Patent Publication Number: US-10775598-B2

Title: Scanning microscope

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a Divisional application of U.S. application Ser. No. 15/230,213, filed Aug. 5, 2016, which is based on and claims the benefit of priority from prior Japanese Patent Application No. 2015-167266, filed Aug. 26, 2015, the entire contents of both of which are incorporated herein by this reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present invention relates to a technology for a scanning microscope and particularly relates to a technology for a scanning microscope that includes a varifocal lens. 
     Description of the Related Art 
     Scanning microscopes such as confocal laser scanning microscopes and multiphoton-excitation laser scanning microscopes are known as apparatuses for observing a three-dimensional image of an object. 
     For directions orthogonal to the optical axis of an objective (x direction and y direction), a scanning microscope is capable of performing a scan at a high frequency of hundreds to thousands Hertz using a galvanometer scanner or a resonance scanner. Meanwhile, a scan in an optical-axis direction (z-axis direction) is performed by moving the objective or a stage in the optical-axis direction typically using a piezoelectric transducer (hereinafter referred to as a piezo element) and another actuator. U.S. Pat. No. 8,553,324 describes a technique for achieving a scan in an optical-axis direction by moving a tube lens in the optical-axis direction. 
     However, a method of mechanically moving structures such as an optical system and a stage using an actuator involves a time for the moving and a time before oscillations caused by the moving stop. Hence, it is difficult to perform a fast scan in an optical-axis direction. As a result, it takes a long time to obtain a three-dimensional image. 
     Japanese Laid-open Patent Publication No. 2004-317704 describes a varifocal lens as means for solving such a technical problem. Use of the varifocal lens allows a focal length to be rapidly changed by changing an applied current or voltage. This enables a fast scan in an optical-axis direction. Recently, varifocal lenses have been developed that vary a focal length more greatly than those in the prior art. Hence, usability of a varifocal lens as scan means has been enhanced. 
     SUMMARY OF THE INVENTION 
     An aspect of the present invention provides a scanning microscope that includes: a varifocal lens that scans an object in an optical-axis direction of an objective; a scanner that scans the object in a direction orthogonal to the optical axis of the objective; and a controller that controls the varifocal lens and the scanner, wherein the controller controls the scanner according to scan control data for correcting deviation of a scan position that is caused by the varifocal lens. 
     Another aspect of the invention provides a scanning microscope that includes: a varifocal lens that scans an object in an optical-axis direction of an objective; a scanner that scans the object in a direction orthogonal to the optical axis of the objective; and a controller that controls the varifocal lens and the scanner, wherein the controller controls the scanner so as to correct deviation of a scan position that is caused by the varifocal lens. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention will be more apparent from the following detailed description when the accompanying drawings are referenced. 
         FIG. 1  illustrates the configuration of a microscope  100  in accordance with embodiment 1; 
         FIG. 2  is a flowchart illustrating a method of generating scan control data; 
         FIG. 3  illustrates a method of correcting a lateral magnification; 
         FIG. 4A  illustrates a method of correcting pincushion distortion; 
         FIG. 4B  illustrates a method of correcting barrel distortion; 
         FIG. 5  illustrates a method of correcting a center-position deviation of a scan range; 
         FIG. 6  illustrates a relationship between a scanner and a scan position; 
         FIG. 7A  illustrates a method of correcting a field curvature, where the field curvature has not been corrected yet; 
         FIG. 7B  illustrates a method of correcting a field curvature, where the field curvature has been corrected; 
         FIG. 8  is a flowchart of a process of generating three-dimensional-image data performed by a microscope  100  in accordance with embodiment 1; 
         FIG. 9  illustrates the configuration of a microscope  200  in accordance with embodiment 2; 
         FIG. 10  illustrates the configuration of a microscope  300  in accordance with embodiment 3; 
         FIG. 11  illustrates the configuration of a microscope  400  in accordance with embodiment 4; and 
         FIG. 12  illustrates the configuration of a microscope  500  in accordance with embodiment 5. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Performing a scan in an optical-axis direction using a varifocal lens changes the projection magnification of an optical system, including the varifocal lens, due to a change in the focal length of the varifocal lens, as described in Japanese Laid-open Patent Publication Nos. 2001-257932 and 2004-029685. This also changes an aberration caused by the optical system. Hence, in the prior art, it is difficult to obtain an accurate three-dimensional image of an object. 
     The following describes embodiments of the invention. 
     Embodiment 1 
       FIG. 1  illustrates the configuration of a microscope  100  in accordance with the present embodiment. The microscope  100  is a confocal laser scanning microscope. The description is based on an exemplary situation in which the microscope  100  is a fluorescence microscope that detects fluorescence from a sample S. However, the microscope  100  is not limited to a fluorescence microscope but may be, for example, an industrial confocal laser scanning microscope that detects reflected light. 
     The microscope  100  includes a laser  1  that emits laser light and a stage  9  on which a sample S, i.e., an object, is placed. The microscope  100  includes a beam expander  19 , a dichroic mirror  2 , a scanner  3 , a relay lens  4 , a varifocal lens  5 , a relay lens  6 , a mirror  7 , and an objective  8 , all of which are provided on a path of illumination light between the laser  1  and the stage  9 . When the microscope  100  is an industrial confocal laser scanning microscope, the microscope  100  may include a polarization beam splitter, rather than the dichroic mirror  2 , and may include a λ/4 plate between the polarization beam splitter and the objective. The microscope  100  may include a λ/2 plate between the polarization beam splitter and the laser  1 . 
     The beam expander  19  is an optical system that changes the light flux diameter (beam diameter) of laser light from the laser  1  in accordance with, for example, the pupil diameter of an objective in use. Controlling the light flux diameter of laser light entering the pupil of the objective  8  using the beam expander  19  allows various aberrations that occur at the objective  8  (e.g., spherical aberrations) to be suppressed. For example, a scattering of excitation light can be suppressed in observing a portion deep within a cell. 
     The dichroic mirror  2  has properties of reflecting laser light and allowing passage of fluorescence. The dichroic mirror  2  is a splitter that separates entering light according to a wavelength. The scanner  3  scans a sample S in a direction orthogonal to the optical axis of the objective  8 . The scanner  3  is located at, or near, a position optically conjugate to the pupil of the objective  8  (hereinafter referred to as a pupil-conjugate position). The scanner  3  includes a scanner that performs a scan in an x direction orthogonal to the optical axis of the objective  8 , and a scanner that performs a scan in a y direction orthogonal to the optical axis of the objective  8  and orthogonal to the x direction. The scanners are, for example, galvanometer scanners or resonance scanners. 
     The relay lens  4  projects the scanner  3  onto the varifocal lens  5 . The varifocal lens  5  changes the focal length thereof so as to scan the sample S in the optical axis direction of the objective  8 . The varifocal lens  5  changes the focal length by, for example, changing the lens shape. The difference between light fluxes represented by dotted lines in  FIG. 1  and light fluxes represented by solid lines in  FIG. 1  results from a change in the focal length of the varifocal lens  5 . The varifocal lens  5  is located at, or near, the pupil-conjugate position of the objective  8 . 
     The relay lens  6  projects the pupil of the objective  8  onto the varifocal lens  5 . The objective  8  is mounted on a revolver (not illustrated). In the microscope  100 , a plurality of objectives, including the objective  8 , are switched between in accordance with, for example, an observation magnification. The stage  9  is an electric stage moved by an actuator (not illustrated) in the optical axis direction of the objective  8 . 
     The microscope  100  includes a photodetector  13  that detects fluorescence from a sample S. The microscope  100  further includes a lens  10 , a pinhole plate  11 , and a lens  12 , all of which are provided on a path of detected light between the dichroic mirror  2  and the photodetector  13 . 
     The lens  10  gathers fluorescence that has passed through the dichroic mirror  2 . The pinhole plate  11  is a confocal diaphragm that has a pinhole formed at a position conjugate to a front focal position of the objective  8 . The lens  12  guides, to the photodetector  13 , fluorescence that has passed through the pinhole plate  11 . The photodetector  13  outputs a signal that depends on the intensity of detected fluorescence, and is, for example, a Photomultiplier Tube (PMT). 
     The microscope  100  further includes a control unit  14  that includes a processor and a storage unit  15 . The control unit  14  is a controller that controls operations of the entirety of the microscope  100 . The control unit  14  controls the scanner  3 , the varifocal lens  5 , and the stage  9 . The control unit  14  may change the oscillation angle of a mirror included in the scanner  3 , the focal length of the varifocal lens  5 , and the position of the stage  9  in an optical-axis direction. The control unit  14  may control the beam expander  19  and change the light flux diameter of laser light emitted from the beam expander  19 . The storage unit  15  is, for example, a hard disk apparatus, a flash memory, or another memory, and stores scan control data for correcting deviation of a scan position that is caused by the varifocal lens  5 . Scan control data is used to control both, or one of, the scanner  3  and the varifocal lens  5  and is stored in the storage unit  15  for each objective. Scan control data is generated using a method described hereinafter with reference to  FIG. 2  and stored in the storage unit  15 . 
     In the microscope  100  configured as described above, laser light emitted from the laser  1  is reflected at the dichroic mirror  2 , and is focused onto the sample S after entering the objective  8  via the scanner  3 , the relay lens  4 , the varifocal lens  5 , the relay lens  6 , and the mirror  7 . Changing the oscillation angle of the scanner  3  changes a focused-on position of the light (hereinafter referred to as a scan position) with respect to a direction orthogonal to the optical axis (xy direction), and changing the focal length of the varifocal lens  5  changes the scan position with respect to the optical-axis direction (z direction). Accordingly, the control unit  14  may control the scanner  3  and the varifocal lens  5  so as to three-dimensionally scan the sample S. The amount of a variation in the focal length of the varifocal lens  5  is limited, and hence the varifocal lens  5  performs a scan in the optical-axis direction after the sample S has come close to the focal position of the objective  8  through the movement of the stage  9 . 
     Fluorescence is generated from the sample S irradiated with laser light and enters the dichroic mirror  2  via the objective  8 , the mirror  7 , the relay lens  6 , the varifocal lens  5 , the relay lens  4 , and the scanner  3 . The fluorescence passes through the dichroic mirror  2  and is focused onto the pinhole plate  11  by the lens  10 . Fluorescence generated from the scan position, i.e., the position onto which the laser light has been focused, passes through the pinhole formed on the pinhole plate  11  and enters the photodetector  13  via the lens  12 ; the fluorescence is detected by the photodetector  13 . Meanwhile, fluorescence generated from positions other than the scan position is blocked by the pinhole plate  11 . Accordingly, the photodetector  13  generates a signal that depends on the intensity of the fluorescence generated from the scan position. 
     While the scanner  3  is performing scanning, the microscope  100  samples the output signal from the photodetector  13  so as to obtain a two-dimensional image (xy cross-sectional image) of the sample S, thereby generating xy-cross-sectional-image data. This process is performed every time a scan based on the varifocal lens  5  is performed in the optical-axis direction. The microscope  100  generates three-dimensional-image data according to a plurality of pieces of xy-cross-sectional-image data, each of which is generated in the described manner and corresponds to a different z coordinate. 
     In the microscope  100 , the control unit  14  is configured to control the scanner  3 , or both the scanner  3  and the varifocal lens  5  so as to correct, according to scan control data stored in the storage unit  15 , deviation of a scan position that is caused by the varifocal lens  5 . This enables, for example, a magnification error or aberration caused by the varifocal lens  5  to be suppressed so that the microscope  100  can obtain an accurate three-dimensional image. The microscope  100  scans the sample S in the optical-axis direction by changing the focal length of the varifocal lens  5 . This enables a fast scan in comparison with a situation in which the objective  8  or the stage  9  is moved in the optical-axis direction. Hence, the microscope  100  can quickly obtain an accurate three-dimensional image. 
       FIG. 2  is a flowchart illustrating a method of generating scan control data to be used for scan control performed by the control unit  14 . The following specifically describes the method of generating scan control data with reference to  FIG. 2 . 
     A user prepares and places a sample for calibration on the stage  9  (step S 1 ). The sample for calibration is, for example, a sample that includes a region having a known shape from which fluorescence is to be emitted. 
     After placing the sample for calibration on the stage  9 , the user adjusts the varifocal lens  5  in a manner such that a parallel light flux enters the objective  8  (step S 2 ). After adjusting the varifocal lens  5 , the user moves the stage  9  in an optical-axis direction so as to adjust an interval between the sample and the objective  8  in a manner such that the sample is focused (step S 3 ). In this example, the user may adjust the position of the stage  9  while using an observation apparatus (not illustrated) of the microscope  100  so as to check whether the sample S is focused. 
     The microscope  100  obtains an xy cross-sectional image corresponding to a plane of the sample in accordance with an instruction from the user (step S 4 ). In this example, the control unit  14  controls the scanner  3  without using scan control data. That is, xy cross-sectional-image data is generated by the control unit  14  controlling the scanner  3 , as in the case of a situation in which the varifocal lens  5  is not provided. 
     The microscope  100  determines whether all xy cross-sectional images within a depth of focus have been obtained (step S 5 ). In this example, the microscope  100  determines, for example, whether all xy cross-sectional images for a region extending from the plane focused in step S 3  within the depth of focus have been obtained at certain intervals. 
     When it is determined that not all of the images have been obtained (NO in step S 5 ), the microscope  100  changes the distance between the sample and the objective  8  by moving the stage  9  in the optical-axis direction without deviating from the region within the depth of focus (step S 6 ). Subsequently, the process of obtaining xy cross-sectional images (step S 4 ) and the determining process (step S 5 ) are performed again. 
     When it is determined that all of the images have been obtained (YES in step S 5 ), the microscope  100  determines whether xy cross-sectional images have been obtained for all of the focal lengths of the varifocal lens  5  (step S 7 ). “All of the focal lengths” means, for example, all of the focal lengths that fall within the range of variation in focal length of the varifocal lens  5 , the focal lengths being obtained at certain intervals. 
     When it is determined that xy cross-sectional images have not been obtained for all of the focal lengths (NO in step S 7 ), the microscope  100  changes the focal length of the varifocal lens  5  (step S 8 ). Subsequently, the focusing process and the following steps (steps S 3 -S 7 ) are performed again. 
     When it is determined that xy cross-sectional images have been obtained for all of the focal lengths (YES in step S 7 ), the microscope  100  generates scan control data (step S 9 ) and ends the process of generating scan control data. In this example, the microscope  100  first generates three-dimensional-image data of the sample for each focal length from a plurality of pieces of xy cross-sectional-image data each generated for a certain focal length of the varifocal lens  5 . According to the three-dimensional-image data generated for each focal length and information on the sample for calibration, the microscope  100  then locates the deviation of a scan position that has been caused by the varifocal lens  5 . The microscope  100  finally generates scan control data to correct the located deviation of the scan position, and stores this scan control data in the storage unit  15 . The flowchart depicted in  FIG. 2  is intended to obtain scan control data for one wavelength, but scan control data may be obtained for each wavelength used for an observation. Scan control data may be generated by the control unit  14 . 
     With reference to factors that could lead to deviation of a scan position (error in lateral magnification, distortion, center-position deviation, and field curvature), the following describes in detail a method of generating scan control data for correcting the deviation of a scan position by referring to  FIGS. 3-7 . 
       FIG. 3  illustrates a method of correcting a lateral magnification.  FIG. 3  depicts a change in scan range caused by an error in lateral magnification. As depicted in  FIG. 3 , when an error is caused in the lateral magnification, a scan range F 1 , which is a scan range in an xy direction that is included in three-dimensional-image data, is enlarged or reduced (in this example, reduced) relative to a scan range F 0 , which is a scan range in the xy direction that is scanned in the absence of an error. Accordingly, when the scan range is reduced due to the varifocal lens  5 , the microscope  100  may generate scan control data to designate a scan range that is wider than a scan range that is designated in the absence of an error (e.g., scan range F 0 ). When the scan range is enlarged due to the varifocal lens  5 , the microscope  100  may generate scan control data to designate a scan range that is narrower than a scan range that is designated in the absence of an error (e.g., scan range F 0 ). 
       FIG. 4A  illustrates a method of correcting pincushion distortion.  FIG. 4B  illustrates a method of correcting barrel distortion.  FIGS. 4A and 4B  depict a change in scan range caused by distortion. As depicted in  FIGS. 4A and 4B , when distortion occurs, the shape of a scan range in the xy direction that is included in three-dimensional-image data (scan range F 2  or F 3 ) becomes a scan range F 0  vertically and laterally distorted, the scan range F 0  being a scan range in the xy direction that is scanned in the absence of distortion. Hence, as the distance from the center of the scan range F 2  (or scan range F 3 ) increases, deviation of the scan position becomes greater; in particular, as the distance from the center of the scan range F 2  (or scan range F 3 ) increases in the x direction (y direction), deviation of the scan position in the x direction (y direction) becomes greater. Accordingly, when distortion occurs due to the varifocal lens  5 , the microscope  100  may generate scan control data that designates a scan position obtained by making a correction in the xy direction more greatly, as the distance from the center of the scan range becomes greater, with respect to a scan position that is designated in the absence of the distortion. 
       FIG. 5  illustrates a method of correcting a center-position deviation of a scan range.  FIG. 5  depicts a change in a scan range caused by deviation of the center position of the scan range. The center-position deviation of the scan range may occur when, for example, the optical-axis direction of the varifocal lens  5  changes due to a change in the focal length of the varifocal lens  5 . As depicted in  FIG. 5 , when center-position deviation occurs, a scan range F 4  in the xy direction that is included in three-dimensional-image data becomes a range obtained as a result of a shift (parallel displacement) of a scan range F 0  in the xy direction that is scanned in the absence of center-position deviation. The shift occurs in a direction in which the center position deviates and by a distance by which the center position is deviated. Accordingly, when center-position deviation occurs due to the varifocal lens  5 , the microscope  100  may generate scan control data for designating a scan range obtained by shifting a scan range that is designated in the absence of center-position deviation in a direction opposite to the direction of the deviation and by the same distance as the deviation. 
       FIG. 6  illustrates an optical system in which a scanner SC is located at a pupil conjugate position of an objective OB formed by a relay lens RL. In such an optical system, changing the oscillation angle of the scanner SC (angle formed by the reflection surface of the scanner SC and an optical axis) changes the angle of a principal ray incident on a pupil position of the objective OB. The objective OB converts this angle change into a change in a focused-on position of the light (scan position) in the xy direction, so that the focused-on position of the light can be changed by changing the oscillation angle of the scanner SC.  FIG. 6  depicts an example in which a scan position has been moved from position P 1  to position P 2  by changing the oscillation angle of the scanner SC. 
     The optical system of the microscope  100  functions in the same manner as the optical system depicted in  FIG. 6 , and hence changing the oscillation angle of the scanner  3  in the microscope  100  can change a scan position in the xy direction. The factors described above with reference to  FIGS. 3-5  (error in lateral magnification, distortion, and center-position deviation) and their combinations all lead to deviation of a scan position in the xy direction that is caused by the varifocal lens  5 . Accordingly, when the varifocal lens  5  has caused an error in lateral magnification, distortion, or center-position deviation and thus has deviated a scan position, the microscope  100  may correct the deviation of the scan position simply via the control unit  14  controlling the scanner  3  according to scan control data. In this situation, for all of the factors above, the scan control data is intended to control the scanner  3 . In particular, the scan control data is intended to control the scanner  3  in accordance with the focal length of the varifocal lens  5 , e.g., is intended to designate an oscillation angle for the scanner  3  for each of target xyz coordinates. That is, the control unit  14  controls the scanner  3  in accordance with the focal length of the varifocal lens  5 , thereby allowing the microscope  100  to correct deviation of a scan position that is caused by the varifocal lens  5 . 
       FIGS. 7A and 7B  illustrate a method of correcting a field curvature, and a change in a scan range that is caused by the field curvature.  FIG. 7A  depicts a state before correction of the field curvature.  FIG. 7B  depicts a state after correction of the field curvature. The optical system between the varifocal lens  5  and a sample (relay lens  6 , mirror  7 , and objective  8 ) is not illustrated in  FIGS. 7A and 7B . As depicted in  FIG. 7A , when a field curvature occurs, a scan range F 5  included in three-dimensional data is warped in a z direction, and hence the z coordinate of a scan position close to the center of a scan range becomes different from the z coordinate of a scan position distant from the center. Accordingly, when the field curvature occurs due to the varifocal lens  5 , the microscope  100  may generate scan control data that designates a scan position obtained by making a correction in the z direction more greatly, as the distance from the center of the scan range becomes greater, with respect to a scan position that is designated in the absence of distortion. 
     As depicted in  FIG. 7B , the z coordinate of the scan position may be changed by changing the lens shape so as to change the focal length of the varifocal lens  5 . A field curvature causes a scan position to deviate in the z direction due to the varifocal lens  5 . Hence, when the varifocal lens  5  causes a field curvature and thus deviates a scan position, the microscope  100  may correct the deviation of the scan position via the control unit  14  simply controlling the varifocal lens  5  according to scan control data. In this situation, the scan control data is intended to control the varifocal lens  5 . In particular, the scan control data is intended to control the varifocal lens  5  in accordance with a focal length calculated for the varifocal lens  5  from a target z coordinate (information on a scan position) and an oscillation angle calculated for the scanner  3  from target xy coordinates (information on the scan position). For example, the scan control data may be intended to designate a focal length for the varifocal lens  5  for each of the target xyz coordinates. That is, the control unit  14  controls the varifocal lens  5  in accordance with a focal length calculated for the varifocal lens  5  from a target z coordinate and an oscillation angle calculated for the scanner  3  from target xy coordinates. This allows the microscope  100  to correct deviation of a scan position caused by the varifocal lens  5 . 
     In the microscope  100 , when the varifocal lens  5  deviates a scan position in both the xy direction and the z direction (e.g., when a field curvature and distortion occur), the control unit  14  controls the scanner  3  and the varifocal lens  5  according to scan control data. This may correct the deviation of the scan position. In this case, the scan control data includes data for controlling the scanner  3  in accordance with a focal length calculated for the varifocal lens  5  from a target z coordinate. The scan control data further includes data for controlling the varifocal lens  5  in accordance with the focal length calculated for the varifocal lens  5  from the target z coordinate and an oscillation angle calculated for the scanner  3  from target xy coordinates. The scan control data is intended to, for example, designate the oscillation angle of the scanner  3  and the focal length of the varifocal lens  5  for each of the target xyz coordinates. That is, the control unit  14  controls the scanner  3  in accordance with a focal length calculated for the varifocal lens  5  from the target z coordinate, and controls the varifocal lens  5  in accordance with the focal length calculated for the varifocal lens  5  from the target z coordinate and an oscillation angle calculated for the scanner  3  from target xy coordinates. This allows the microscope  100  to correct deviation of a scan position caused by the varifocal lens  5 . Deviation of a scan position is different for each wavelength used for an observation, and hence scan control data may be provided for each wavelength. 
       FIG. 8  is a flowchart of a process of generating three-dimensional-image data performed by the microscope  100 . With reference to  FIG. 8 , the following specifically describes a method of generating three-dimensional-image data using scan control data. The three-dimensional-image-data generating process depicted in  FIG. 8  starts after a user has put a lens in focus on a sample S placed on the stage  9  with the focal length of the varifocal lens  5  adjusted in a manner such that a parallel light flux enters the objective  8 . 
     The microscope  100  makes initial settings (step S 11 ). In this example, the microscope  100  designates information such as an objective to be used, scan ranges (a scan range in the xy direction and a scan range in the z direction), and intervals between z coordinates at which xy cross-sectional images are to be obtained (hereinafter referred to as z intervals). These pieces of information may be input to the microscope  100  by the user, or may be read from a setting file stored in advance in the storage unit  15 . 
     The microscope  100  reads scan control data (step S 12 ). In this example, the control unit  14  reads apiece of scan control data corresponding to the objective to be used (objective  8 , in this example) from scan control data stored in the storage unit  15  for each objective. 
     After reading scan control data, the microscope  100  obtains xy cross-sectional images according to the read scan control data (step S 13 ). In this example, when the varifocal lens  5  has caused deviation of a scan position in the xy direction, the control unit  14  controls the scanner  3  according to the scan control data that depends on the objective  8 . When the varifocal lens  5  has caused deviation of a scan position in the xy direction and the z direction, the control unit  14  controls the scanner  3  and the varifocal lens  5  according to the scan control data that depends on the objective  8 . Consequently, xy cross-sectional images are obtained, and xy-cross-sectional-image data is generated. 
     The microscope  100  determines whether xy cross-sectional images have been obtained for all z coordinates (step S 14 ). In this example, the microscope  100  determines, for example, whether scans have been performed completely at the z intervals within the scan range in the z direction that has been designated in step S 11 . 
     When it is determined that all images have not been obtained (NO in step S 14 ), the microscope  100  changes the z coordinate by changing the focal length of the varifocal lens  5  (step S 15 ). In this example, the control unit  14  changes the focal length of the varifocal lens  5  in such a manner as to change the z coordinate of the scan position by the length of one z interval. Then, the obtaining process (step S 13 ) and determining process (step S 14 ) on xy cross-sectional images are performed again. 
     When it is determined that all images have been obtained (YES in step S 14 ), the microscope  100  generates three-dimensional-image data (step S 15 ) and ends the three-dimensional-image-data generating process. In this example, the microscope  100  generates three-dimensional-image data from pieces of data generated in step S 13 , i.e., a plurality of pieces of xy-cross-sectional-image data corresponding to different z coordinates. Although a piece of three-dimensional-image data is generated using one wavelength in the flowchart depicted in  FIG. 8 , a piece of three-dimensional-image data may be generated for each wavelength used for an observation so as to generate a piece of multicolored three-dimensional-image data by mixing pieces of generated three-dimensional-image data. Three-dimensional-image data may be generated by controlling the varifocal lens and the scanner according to color aberration information of the optical system in such a manner that an identical three-dimensional-image position is achieved for various wavelengths. 
     Embodiment 2 
       FIG. 9  illustrates the configuration of a microscope  200  in accordance with the present embodiment. The microscope  200  is a confocal laser scanning microscope. The microscope  200  is similar to the microscope  100  except for a difference in order in which the scanner  3  and the varifocal lens  5  are arranged. As with the microscope  100  in accordance with embodiment 1, the microscope  200  can quickly obtain an accurate three-dimensional image by scanning a sample S according to scan control data. 
     Embodiment 3 
       FIG. 10  illustrates the configuration of a microscope  300  in accordance with the present embodiment. The microscope  300  is a confocal laser scanning microscope. The microscope  300  is similar to the microscope  200  except for the fact that, in the microscope  300 , the varifocal lens  5  is located at, or near, a pupil position of the objective  8 . As with the microscopes in accordance with embodiments 1 and 2, the microscope  300  can quickly obtain an accurate three-dimensional image by scanning a sample S according to scan control data. This configuration may be used when the pupil position of the objective  8  is located outside the objective  8 . 
     Embodiment 4 
       FIG. 11  illustrates the configuration of a microscope  400  in accordance with the present embodiment. The microscope  400  is a confocal laser scanning microscope. The microscope  400  is different from the microscope  100  in the sense that the microscope  400  includes an actuator  16  that moves the objective  8  in an optical-axis direction. The microscope  400  may change a distance between a sample S and the objective  8  via the control unit  14  causing the actuator  16  to move the objective  8 . For the other features, the microscope  400  is similar to the microscope  100 . As with the microscopes in accordance with embodiments 1-3, the microscope  400  can quickly obtain an accurate three-dimensional image by scanning a sample S according to scan control data. 
     In the microscope  400 , moving the objective  8  changes a position relationship between a pupil conjugate position of the objective  8  and the varifocal lens  5 . Hence, deviation of a scan position caused by the varifocal lens  5  may change with the position of the objective  8 . In consideration of this fact, scan control data may be stored in the storage unit  15  for each objective position. The control unit  14  may control the scanner  3  or both the scanner  3  and the varifocal lens  5  according to scan control data that depends on an objective position. When the varifocal lens  5  has been displaced greatly from the pupil conjugate position of the objective  8  due to movement of the objective  8 , deviation of a scan position may become too large to be adjusted using the varifocal lens  5 . The storage unit  15  may additionally store offset data for offsetting a scan position in the z direction that is used in such a situation. 
     Embodiment 5 
       FIG. 12  illustrates the configuration of a microscope  500  in accordance with the present embodiment. The microscope  500  is a confocal laser scanning microscope. The microscope  500  is different from the microscope  400  in the sense that the microscope  500  includes a relay lens  17  and a wavefront modulator  18  between the scanner  3  and the dichroic mirror  2 . The relay lens  17  projects the wavefront modulator  18  onto the scanner  3 . Thewavefront modulator  18  is awavefront aberration correcting apparatus for correcting a wavefront aberration, and is located at a pupil conjugate position of an objective. 
     As with the microscopes in accordance with embodiments 1-4, the microscope  500  can quickly obtain an accurate three-dimensional image by scanning a sample S according to scan control data. In the microscope  500 , the wavefront modulator  18  can correct an aberration that cannot be corrected by the scanner  3  and the varifocal lens  5  (e.g., spherical aberration). This allows a more accurate three-dimensional image to be obtained. 
       FIG. 12  depicts the wavefront modulator  18 , which is a transmission-type modulator, but a reflection-type wavefront modulator, e.g., an LCOS or a deformable mirror, may be used. In  FIG. 12 , the wavefront modulator  18  is located between the scanner  3  and the dichroic mirror  2  (not illustrated). However, the wavefront modulator  18  only needs to be located at a pupil conjugate position of the objective  8 . 
     The described embodiments indicate specific examples for facilitating understanding of the invention, and embodiments of the invention are not limited to them. Various modifications and changes can be made to the scanning microscopes without departing from the invention defined in the claims. A single embodiment may be achieved by combining some features from contexts of individual embodiments described herein. 
     The described embodiments are based on an exemplary situation in which the microscope is a confocal laser scanning microscope. However, as long as the microscope is a scanning microscope, it may be, for example, a multiphoton-excitation laser scanning microscope. The light source is not limited to a laser, and the scanning microscope may include a non-laser light source, e.g., a super luminescent diode (SLD) or a mercury lamp. 
     The described embodiments are based on an exemplary situation in which the storage unit  15  stores scan control data for each objective, but the storage unit  15  may store scan control data for each objective and for each light flux diameter of laser light emitted from the beam expander  19 . An aberration caused by an objective changes with the light flux diameter of laser light that enters a pupil of the objective. Hence, use of such scan control data may correct not only deviation of a scan position caused by the varifocal lens but also deviation of the scan position caused by a change in aberration made by the objective.