the specifying means;
Have
Irradiating the region of the three-dimensional specimen corresponding to the cross-sectional region acquired by the region acquisition means with the excitation light or stimulation light;
Confocal observation system characterized by that. The area acquisition means is obtained by interpolation from the cross-sectional area obtained from the optical cross-sectional image obtained by the image acquisition means based on the three-dimensional area, or the optical cross-sectional image obtained by the image acquisition means. Obtaining the cross-sectional area as a cross-sectional area for irradiating the excitation light or stimulation light,
The confocal observation system according to claim 1. The excitation light or stimulation light is pulsed laser light for exciting the three-dimensional specimen by a multiphoton excitation phenomenon.
The confocal observation system according to claim 1 or 2, wherein The system independently includes a first optical system for acquiring an optical cross-sectional image of the three-dimensional specimen and a second optical system for irradiating the excitation light or stimulation light.
The confocal observation system according to any one of claims 1 to 3, wherein Obtain an optical cross-sectional image of a three-dimensional specimen,
Constructing a three-dimensional image from the acquired optical cross-sectional image,
Specify the desired 3D region in the constructed 3D image,
Obtain a cross-sectional area for irradiating excitation light or stimulation light based on the designated three-dimensional area,
Irradiating the region of the three-dimensional specimen corresponding to the acquired cross-sectional region with the excitation light or stimulation light;
The light irradiation method of the confocal observation system characterized by the above-mentioned. To the computer of the confocal observation system,
A function of acquiring an optical cross-sectional image of a three-dimensional specimen;
A function of constructing a three-dimensional image from the acquired optical cross-sectional image;
A function for designating a desired three-dimensional region in the constructed three-dimensional image;
A function of acquiring a cross-sectional area for irradiating excitation light or stimulation light based on the designated three-dimensional area;
A function of irradiating the excitation light or stimulation light to the region of the three-dimensional specimen corresponding to the acquired cross-sectional region;
Light irradiation program to realize

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