JP6178656B2 - Method for setting adaptive optical element and microscope - Google Patents

Description

  The present invention relates to a setting method of an adaptive optical element and a microscope.

  The scanning microscope is configured to irradiate an observation specimen with a laser beam through an objective lens, thereby collecting and detecting fluorescence generated from the observation specimen again with the objective lens. In such a scanning microscope, in order to obtain high-energy fluorescence, it is necessary to concentrate excitation light at a scanning point on the specimen, and the aberration of the optical system including the objective lens is minimized. In addition, when observing a biological specimen, strictly speaking, the refractive index of the biological specimen itself and its three-dimensional distribution also affect the concentration of excitation light, so the refractive index of the biological specimen itself and its three-dimensional distribution are also taken into consideration. It is necessary to correct the aberration of the optical system. However, since the refractive index and its three-dimensional distribution of the biological specimen itself are unknown and differ depending on the biological specimen, the refractive index of all biological specimens and aberrations of the optical system taking into account the three-dimensional distribution are accurately corrected in advance. It is difficult to do. For this reason, there is known a method for compensating for aberrations in an optical system by using a compensation optical element (for example, a deformable mirror) that can control the wavefront of incident light (for example, see Patent Document 1). Here, the control parameter of the compensation optical element is the drive amount of the drive mechanism that drives the compensation optical element, and when the compensation optical element is a deformable mirror, the drive parameter of the piezo element that drives the mirror.

Japanese Patent Application Laid-Open No. 11-101942

  However, when a biological specimen is observed using an optical system including such a compensation optical element, if the control parameter output to the compensation optical element is changed, the focal position of the objective lens is shifted in the z direction (optical axis direction). May end up. In that case, if the refractive index of the biological specimen and its three-dimensional distribution are completely known, the focal position displacement amount in the z direction of the objective lens in accordance with the change of the control parameter is predicted, and the displacement amount is also corrected. Although it is possible to prevent the focal position shift of the objective lens in the z direction by controlling the wavefront aberration parameter in consideration, the refractive index and its three-dimensional distribution are unknown in an actual biological specimen, and individual differences are also For this reason, it is difficult to perform control that predicts the amount of positional deviation.

  The present invention has been made in view of such problems, and even if the sample has an unknown refractive index and its three-dimensional distribution, such as a biological sample, a more accurate control parameter can be set for the compensation optical element. It is an object of the present invention to provide a method for setting a compensation optical element that can be determined and a microscope for setting the compensation optical element by this method.

  In order to solve the above-described problem, a compensation optical element setting method according to the present invention includes a compensation optical element that changes a wavefront of illumination light emitted from a light source unit, and uses an objective lens to transmit illumination light whose wavefront has been changed. A compensation optical element setting method in a microscope, comprising: an illumination optical system that irradiates a specimen via a specimen; a detection optical system that guides light from the specimen to a detection section; and a control section that controls the compensation optical element. The first step of acquiring the sample image when the focal plane of the lens is at a predetermined position of the sample as a reference image, and the control unit controls the compensation optical element so that the wavefront of the illumination light is in two or more different states. A second step of acquiring images of the sample at a plurality of positions in a predetermined range in the optical axis direction including a predetermined position in each state, and corresponding to each of the plurality of positions in each state of the wavefront Sample image A third step of selecting a determination image based on a correlation value with the reference image, and calculating an evaluation value of a predetermined region for each of the determination image in each state of the reference image and the wavefront, and the evaluation value and the wavefront A fourth step of newly determining a wavefront state based on each of the above states, and a fifth step of setting the adaptive optics so that the determined wavefront state is obtained. .

  In such a setting method of the compensating optical element, in the second step, the state change of the wavefront of the illumination light is compensated by the control parameter reflecting the coefficient change of each term of the Zernike polynomial corresponding to the desired optical aberration. It is preferably realized by controlling the element.

  In addition, such a setting method of the compensation optical element includes the second step, the third step, the fourth step, and the fifth step for each coefficient of each term of the Zernike polynomial corresponding to each of the plurality of optical aberrations. It is preferable to execute the steps.

  In such a setting method of the adaptive optical element, it is preferable that the correlation value in the third step is calculated by a phase-only correlation method.

  In such a setting method of the compensation optical element, the evaluation value in the fourth step is at least one of an integrated value, a maximum value, an average value, and a contrast value of the luminance values of the pixels in the predetermined region. It is preferable.

  In such a setting method of the compensation optical element, it is preferable that the sample image is an image of at least one of fluorescence, second harmonic, and third harmonic.

  In such a setting method of the adaptive optical element, it is preferable that the predetermined area in the fourth step is an area where the specimen is desired to be observed.

  In such a setting method of the adaptive optical element, it is preferable that the predetermined region in the fourth step is a region outside the region where the specimen is to be observed. In this case, it is preferable to irradiate illumination light only to the outer region when acquiring the specimen image.

  In addition, the microscope according to the present invention includes an compensation optical element that changes the wavefront of the illumination light emitted from the light source unit, and an illumination optical system that irradiates the specimen with the illumination light with the changed wavefront via the objective lens; A detection optical system that guides light from the specimen to the detection unit; and a control unit that controls the compensation optical element. The control unit sets the compensation optical element by any one of the above-described compensation optical element setting methods. It is characterized by that.

  When the present invention is configured as described above, a compensation optical element setting method capable of determining a control parameter with higher accuracy for the compensation optical element and a microscope for setting the compensation optical element by this method are provided. Thus, the image quality and image acquisition efficiency of the entire observation (experiment) using this microscope can be improved.

It is explanatory drawing for demonstrating the structure of a scanning microscope. It is a flowchart which shows the whole flow of the setting method of an adaptive optics element. It is a flowchart which shows the flow of an optimal coefficient calculation process. It is a flowchart which shows the flow of a wavefront update determination process. It is explanatory drawing for demonstrating the relationship between a reference | standard image and ROI. It is explanatory drawing for demonstrating the relationship of the correlation value of a stack image and this stack image, and a reference | standard image. It is explanatory drawing which shows the relationship between a Zernike coefficient and an evaluation function. It is explanatory drawing for demonstrating the outline | summary of the setting method of an adaptive optics element. It is explanatory drawing for demonstrating the sub area | region in a peripheral area | region. It is explanatory drawing for demonstrating the relationship between ROI and a peripheral region.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the optical system of the scanning microscope will be described with reference to FIG. The scanning microscope 10 includes a scanning optical system 30 that scans a sample I0 placed on a stage with laser light (illumination light or excitation light) emitted from a light source device 20 via an objective lens 36; The wavefront compensation optical system 40 that changes the wavefront of the laser beam, the first detection unit 50 and the second detection unit 60 that detect observation light (for example, fluorescence) from the sample collected by the objective lens 36, and the sample And a third detection unit 70 for detecting light transmitted through the light source. In the following description, the optical axis direction of the scanning optical system 30 is defined as the z axis, and the directions orthogonal to each other in a plane orthogonal to the z axis are defined as the x axis and the y axis, respectively.

  In the scanning microscope 10, the light source device 20 includes a light source 21 configured to be able to emit a plurality of laser beams having different wavelengths simultaneously or individually, and an intensity of the passing laser beam of 0 to 100%. And an AOM (Acousto-Optic Modulator) 22 that changes (intensity modulation) between them. In the following description, it is assumed that two types of laser light, infrared light and visible light, are emitted from the light source 21. In addition, the infrared light emitted from the light source 21 is a very short pulse-like light emitted at a predetermined cycle for inducing multiphoton excitation of the specimen (for example, a pulse of about 100 femtoseconds). Hereinafter referred to as “IR pulsed light”).

  The scanning optical system 30 includes a first dichroic mirror 31, a scanning unit 32, a scan lens 33, a second objective lens 34, a second dichroic mirror 35, and an objective lens 36 in order from the light source device 20 side. ing. The first dichroic mirror 31 transmits laser light (illumination light or excitation light) emitted from the light source device 20 and transmits observation light that transmits observation light (fluorescence or second harmonic) described later. It is configured. In addition, the second dichroic mirror 35 is detachably arranged, and when the optical path is inserted, the laser light (illumination light or excitation light) is transmitted, and among the observation light generated in the specimen, observation light shorter than a predetermined wavelength ( (Fluorescence and second harmonic) are reflected.

  The wavefront compensation optical system 40 is disposed between the scan lens 33 and the second objective lens 34 of the scanning optical system 30, and sequentially from the light source device 20 side, the first relay lens 41, the compensation optical element 42, and The second relay lens 43 is included. In this wavefront compensation optical system 40, the compensation optical element 42 can change the wavefront of incident light when reflecting (or transmitting) the incident light (for example, a deformable mirror as a reflective element). And a reflective liquid crystal element, and a transmissive liquid crystal element as a transmissive element. Reflective liquid crystal elements and transmissive liquid crystal elements can independently determine, set, and implement the coefficient values of each term of the Zernike polynomial corresponding to the optical aberrations (spherical aberration, coma aberration, etc.) to be corrected. Is possible. On the other hand, due to the mirror control characteristics of deformable mirrors, the coefficient values of each term of the Zernike polynomial corresponding to the optical aberration to be corrected (spherical aberration, coma aberration, etc.) are subject to interaction (the coefficient of a certain term). If this is changed, the coefficients of other terms are also dragged and changed), and it is often difficult to determine, set and implement them independently.

In the present embodiment, a deformable mirror that is a reflective element will be described as an example of the adaptive optical element 42. The illumination light for the sample and the reference light for monitoring the shape of the deformable mirror are simultaneously incident on the deformable mirror. The illumination light is incident on the deformable mirror at an angle, and the reference light is incident from the front. By controlling the deformable mirror in an elliptical shape according to the incident angle, a specified shape is expressed in the illumination light. Note that the incident angle of the illumination light to the deformable mirror is preferably limited to an angle range in which the influence of the ovalization of the light beam is small and the influence on the asymmetric aberration due to the oblique incidence is small, for example, within 45 degrees, and more preferably Set within 30 degrees. In order to detect the state of the wavefront of the emitted light subjected to the action of the compensation optical element 42, the reference laser device 44, the first collimator lens 45, the half mirror 46, the relay lens 47 (the third relay lens 47a and the fourth relay). A lens 47b) and a wavefront sensor 48 are provided. An optical fiber 49 is provided to guide the reference light from the reference laser device 44 to the first collimator lens 45. Specifically, the state of the wavefront of the emitted light that has been subjected to the action of the compensation optical element 42 is detected by a wavefront sensor 48 (for example, Shack-Hartmann method, interference method) via the half mirror 46 and the relay lens 47 and detected. If the difference value is equal to or greater than a threshold value, the amount of feedback to the adaptive optical element 42 is calculated. , Feedback to the compensation optical element 42. These series of processes are performed using, for example, the following known methods.
Adaptive control of a micromachined continuous-membrane deformable mirror for aberration compensation (APPLIED OPTICS y Vol. 38, No. 1 y 1 January 1999),
Methods for the characterization of deformable membrane mirrors (August 2005 Vol. 44, No. 24 APPLIED OPTICS)

  The first detection unit 50 is a reflection NDD (Non Descanned Detector), and is arranged in a direction in which the observation light of the second dichroic mirror 35 is reflected, and in order from the second dichroic mirror 35 side, the third dichroic is arranged. A mirror 51 and a first condenser lens 52 and a first photodetector 53 in a direction in which light passes through the third dichroic mirror 51, and a second light collection in a direction in which light is reflected by the third dichroic mirror 51. An optical lens 54 and a second photodetector 55 are included. The third dichroic mirror 51 of the first detection unit 50 is configured to transmit light (for example, fluorescence) having a predetermined wavelength or longer and reflect light (for example, second harmonic) having a wavelength shorter than the predetermined wavelength. Then, the first and second photodetectors 53 and 55 can detect the intensities of light having different wavelengths (for example, the first photodetector 53 detects fluorescence and the second photodetector 55 detects the second intensity). The second harmonic can be detected).

  The second detection unit 60 is arranged in a direction in which the observation light from the first dichroic mirror 31 is transmitted. The third condenser lens 61 and the sample of the objective lens 36 are sequentially arranged from the first dichroic mirror 31 side. A light shielding plate 62, a second collimator lens 63, a fourth dichroic mirror 64, and a fourth condensing lens in a direction in which light passes through the fourth dichroic mirror 64. 65 and a third photodetector 66, and a fifth condenser lens 67 and a fourth photodetector 68 in the direction in which light is reflected by the fourth dichroic mirror 64. The light shielding plate 62 is provided with a pinhole 62a, and the pinhole 62a is disposed so as to include the optical axis of the scanning optical system 30. Further, in order to guide the light that has passed through the pinhole 62 a of the light shielding plate 62 to the third and fourth photodetectors 66 and 68, a relay optical system 69 is provided between the light shielding plate 62 and the second collimator lens 63. (Fifth and sixth relay lenses 69a and 69b and an optical fiber 69c) are provided. Also in the second detection unit 60, the third and fourth photodetectors 66 and 68 can detect the intensity of light of different wavelengths by the fourth dichroic mirror 64.

  The third detection unit 70 is a transmission NDD, and is disposed on the opposite side of the objective lens 36 across the sample surface I0. The condenser lens 71 and the fifth dichroic mirror are sequentially arranged from the sample surface I0 side. 72 and a sixth condensing lens 73 and a fifth photodetector 74 in the direction in which the light passes through the fifth dichroic mirror 72, and a seventh condensing in the direction in which the light is reflected by the fifth dichroic mirror 72. A lens 75 and a sixth photodetector 76; Also in the third detection unit 70, the fifth and sixth photodetectors 74 and 76 can detect the intensity of light having different wavelengths by the fifth dichroic mirror 72.

  The deflecting element of the scanning unit 32 and the reflecting surface (or transmitting surface) of the compensating optical element 42 are preferably arranged at a position substantially conjugate with the exit pupil of the objective lens 36, and therefore the scanning lens 33 and the first In addition, the image of the exit pupil P0 is relayed by the second relay lenses 41 and 43, and the scanning unit 32 and the compensation optical element 42 are arranged so as to coincide with this image (in the example of FIG. 1, the image of the exit pupil). P1 and the compensation optical element 42 substantially coincide, and the exit pupil image P2 and the scanning unit 32 substantially coincide). In FIG. 1, the wavefront compensation optical system 40 (compensation optical element 42) is disposed between the scanning unit 32 and the objective lens 36, but this wavefront compensation optical system is disposed between the scanning unit 32 and the light source device 20. It is also possible to arrange 40.

  In the scanning optical system 30, three mirrors 11, 12, and 13 are used to guide the laser light transmitted through the AOM 22 of the light source device 20 to the first dichroic mirror 31, and a pinhole 62 a of the light shielding plate 62 is used. The mirror 17 and the relay optical system 69 (the fifth and sixth relay lenses 69a and 69b and the optical fiber 69c) are used to guide the light that has passed through the second collimator lens 63, and the wavefront compensation optical system 40 is further used. 3, three mirrors 14, 15, and 16 are used to guide the laser light and the observation light to the compensation optical element 42, and an optical fiber to guide the reference light from the reference laser device 44 to the first collimator lens 45. 49 are used, but these are used for the purpose of bending the optical path in order to arrange each optical element. It is not limited to this configuration.

  Further, as the first to sixth photodetectors 53, 55, 66, 68, 74, and 76, for example, PMT (Photo Multiplier Tube) is used.

  The scanning microscope 10 also selects the wavelength of the laser light emitted from the light source 21, controls the operation of the AOM 22 and the scanning unit 32, and also scans the laser light with the scanning unit 32 (on the specimen). A control unit 80 is provided for processing the values detected by the scanning plane (coordinates within the specimen plane) and the first to sixth photodetectors 53, 55, 66, 68, 74, and 76. The controller 80 is also configured to control the adaptive optical element 42 while monitoring the state of the wavefront detected by the wavefront sensor 48. Specifically, the reference light emitted from the reference laser 44 is converted into a substantially parallel light beam by the first collimator lens 45, partially reflected by the half mirror 46, and vertically incident on the reflection surface of the compensation optical element 42. The reference light vertically reflected by the reflecting surface is partially transmitted by the half mirror 46, reflected by the mirror 18, and then incident on the wavefront sensor 48 via the relay lens 47. Further, the control unit 80 controls the actuator 37 to move the objective lens 36 or the stage in the optical axis direction, thereby changing the position of the focal plane (sample surface I0) of the objective lens 36 in the sample (z direction). It is configured as follows. The control unit 80 includes a display device 81 for displaying the acquired specimen image, an input device 82 for setting various information and operating the objective lens 36 or the stage, and the acquired image. A storage device 83 for storage is connected.

  In the scanning microscope 10, laser light (illumination light or excitation light) that is a substantially parallel light beam emitted from the light source 21 of the light source device 20 passes through the AOM 22 and is then reflected by the three mirrors 11, 12, and 13. Then, the light enters the first dichroic mirror 31, is reflected by the first dichroic mirror 31, and enters the scanning unit 32. The scanning unit 32 scans the laser light two-dimensionally in a direction orthogonal to the optical axis (the above-described x direction and y direction). For example, the scanning unit 32 reflects the laser light to reflect the laser light on the optical axis. A first deflecting element that deflects in a predetermined direction (this direction is assumed to be the x direction) in a plane orthogonal to the direction, and a direction substantially orthogonal to the deflection direction of the first deflecting element in a plane orthogonal to the optical axis It is composed of two deflecting elements composed of a second deflecting element that deflects in this direction (y direction). The laser light (substantially parallel light beam) emitted from the scanning unit 32 is condensed on the secondary image plane I2 by the scan lens 33, converted to a substantially parallel light beam by the first relay lens 41, and reflected by the mirror 14. Then, the light enters the adaptive optics element 42. As described above, the compensation optical element 42 is configured to change the wavefront when the incident laser light is reflected. The laser light reflected by the compensation optical element 42 is the second relay lens 43. Is reflected by the mirror 15 and then condensed on the primary image plane I1 and further reflected by the mirror 16 and passes through the second objective lens 34 to become a substantially parallel light beam again, which passes through the second dichroic mirror 35. Then, the light is condensed by the objective lens 36 onto the focal plane (sample surface I0) of the objective lens 36 on the sample. The laser beam condensed on the sample is a point image, and the diameter of the point image is determined by the numerical aperture (NA) of the objective lens 36.

  Here, in the scanning microscope 10 according to the present embodiment, at the time of observation of the sample, either one of the IR pulse light and the visible light among the laser light emitted from the light source 21 is imaging for obtaining an image of the sample. Used as illumination light (excitation light).

  For example, when IR pulse light is used as illumination light (excitation light), visible light is not used at all or is used for light stimulation of a specimen. In this case, when the sample is irradiated with IR pulse light, fluorescence is generated from the sample by multiphoton excitation, and this fluorescence becomes observation light and is incident on the objective lens 36, and becomes a substantially parallel light beam by the objective lens 36. The light is reflected by the second dichroic mirror 35 and enters the first detector 50. When this observation light passes through the third dichroic mirror 51, it is collected and detected by the first condenser lens 52 on the light receiving surface of the first photodetector 53, and when it is reflected by the third dichroic mirror 51. The light is condensed on the light receiving surface of the second photodetector 55 by the second condenser lens 54 and detected. The first detection unit 50 is provided with an IR cut filter (not shown) in order to remove the IR pulse light reflected by the specimen, the objective lens 36, etc. and incident on the first detection unit 50 together with the observation light. May be.

  On the other hand, when using visible light as illumination light (excitation light), IR pulse light may be used for light stimulation of the specimen. In this case, when the specimen is irradiated with illumination light, fluorescence is generated from the specimen, and this fluorescence becomes observation light and is incident on the objective lens 36. The objective lens 36 becomes a substantially parallel light beam and the second dichroic mirror 35. Transparent. Then, this observation light is condensed on the primary image plane I1 by the second objective lens 34, and is then made into a substantially parallel light flux by the scan lens 33 along the reverse path to the illumination light described above, and is incident on the scanning unit 32. Then, it is descanned and emitted by the scanning unit 32, further passes through the first dichroic mirror 31, and is condensed on the pinhole 62 a of the light shielding plate 62 by the third condenser lens 61. Only light that has passed through the pinhole 62a of the light shielding plate 62 reaches the third or fourth photodetectors 66 and 68 of the second detector 60 and is detected.

  As described above, the pinhole 62a of the light shielding plate 62 is conjugate with the point image of the laser beam collected on the scanning surface (sample surface I0) on the sample, and the irradiation area on the sample (the focal plane of the objective lens 36). The observation light (fluorescence) emitted from (sample surface I0)) can pass through this pinhole 62a. On the other hand, most of the light emitted from other areas on the specimen is not condensed on the pinhole 62a and cannot pass therethrough. Therefore, an image having a high resolution in the optical axis direction can be acquired.

  Further, the illumination light transmitted through the specimen or the observation light (for example, second harmonic) generated by the illumination light is converted into a substantially parallel light beam by the condenser lens 71, reflected by the mirror 17, and reflected by the third detection unit 70. Is incident on. When this observation light passes through the fifth dichroic mirror 72, it is collected and detected by the sixth condenser lens 73 on the light receiving surface of the fifth photodetector 74, and when it is reflected by the fifth dichroic mirror 72. The light is condensed on the light receiving surface of the sixth photodetector 76 by the seventh condenser lens 75 and detected.

  As described above, the scanning optical system 30 includes the illumination optical system and the scanning unit that scans the sample with the illumination light emitted from the light source device 20 that is the light source unit, and the first and second detection units 50, It has the function of the light collecting means leading to 60. The control unit 80 processes the optical signals detected by the first to third detection units 50, 60, and 70 in synchronization with the scanning of the scanning unit 32, so that the scanning position of the laser light on the specimen and the optical signal are processed. Can be used to obtain a two-dimensional image on the sample scanning plane (sample plane I0). As a result, the scanning microscope 10 can obtain an image of the specimen with high resolution. Thus, the scanning microscope 10 according to the present embodiment can be used as both a scanning multiphoton microscope and a scanning confocal microscope.

  A method of correcting the aberration of the optical system by controlling the compensation optical element 42 of the wavefront compensation optical system 40 in the scanning microscope 10 having the above-described configuration will be described. In the control method of the wavefront compensation optical system 40 according to the present embodiment, the Zernike polynomial corresponding to the optical aberration (spherical aberration, coma aberration, etc.) to be corrected is compensated for the compensation optical element 42. A coefficient is selected, an optimum value that can obtain an optimum image for each coefficient is determined, and the adaptive optical element 42 is finally controlled by a control parameter that realizes the optimum value of the coefficient of each term. On the other hand, it is configured to give an optimum wavefront. The Zernike polynomial is easy to understand intuitively because each term of the coefficient corresponds to the optical aberration, and because each coefficient is orthogonal, each term can be handled independently. In addition, since the relationship (correspondence relationship) between each optical aberration and the coefficient of the Zernike polynomial is a well-known technique, description is abbreviate | omitted in this embodiment. Further, in the following description, a case where multiphoton fluorescence observation (especially two-photon fluorescence observation) of a specimen is performed using the scanning microscope 10 according to the present embodiment will be described.

  First, the observer moves at least one of the objective lens 36 and the stage in the z direction (optical axis direction), and the position of the objective lens 36 with respect to the specimen to be observed (the z-direction position, which is the objective lens 36 in the specimen). The focal plane). Hereinafter, a case where the stage on which the sample is placed is fixed and the objective lens 36 is moved in the z direction will be described. However, the present invention is not limited to this. Then, when setting processing of an optimal control parameter (hereinafter referred to as “optimum value”) for controlling the adaptive optical element 42 is started in this state, the control unit 80, as shown in FIG. An image of the sample surface I0 at the position of the objective lens 36 with respect to the sample in the z direction (hereinafter referred to as “reference position”) is acquired and stored in the storage device 83 (step S100). Then, the image acquired in step S100 is read from the storage device 83 and displayed on the display device 81 (step S110). In this state, the control unit 80 causes the observer to select a region to be observed (hereinafter referred to as “ROI (Region Of Interest)”) in the image displayed on the display device 81, and the reference position and the ROI. Is stored (step S120). For example, as shown in FIG. 5, the image G of the entire field of view acquired by the scanning microscope 10 is displayed on the display device 81, and the ROI is selected using the input device 82.

  Next, the control unit 80 selects one of optical aberrations to be corrected (Zernike coefficients) (step S130), and executes an optimum coefficient calculation process (step S140). Here, the optical aberration (Zernike coefficient) to be corrected may be set in the control unit 80 in advance, or may be selected by the observer before this control is executed. As shown in FIG. 3, in the optimum coefficient calculation process S140, the control unit 80 moves the objective lens 36 to the reference position in the z direction, acquires an image of the sample at this reference position, and stores it in the storage device 83 (step S3). S1401). An image at this reference position is referred to as a “reference image”. Then, the control unit 80 reads the image acquired in step S1401 from the storage device 83 (step S1402), and calculates an evaluation value for evaluating this image in the ROI in the read image (step S1403). ). Here, in this embodiment, the value obtained by integrating the luminance of each pixel in the ROI is used as the evaluation value. When performing multi-photon fluorescence observation, for example, two-photon fluorescence observation, in the scanning microscope 10 described above, the fluorescence intensity is proportional to the square of the excitation light, so that the sensitivity is good and suitable for evaluating the acquired image. Because. Of course, the evaluation function is not limited to the luminance value, and contrast in the ROI (for example, assuming that the maximum value among the luminance values of each pixel is Lmax and the minimum value is Lmin, (Lmax−Lmin) / (Lmax + Lmin), Lmax / Lmin) may be used. Further, the present invention is not limited to the integration of the luminance values of the respective pixels in the ROI, and may be the average value of the luminance values of the respective pixels in the ROI or the maximum value among the luminance values of the respective pixels. Alternatively, a plurality of evaluation values such as a luminance value and contrast may be used.

  Further, the control unit 80 compensates the control parameter (value) based on the Zernike polynomial that is changed by adding a predetermined value to the current value of the selected Zernike coefficient (the value when the reference image is acquired). It outputs to the element 42 (step S1404). When the compensation optical element 42 is controlled in this way, the illumination light (excitation light) whose wavefront aberration is corrected is irradiated onto the specimen (specimen plane I0) through the objective lens 36. However, the focal position of the objective lens 36 is shifted in the z direction from the reference position. Then, the control unit 80 moves the objective lens 36 by a predetermined step size within a predetermined range in the z direction including the reference position, acquires a sample image at each position, and stores it in the storage device 83 (step S1405). ). These images are called “stack images”. The range and step size in the z direction for acquiring this stack image may be determined in advance, or may be changed based on the currently selected optical aberration (Zernike coefficient). For example, if the Zernike coefficient corresponding to spherical aberration increases the defocus amount, scan a wide range, and changing the Zernike coefficient corresponding to astigmatism reduces the defocus amount It is possible to scan a narrow range with a fine step size.

  The control unit 80 reads the stack image acquired in this way from the storage device 83 (step S1406), calculates a correlation value between each image and the reference image acquired in step S1401, and images having the largest correlation value. Is selected (step S1407). This image is called an “addition side determination image”. Here, the reason why the image having the largest correlation value with the reference image is selected is to find an image that would have been acquired at the reference position by the illumination light whose wavefront aberration is corrected, from the stack image. In the present embodiment, the phase only correlation method is used as the correlation value calculation method. In the phase-only correlation method, only the phase component of an image (which corresponds to shape information) is extracted by Fourier transforming the image, and the similarity is measured by obtaining the correlation value of the phase component. This is a pattern matching technique. Since it is not affected by the shade information of the image, the correlation focused on the “shape” can be obtained with high accuracy, and the positional deviation of the image (the deviation occurring in the xy plane with the movement of the objective lens 36 and the stage, It is not affected by the fine movement of the biological specimen itself. As described above, when the wavefront of the incident light is changed by the adaptive optics 42 whose control parameter has been changed, the focal plane of the objective lens 36 is moved (defocused) from the reference position accordingly. An image in a predetermined range including the position is obtained, and a correlation value with the reference image is obtained to select an image most similar to the reference image (“addition side determination image”). The relationship between the correlation value and the z-direction position is, for example, as shown in FIG. Here, the step size is expressed as Δz. Then, the control unit 80 calculates an evaluation value in the ROI in the selected addition side determination image by the same method as in step S1403 (step S1408).

  In addition, the control unit 80 subtracts a predetermined value from the selected Zernike coefficient, and outputs a control parameter (value) based on the Zernike polynomial to the adaptive optical element 42 (step S1409). Then, similarly to step S1405, the control unit 80 moves the objective lens 36 by a predetermined step size within a predetermined range in the z direction including the reference position, acquires a sample image at each position, and stores it in the storage device. 83 (step S1410), the stack image acquired in this way is read from the storage device 83 (step S1411), the correlation value between each image and the reference image acquired in step S1401 is calculated, and the most correlated An image having a large value is selected (step S1412). This image is called a “subtraction side determination image”. The reason for selecting the image having the largest correlation value with the reference image is as described above. In addition, the control unit 80 calculates an evaluation value in the ROI in the selected subtraction-side determination image by the same method as in step S1403 (step S1413).

  When the above processing is executed, the evaluation value in each ROI of the reference image, the addition side determination image, and the subtraction side determination image (that is, the Zernike coefficient currently selected is changed (positive direction and negative direction centering on the reference image). 7), the controller 80 obtains the evaluation value when the evaluation value becomes the largest as shown in FIG. 7 by performing secondary fitting on these values. An optimum value of the Zernike coefficient (the addition side is positive and the subtraction side is negative) can be calculated (step S1414).

  Returning to FIG. 2, when the optimum value of the Zernike coefficient selected in step S140 is calculated, the control unit 80 determines whether or not the control parameter needs to be updated (step S150). That is, it is determined whether or not the value of the selected Zernike coefficient needs to be updated to the optimum value, and when the control unit 80 determines that the control parameter needs to be updated, the update improves the aberration. It is determined by verifying whether or not it is performed (step S160).

  In this control parameter update determination process S160, as shown in FIG. 4, the control unit 80 moves the objective lens 36 to the reference position in the z direction in a state before updating the control parameter, and again at this reference position. The sample image (reference image) is acquired and stored in the storage device 83 (step S1601). The control unit 80 reads out the reference image acquired in step S1601 from the storage device 83 (step S1602), and calculates the same evaluation value as in step S1403 for the ROI in the read reference image (step S1603). . Next, the control unit 80 updates the selected Zernike coefficient to the optimum value described above, and outputs a control parameter (value) based on the Zernike polynomial to the adaptive optics element 42 (step S1604). Then, similarly to step S1405, the control unit 80 moves the objective lens 36 by a predetermined step size within a predetermined range in the z direction including the reference position, acquires a sample image at each position, and stores it in the storage device. 83 (step S1605), the stack image acquired in this way is read from the storage device 83 (step S1606), the correlation value between each image and the reference image acquired in step S1601 is calculated, and the most correlated An image having a large value is selected (step S1607). This image is called an “optimum value determination image”. In addition, the control unit 80 calculates an evaluation value in the ROI in the selected optimum value determination image by the same method as in step S1403 (step S1608). Further, the control unit 80 determines whether or not the correction of the optical aberration (aberration when the optimum value is added to the Zernike coefficient) is improved from the evaluation value in step S1608 (step S1609), and in step S1603. The evaluation value after the control parameter is updated with the optimal value calculated in step S1608 is compared with the calculated evaluation value (the state of the image before the control parameter is updated with the optimal value). When it is determined that the image has improved, the control parameter of the adaptive optics 42 is updated with the optimum value (step S1610), and when it is determined that the image has not been improved, the control parameter is not updated (step S1610). Step S1611).

  Returning to FIG. 2, the control unit 80 determines whether or not the above-described processing has been executed for all aberrations (step S <b> 170), and selects the next aberration (Zernike coefficient) if not executed for all aberrations. Then, returning to Step S140, Steps S140 to S170 are repeated (Step S180), and when it is determined that correction has been completed for all aberrations, this process ends.

  Note that the operations of steps S1401 to S1403 and S1601 to S1603 can be omitted as follows. That is, in the series of sequences, the optimum image Image-best and the evaluation function value Value-best in the immediately previous state are stored, and each time the control parameter is updated in step S1610, the respective values are replaced with the latest values. . More specifically, first, in step S120, the image and the evaluation function at the reference position are stored as the optimum image Image-Best and the evaluation function value Value-best. Next, instead of performing the operations of steps S1401 to S1403 in the initial loop of steps S1401 to S1403, the stored values of the optimal image Image-Best and the evaluation function value Value-best are used instead of the evaluation function. In the second and subsequent loops, when the control parameter is updated in step S1610, the image and evaluation function selected in step S1608 are updated as the optimum image Image-best and evaluation function value Value-best in the immediately preceding loop. If the control parameter is not updated in step S1610, it is not updated. In the next loop, the values of the optimum image Image-best and the evaluation function value Value-best are used again instead of performing the operations of S1401 to S1403. Note that the method of always storing the optimal image Image-best and the evaluation function value Value-best, and updating each value every time the control parameter is updated, compensates for the signal intensity from the sample in a series of sequences. The present invention can be applied to cases where there is no fear of fluctuation due to factors other than changing the control parameter of the optical element 42 or when there is little concern. For example, the present invention can be applied to an observation method that does not worry about sample fading such as second harmonic generation (SHG) and third harmonic generation (THG). It is possible to shorten the time. On the other hand, for example, when a multiphoton fluorescence image is acquired in a sample with intense amber color and the present invention is applied, there is a concern about the influence of the sample's amber color by acquiring the image many times during the sequence. In such a case, it is desirable to re-acquire an image for calculating the evaluation function immediately before determining whether to update the control parameter as in the embodiment without omitting the steps. Note that it is desirable that the user can select whether or not to omit the step according to the specimen and the acquisition conditions.

  In this embodiment, every time a Zernike coefficient is selected and the optimum coefficient is calculated, the control parameter is updated and the control parameter is updated in step S160. In this way, the wavefront aberration is sequentially performed. The method of updating the value tends to be influenced by the coefficient value of each term of the Zernike polynomial corresponding to the optical aberration (spherical aberration or coma aberration) to be corrected, like a deformable mirror. This is particularly effective when an adaptive optical element is used, or when the aberration to be corrected is complex and the amount of aberration is large, such as observation at a deep part of a biological specimen. By selecting the order of the Zernike coefficient terms for calculating and updating the optimum values according to the response characteristics of the adaptive optics used and the aberration characteristics of the biological specimen, it is possible to achieve a better optimum in a shorter time with higher accuracy. The value can be found.

  As described above, when a reflective liquid crystal element or a transmissive liquid crystal element is used as the compensation optical element 42, each Zernike polynomial corresponding to an optical aberration (spherical aberration, coma aberration, etc.) to be corrected from the characteristics thereof is used. Since the value of the coefficient of the term can be determined / set / implemented independently, the setting method of the adaptive optics element shown in FIG. 2 omits steps S150 to S170 and loops from step S140 to step S180. , And after performing all the optimum coefficient calculation processing of the optical aberration (Zernike coefficient) to be corrected, step S150 and step S160 may be performed using all the calculated Zernike coefficients.

  In the scanning microscope 10 according to the present embodiment, if the compensation optical element 42 is controlled as described above, even if the focal plane (sample surface I0) of the objective lens 36 is moved in the z direction by changing the wavefront. As shown in FIG. 8, a stack image is acquired with a predetermined step size in a predetermined range centered on the reference position, a correlation value with the reference image is calculated, and an image closest to the reference image is selected. , An image of the focal plane before moving can be selected. In addition, since the plurality of optical aberrations are controlled by the coefficients of the terms of the Zernike polynomial, the orthogonality can be used. Therefore, even if control is performed for each optical aberration (Zernike coefficient) (step S140 described above). Since each optical aberration can be controlled (even if S180 is repeated), the control of the compensation optical element 42 can be simplified. As described above, by performing the above-described processing, it becomes possible to determine a control parameter with higher accuracy of the compensation optical element, and it is possible to shorten the convergence speed to the optimum value. It is possible to perform observation with high accuracy even for a sample whose three-dimensional distribution is unknown (for example, a biological sample).

  In the above description, the case of performing multiphoton fluorescence observation (two-photon fluorescence observation) of a specimen using the scanning microscope 10 according to the present embodiment has been described. However, the present invention is limited to this observation method. There is no. As described above, the scanning microscope 10 according to the present embodiment has not only the first detection unit 50 that detects the observation term from the specimen without descanning by the scanning unit 32, but also after the descanning by the scanning unit 32, It has the 2nd detection part 60 detected via the pinhole 62a of the light-shielding plate 62, and the 3rd detection part 70 which detects the light which permeate | transmitted the sample, and each detection part 50,60,70 has a different wavelength. It is configured to detect light. For this reason, for example, one-photon fluorescence in a normal confocal microscope, second harmonic generated by second harmonic generation generated by second harmonic generation generated along with the fluorescence by irradiating excitation light, and third harmonic. Even when detecting and observing the third harmonic generated by Third Harmonic Generation or the observation light due to the autofluorescence of the specimen, the wavefront of the illumination light (excitation light) is changed by the above method. Aberration can be reduced and an accurate image can be obtained.

  Further, in the above description, the multiphoton fluorescence observation by the scanning microscope 10 is described as an example as an observation method capable of acquiring an optical tomographic image in the depth direction of the specimen, but the present invention is not limited to this, For example, other observation methods and microscopy methods capable of acquiring an optical tomographic image of a selected depth, such as a sheet illumination microscope called Selective Plane Illumination Microscope (SPIM), may be used. SPIM uses a separate illumination optical system to illuminate only the vicinity of the focal plane of the objective lens with sheet-like illumination light from a direction substantially perpendicular to the optical axis of the objective lens for observing the specimen. An optical tomographic image is obtained, and more specifically, for example, disclosed in Japanese Patent Application Publication No. 2006-509246.

  Further, in the above description, the case where the optimum value is calculated for a single optical aberration (Zernike coefficient) is calculated using a reference image and an image in three states consisting of images obtained by adding / subtracting a predetermined value to / from the coefficient. However, the Zernike coefficient may be changed to four or more states, and the optimum value may be determined from the images obtained in those states.

[Modification]
In the above description, the method for determining the optimum value of the aberration correction optical element 42 using the ROI has been described. However, an area outside the ROI (hereinafter referred to as “peripheral area A”) may be used. As described above, the excitation light (illumination light of the second wavelength) for SHG observation or THG observation can suppress the deterioration of the specimen, but when determining the optimum value of the aberration correction optical element 42, By using the peripheral region A, it is possible to avoid irradiating the observation region (ROI) with excitation light, and to further suppress deterioration of the specimen in the observation region. Hereinafter, a method for determining the optimum value of the aberration correcting optical element 42 using the peripheral region A will be described. In the flowcharts shown in FIGS. 2 to 4, different portions will be mainly described.

  First, ROI determination (step S120) in the case of using the peripheral region A is performed using a sample image acquired by two-photon fluorescence observation in step S100. Note that since the observation of the specimen in step S100 is two-photon fluorescence observation, it is necessary to perform an operation so as to minimize the irradiation time of the laser light (illumination light) on the specimen. Then, the evaluation value calculation (steps S1403, S1408, S1413, S1414, S1603, and S1608) in the subsequent processing is performed on the peripheral area A instead of the ROI, as described above.

  In the calculation of the evaluation value, as shown in FIG. 9, the peripheral area A is divided into a plurality of sub areas An (n is the number of each sub area), and the luminance values in each sub area An are integrated. The evaluation value. As shown in FIG. 9, when the medium configuration in the peripheral region A is different, the refractive index and the like differ depending on the properties of the medium. That is, the above-described evaluation value is calculated for each of the sub-regions An of the peripheral region A. Similarly, in step S1414, the optimum value of the Zernike coefficient when the evaluation value becomes the largest is calculated by performing a second-order fitting on these values for each sub-region An to obtain the optimum value for each sub-region An. The average of the values is calculated and determined as the optimum value for observing the ROI. Further, the determination in step S1609 determines from the evaluation value in step S1608 whether or not the correction of the optical aberration (aberration when an optimal value is set for the Zernike coefficient) is improved for each sub-region An, When the evaluation value after the control parameter is updated with the optimum value calculated in step S1608 is compared, and the difference is larger than a predetermined threshold in at least one sub-region An, and it is determined that the image has been improved. Is configured to update the control parameter of the adaptive optics 42 with the optimum value (step S1610) and not to update the control parameter when it is determined that no improvement has been made in any of the sub-regions An (step S1611).

  As described above, when the optimum value of the aberration correcting optical element 42 is determined using the peripheral region A, the ROI is determined by two-photon fluorescence observation as shown in FIG. As shown in FIG. 5, the peripheral region A is used to determine the control parameter of the aberration correction optical element 42. When the sample is irradiated with the excitation light for performing the above-described two-photon fluorescence observation, the sample may be deteriorated. When acquiring an image for determining the optimum value of the compensation optical element 42, the excitation light is applied only to the peripheral region A. By irradiating, it is possible to suppress the deterioration of the sample in the ROI that is the actual observation region shown in FIG. Although FIG. 9 shows the case where the peripheral area A is divided into eight sub-areas A1 to A8, the number of divisions is not limited to eight. Also, instead of the average value of the Zernike coefficients of the sub-region An, a coefficient determined by multiplying a different weighting coefficient for each region of An may be used. It is also possible to determine the optimum value of the Zernike coefficient by calculating the evaluation value for the entire peripheral area A without being divided into sub-areas An.

DESCRIPTION OF SYMBOLS 10 Scanning microscope 20 Light source device 30 Scanning optical system 32 Scanning unit 36 Objective lens 40 Wavefront compensation optical system 42 Compensation optical element 50 1st detection part 80 Control part

Claims (10)

An illumination optical system that includes a compensation optical element that changes a wavefront of illumination light emitted from the light source unit, and that irradiates the specimen with the illumination light with the wavefront changed via an objective lens;
A detection optical system for guiding light from the specimen to a detection unit;
A control unit for controlling the compensation optical element, and a setting method of the compensation optical element in a microscope having:
A first step of acquiring, as a reference image, an image of the specimen when the focal plane of the objective lens is at a predetermined position of the specimen;
The control unit controls the compensation optical element to change the wavefront of the illumination light into two or more different states, and in each of the states, a plurality of predetermined ranges in the optical axis direction including the predetermined position. A second step of acquiring an image of the specimen at a position of
A third step of selecting a determination image based on a correlation value between the image of the sample corresponding to each of the plurality of positions and the reference image in each state of the wavefront;
An evaluation value of a predetermined region is calculated for each of the determination image in each state of the reference image and the wavefront, and a state of the wavefront is newly determined based on the evaluation value and each state of the wavefront. A fourth step;
And a fifth step of setting the compensation optical element so as to be in the determined wavefront state.   In the second step, the state change of the wavefront of the illumination light is realized by controlling the compensating optical element with a control parameter reflecting the coefficient change of each term of the Zernike polynomial corresponding to the desired optical aberration. The method for setting an adaptive optical element according to claim 1, wherein:   Performing the second step, the third step, the fourth step, and the fifth step for each coefficient of each term of the Zernike polynomial corresponding to each of a plurality of optical aberrations. The method for setting an adaptive optics element according to claim 2.   The method for setting an adaptive optical element according to claim 1, wherein the correlation value in the third step is calculated by a phase-only correlation method.   5. The evaluation value in the fourth step is at least one of an integrated value, a maximum value, an average value, and a contrast value of luminance values of each pixel in the predetermined region. The setting method of the adaptive optics element as described in any one of these.   The method of setting an adaptive optical element according to claim 1, wherein the image of the specimen is an image of at least one of fluorescence, second harmonic, and third harmonic.   The method for setting an adaptive optical element according to any one of claims 1 to 6, wherein the predetermined region in the fourth step is a region where the specimen is desired to be observed.   The method for setting an adaptive optical element according to claim 1, wherein the predetermined region in the fourth step is a region outside a region where the specimen is to be observed. .   9. The method of setting an adaptive optical element according to claim 8, wherein when the image of the specimen is acquired, the illumination light is irradiated only on the outer region. An illumination optical system that includes a compensation optical element that changes a wavefront of illumination light emitted from the light source unit, and that irradiates the specimen with the illumination light with the wavefront changed via an objective lens;
A detection optical system for guiding light from the specimen to a detection unit;
A control unit for controlling the compensation optical element,
The microscope according to claim 1, wherein the control unit sets the compensation optical element by the setting method of the compensation optical element according to claim 1.

Images