Patent Publication Number: US-11042017-B2

Title: Point-spread-function measurement device and measurement method, image acquisition apparatus, and image acquisition method

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a Continuation Application of International Application No. PCT/JP2016/058012 filed on Mar. 14, 2016, the content of which is incorporated herein by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     The present invention relates to point-spread-function measurement devices and measurement methods, image acquisition apparatuses, and image acquisition methods. 
     BACKGROUND ART 
     In an image acquisition apparatus such as a two-photon excitation microscope, the spatial resolution of an image is set solely in accordance with the point spread function of an excitation laser. In particular, when observing a deep section of a sample, since the shape of the point spread function extends in accordance with the refractive index of the sample or scattering, the spatial resolution of the image deteriorates. 
     A known method involves ascertaining the shape of the point spread function and reducing blurriness of the image by performing deconvolution (for example, see Non Patent Literature 1). 
     This method involves disposing a microscopic fluorescence bead within a sample and moving the fluorescence bead by optical trapping so as to measure the point spread function at a freely-chosen position. 
     CITATION LIST 
     Non Patent Literature 
     NPL 1 
     
         
         J. W. Shaevitz and D. A. Fletcher, “Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function,” J. Opt, Soc. Am. A, Vol. 24, 2622 (2007) 
       
    
     SUMMARY OF INVENTION 
     An aspect of the present disclosure provides a point-spread-function measurement device including: a scanner configured to scan two illumination light beams emitted from a light source; an illumination optical system configured to radiate the two illumination light beams scanned by the scanner onto a sample; a relative-position adjustor configured to change a relative irradiation position, in the sample, between the two illumination light beams radiated by the illumination optical system; a detection optical system configured to detect signal light generated at an overlapping position, in the sample, of the illumination light beams radiated by the illumination optical system; and a calculator configured to calculate a point spread function based on the signal light detected by the detection optical system and the relative irradiation position between the two illumination light beams when the signal light is detected. 
     Another aspect of the present disclosure provides a point-spread-function measurement method including: scanning two illumination light beams emitted from a light source over a sample while changing a relative irradiation position in the sample; detecting signal light generated at an overlapping position, in the sample, of the illumination light beams radiated; and calculating a point spread function based on the signal light detected and the relative irradiation position between the two illumination light beams when the signal light is detected. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  schematically illustrates an image acquisition apparatus according to an embodiment of the present invention. 
         FIG. 2  illustrates a relative position between two laser beams in accordance with a point-spread-function measurement device according to an embodiment of the present invention included in the image acquisition apparatus in  FIG. 1 . 
         FIG. 3  illustrates the fluorescence intensity distribution with respect to the distance between focal points acquired by the measurement device in  FIG. 2 . 
         FIG. 4  illustrates an autocorrelation waveform image of a laser beam acquired by removing an offset component from the fluorescence intensity distribution in  FIG. 3  and rotating the fluorescence intensity distribution. 
         FIG. 5  illustrates the spatial distribution of the PSF of two-photon-excitation fluorescence calculated by using the autocorrelation waveform image in  FIG. 4 . 
         FIG. 6  is a flowchart illustrating an image acquisition method according to an embodiment of the present invention in a case where a PSF-shape measurement position and an image acquisition position are the same. 
         FIG. 7  is a flowchart illustrating a PSF measurement method according to an embodiment of the present invention. 
         FIG. 8  is a flowchart illustrating an image acquisition method in a case where the PSF-shape measurement position and the image acquisition position are different from each other. 
         FIG. 9  illustrates an example of a PSF-shape measurement method of a laser beam in a case where the PSF in a sample is asymmetric. 
         FIG. 10  schematically illustrates a first modification of the image acquisition apparatus in  FIG. 1 . 
         FIG. 11  schematically illustrates a second modification of the image acquisition apparatus in  FIG. 1 . 
         FIG. 12  schematically illustrates a third modification of the image acquisition apparatus in  FIG. 1 . 
         FIG. 13  schematically illustrates a fourth modification of the image acquisition apparatus in  FIG. 1 . 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     A point-spread-function measurement device  25  and measurement method, an image acquisition apparatus  1 , and an image acquisition method according to embodiments of the present invention will be described below with reference to the drawings. 
     As shown in  FIG. 1 , the image acquisition apparatus  1  according to this embodiment is a multiphoton-excitation-type (more specifically, a two-photon-excitation-type) scanning fluorescence microscope and includes: a light source (laser light source)  2 , such as a titanium-sapphire laser, emitting a near-infrared ultra-short pulsed laser beam (referred to as “laser beam” hereinafter); an illumination optical system  3  that radiates the laser beam from the light source  2  onto a sample X; a scanning unit  4  that is disposed at an intermediate position of the illumination optical system  3  and that two-dimensionally scans the laser beam; a detection optical system  5  that detects fluorescence (signal light) generated in the sample X as a result of the sample X being irradiated with the laser beam; and a processing device  6  that calculates a point spread function (referred to as “PSF” hereinafter) based on the intensity of the fluorescence detected by the detection optical system  5  and that reconfigures an image by using the calculated PSF. 
     The illumination optical system  3  includes a beam-diameter adjustment optical system  7  that adjusts the beam diameter of the laser beam from the light source  2 , a half-wave plate  8  that sets the polarization direction of the laser beam to 45°, a first polarization beam splitter  9  that splits the laser beam into two optical paths, an optical-path-length adjustment optical system (timing adjustment unit)  10  provided in one of the optical paths, a second polarization beam splitter  11  that multiplexes the laser beams (first laser beam and second laser beam) traveling along the two optical paths, a quarter-wave plate  12  that allows the multiplexed laser beam to pass therethrough, two pairs of relay lenses  13   a  and  13   b , a beam shaping element (light modulating unit)  14 , a pupil projection lens  15 , an imaging lens  16 , and an objective lens  17 . In the drawing, reference sign  18  denotes a mirror for forming an optical path. 
     The first polarization beam splitter  9  receives the laser beam after the polarization direction thereof is set to 45° by the half-wave plate  8  so as to split the input laser beam into a first laser beam (illumination light beam) L 1  and a second laser beam (illumination light beam) L 2  with an intensity ratio of 1:1 and having polarization directions orthogonal to each other. 
     The second polarization beam splitter  11  is set in a motorized holder (relative-position adjustment unit)  19  whose tilt angle is independently controllable along two axes. When the tilt angle of the second polarization beam splitter  11  is changed by actuating the motorized holder  19 , the first laser beam L 1  transmitted through the second polarization beam splitter  11  is transmitted substantially straight therethrough substantially without being polarized, whereas the polarization angle of the second laser beam L 2  reflected by the first polarization beam splitter  9  is changed. Consequently, the angle of the second laser beam L 2  input to the scanning unit  4  changes, so that the relative irradiation position between the first laser beam L 1  and the second laser beam L 2  in the sample X changes. 
     The optical-path-length adjustment optical system  10  causes a pair of mirrors  20  to move in the direction of the arrow so as to adjust the optical-path length of the second laser beam L 2 , thus causing the pulses of the first laser beam L 1  and the second laser beam L 2  to be synchronous after the laser beams are multiplexed by the second polarization beam splitter  11 . 
     The quarter-wave plate  12  converts the multiplexed first laser beam L 1  and second laser beam L 2  into circularly-polarized light. 
     The scanning unit  4  includes, for example, a two-axis galvanometer mirror  21  and is disposed between the relay lens  13   b  and the pupil projection lens  15 . The scanning unit  4  is disposed at an optically conjugate position with respect to the second polarization beam splitter  11 , the beam shaping element  14 , and the pupil position of the objective lens  17  by means of the two pairs of relay lenses  13   a  and  13   b , the pupil projection lens  15 , and the imaging lens  16 . 
     The detection optical system  5  includes a dichroic mirror  22  that is disposed between the imaging lens  16  and the objective lens  17  and that splits off fluorescence collected by the objective lens  17  from the optical path, a focusing lens  23  that focuses the fluorescence split off by the dichroic mirror  22 , and a photodetector  24 , such as a photomultiplier tube, that detects the focused fluorescence. 
     The processing device  6  includes a calculating unit (not shown) that calculates a PSF and an image processor (not shown) that reconfigures a fluorescence image by using the calculated PSF. The components from the light source  2  to the calculating unit included in the processing device  6  constitute the PSF measurement device  25  according to an embodiment of the present invention. 
     For each relative distance between the first laser beam L 1  and the second laser beam L 2  set by the motorized holder  19 , the calculating unit integrates the intensity of each fluorescence image acquired by actuating the scanning unit  4  and plots the integral value of the fluorescence intensity with respect to the relative distance, so that the fluorescence intensity distribution in  FIG. 3  is obtained. 
     The calculating unit also removes an offset component from the fluorescence intensity distribution in  FIG. 3 . 
     Furthermore, the calculating unit rotates the waveform of the fluorescence intensity distribution, from which the offset component is removed, around an axis extending through the peak so as to create a rotationally symmetric image shown in  FIG. 4 , performs two-dimensional Fourier transformation on the created image, raises the obtained image to the power of ½, then performs two-dimensional inverse Fourier transformation on the obtained image, and raises the obtained image to the power of 2. Consequently, a two-photon-excitation PSF spatial distribution is acquired, as shown in  FIG. 5 . 
     The processing in the calculating unit will be described below by using mathematical expressions. 
     The excitation light intensity including both the first laser beam L 1  and the second laser beam L 2  and the fluorescence intensity generated accordingly are expressed with mathematical expressions. The excitation light intensity I ex  in a case where electrical field amplitudes of the first laser beam L 1  and the second laser beam L 2  are defined as E 1  and E 2  is expressed with expression (1).
 
 I   ex   =|E   1   +E   2 | 2   =|E   1 | 2   +|E   2 | 2   +E*   1   E   2   +E   1   E*   2   =I   1   +I   2   +E*   1   E   2   +E   1   E*   2    (1)
 
     In expression (1), “★” denotes the complex conjugate, and I 1  and I 2  respectively denote intensities of the first laser beam L 1  and the second laser beam L 2 . 
     Since it is assumed that a two-photon-excitation microscope is used in this embodiment, the fluorescence intensity is proportional to the square of the excitation light intensity. The fluorescence intensity is expressed with expression (2) in a case where this proportionality factor is defined as α. 
     
       
         
           
             
               
                 
                   
                     
                       
                         
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     As mentioned above, the first laser beam L 1  and the second laser beam L 2  are converted by the second polarization beam splitter  11  into light beams that are linearly polarized orthogonal to each other and are subsequently converted into circularly-polarized light beams by the quarter-wave plate  12 . In this case, the first laser beam L 1  and the second laser beam L 2  do not interfere with each other, and the fourth term and the fifth term in expression (2) are small enough to be negligible, as compared with other terms. Specifically, expression (2) can be simplified into expression (3).
 
 I   fl_2P =α( I   1   2   +I   2   2 +2 I   1   I   2 )  (3)
 
     The first term and the second term in expression (3) indicate fluorescence components generated by being independently excited by the first laser beam L 1  and the second laser beam L 2 , regardless of the relative position between the focal points of the first laser beam L 1  and the second laser beam L 2 . On the other hand, the third term in expression (3) indicates an intensity integral of the first laser beam L 1  and the second laser beam L 2  and corresponds to overlapping of the focal points. Specifically, when the focal point of the first laser beam L 1  and the focal point of the second laser beam L 2  are in a completely non-overlapped state, the third term is zero. When the focal point of the first laser beam L 1  and the focal point of the second laser beam L 2  are in a completely overlapped state, the third term is a maximum value. 
       FIG. 2  illustrates an example of the positions of the focal point of the first laser beam L 1  and the focal point of the second laser beam L 2 . Reference sign P 1  denotes the focal point of the first laser beam L 1 , and reference sign P 2  denotes the focal point of the second laser beam L 2 . In FIG.  2 , the focal points are not overlapped with each other. It is assumed that an image of the sample X is acquired while changing the relative distance between the focal points P 1  and P 2 . Specifically, the image is acquired while moving the focal point of the second laser beam L 2  in the direction of the arrow in  FIG. 2  by using the motorized holder  19  to change the polarization angle of the second laser beam L 2 . 
     More specifically, in a state where the focal point P 2  of the second laser beam L 2  is located at the position in  FIG. 2 , a two-dimensional fluorescence image of the sample X is acquired by actuating the scanning unit  4 . Then, the focal point P 2  is moved by predetermined steps in the direction of the arrow in  FIG. 2 , and another fluorescence image of the sample X is acquired in this state. By repeating this process, a fluorescence image of the sample X is acquired for each relative distance between the focal points P 1  and P 2 . 
     The fluorescence images acquired in this manner each have a fluorescence intensity in which the state of overlapping of the focal points P 1  and P 2  is reflected. Specifically, the fluorescence intensity changes in accordance with expression (3). In particular, the third term changes in accordance with the overlapping of the focal points P 1  and P 2 . 
     The integral fluorescence intensity distribution in  FIG. 3 , which is obtained by integrating the fluorescence intensities within the respective fluorescence images acquired in this manner and plotting them relative to the relative distance between the focal points P 1  and P 2 , has a shape in which the integral fluorescence intensity changes in accordance with the relative distance between the focal points P 1  and P 2  and particularly has a peak at a position where the relative distance between the focal points P 1  and P 2  is zero. 
     This implies that the overlapping region of the focal points P 1  and P 2  is at a maximum in this state and that the third term in expression (3) is at a maximum. The offset component of the fluorescence intensity distribution in  FIG. 3  does not change even when the relative distance between the focal points P 1  and P 2  changes, and corresponds to the sum of the first term and the second term in expression (3). 
     Next, a process for removing the offset component will be described. One method for removing the offset component involves detecting a signal value when the focal points P 1  and P 2  have a relative distance therebetween and thus do not overlap in the form of an offset value, and then subtracting the offset value from the overall fluorescence intensity distribution. It is clear from expression (3) that, when the light intensities of the focal point P 1  and the focal point P 2  are equal to each other (i.e., when I 1 =I 2 ), the peak value of the waveform is twice as large as the offset value. Thus, it is possible to calculate ½ of the peak value and calculate the difference from the overall waveform. 
     However, if there are fluorescent molecules in a peripheral region of an image, in the direction in which relative scanning of the focal points P 1  and P 2  is performed, the waveform in  FIG. 3  is not properly obtained due to the effect of the distribution of the fluorescence molecules, and the value is not fixed even when the focal points P 1  and P 2  have a relative distance therebetween and thus do not overlap. In such a case, the offset-component removing method described above cannot be applied. 
     In this case, a method of using the optical-path-length adjustment optical system  10  may be applied. Specifically, the fluorescence intensity distribution in  FIG. 3  is acquired by varying the optical-path length of the first laser beam L 1  and the optical-path length of the second laser beam L 2  by adjusting the optical-path-length adjustment optical system  10 . In a state where the optical-path length of the first laser beam L 1  and the optical-path length of the second laser beam L 2  are different from each other, the pulse of the first laser beam L 1  and the pulse of the second laser beam L 2  are not simultaneously radiated onto the sample X, so that the contribution of the third term in expression (3) becomes lost. Specifically, only an offset component constituted of the sum of the first term and the second term in expression (3) is obtained. 
     Accordingly, the process of acquiring the fluorescence intensity distribution in  FIG. 3  in a state where the first laser beam L 1  and the second laser beam L 2  are simultaneously radiated onto the sample X and the process of acquiring the fluorescence intensity distribution in  FIG. 3  in a state where the first laser beam L 1  and the second laser beam L 2  are not simultaneously radiated onto the sample X are performed by switching the optical-path-length adjustment optical system  10 , and the difference is calculated, whereby an offset-removed fluorescence intensity distribution can be acquired. 
     In this offset-component removing method that utilizes the optical-path-length adjustment optical system  10 , the fluorescent molecules to be excited are the same when the pulse of the first laser beam L 1  and the pulse of the second laser beam L 2  are simultaneously radiated onto the sample X and when the pulse of the first laser beam L 1  and the pulse of the second laser beam L 2  are non-simultaneously radiated onto the sample X, so that the effect of the distribution of the fluorescent molecules is also removed when calculating the difference. This is advantageous in that the offset component can be properly removed regardless of the distribution of the fluorescent molecules. 
     The fluorescence intensity distribution from which the offset component is removed in this manner is such that the relative distance between the focal points P 1  and P 2  of the first laser beam L 1  and the second laser beam L 2  in the third term of expression (3) is changed, and can be expressed as the convolution of the two laser beams L 1  and L 2  having the same intensity I, as indicated in expression (4).
 
 G ( u )=∫ −∞   ∞   I ( x )· I ( x−u ) dx   (4)
 
     Subsequently, the fluorescence intensity distribution from which the offset component is removed is rotated around a line extending through the peak, thereby creating a rotationally symmetric image shown in  FIG. 4 . In this image, the cross-sectional profile is an autocorrelation waveform in  FIG. 3  at any angle. This image is a two-dimensionally developed form of expression (4) and can be expressed with expression (5).
 
 G ( u,v )=∫ −∞   ∞∫   −∞   ∞   I ( u,v )· I ( x−u,y−v ) dxdy   (5)
 
     Because a convolution in actual space can be replaced by a product in Fourier space, when expression (5) is two-dimensionally Fourier-transformed, the excitation light intensity I can be expressed as the square of the Fourier-transformed result, as in expression (6). Therefore, as indicated by expression (7), by performing a two-dimensional inverse Fourier transformation by raising expression (6) to the power of ½, the spatial distribution of the excitation light intensity I, that is, the PSF shape of the laser beam at the focal point, can be acquired. Then, by raising this excitation light intensity I to the power of 2, the shape of the two-photon-excitation PSF is obtained.
 
 { G ( u,v )}=[ { I ( u,v )}] 2   (6)
 
 I ( u,v )=   −1 {[ { G ( u,v )}] 1/2 }  (7)
 
     In order to obtain the excitation light intensity I by performing the inverse Fourier transformation by raising expression (6) to the power of ½, as in expression (7), the convolutions have to be of the same excitation light intensity I, as in expression (4). In order to achieve this, the polarization directions of the focal points P 1  and P 2  are set to be orthogonal to each other by the two polarization beam splitters  9  and  11  in  FIG. 1 , whereby the fourth term and the fifth term in expression (2) can be removed. 
     The image processor uses the PSF acquired in the above-described manner to perform deconvolution, thereby improving the image resolution. An example of deconvolution that may be applied includes Wiener deconvolution, which is widely known. 
     A process of observing the sample X involves acquiring an image in a state where the first laser beam L 1  and the second laser beam L 2  are completely overlapped or acquiring an image in a state where only one of the first laser beam L 1  and the second laser beam L 2  is radiated onto the sample X, and performing deconvolution of the acquired image by using the already-determined PSF. 
     As the image acquired in the state where the first laser beam L 1  and the second laser beam L 2  are completely overlapped, an image selected from images acquired while changing the relative position between the focal points P 1  and P 2  may be used. 
     If only one of the first laser beam L 1  and the second laser beam L 2  is to be radiated onto the sample X, for example, the half-wave plate  8  may be rotated so that all components are reflected by or transmitted through the first polarization beam splitter  9 . Accordingly, the PSF-shape measurement and the image acquisition for observing the sample X can be switched therebetween without losing the power of the laser beams. 
       FIG. 6  illustrates an example of flow from PSF-shape measurement to high-resolution-image acquisition of the sample X in accordance with deconvolution. 
     Referring to  FIG. 6 , an image acquisition method according to an embodiment of the present invention includes setting an image acquisition position within the sample X (step S 1 ), measuring the PSF shape at that position (step S 2 ), subsequently acquiring an image of the sample X (image generating step S 3 , image processing step), performing a reconfiguring process (deconvolution) on that image by using the measured PSF (reconfiguring step S 4 , image processing step), and determining whether or not there is another position where the PSF shape is to be measured (step S 5 ). If it is determined that observation is completed for the entire range, the measurement ends. If it is necessary to observe the sample X at a different field of view within the sample X, the process returns to the setting of the image acquisition position from step S 5  so as to repeat the process from step S 1 . The reconfiguring step S 4  does not have to be performed for each image acquisition process at every field of view, and may be performed collectively after the measurement for the entire field of view is completed. 
     Referring to  FIG. 7 , a PSF measurement method according to an embodiment of the present invention includes actuating the optical-path-length adjustment optical system  10  such that two illumination light beams are simultaneously radiated onto the sample X (step S 21 ), setting the polarization angle of the second laser beam L 2  by using the motorized holder  19  (step S 22 ), actuating the scanning unit  4  to scan the two laser beams L 1  and L 2  over the sample X and detecting the intensity of fluorescence generated at each scan position (step S 23 , scanning step, detecting step), and determining whether or not acquisition of a first fluorescence intensity distribution is completed (step S 24 ). If the acquisition is not completed, the process returns to step S 22  where the polarization angle of the second laser beam L 2  is slightly changed by using the motorized holder  19 , and the process thereafter is repeated. Consequently, the first fluorescence intensity distribution with respect to a relative irradiation position can be acquired. 
     Subsequently, when the acquisition of the first fluorescence intensity distribution is completed, the optical-path-length adjustment optical system  10  is actuated such that two illumination light beams are non-simultaneously radiated onto the sample X (step S 25 ), the polarization angle of the second laser beam is set by the motorized holder  19  (step S 26 ), and the two laser beams are scanned over the sample X by actuating the scanning unit  4 , and the intensity of fluorescence generated at each scan position is detected (step S 27 , scanning step, detecting step). Then, it is determined whether or not acquisition of a second fluorescence intensity distribution is completed (step S 28 ). If the acquisition is not completed, the process returns to step S 26  where the polarization angle of the second laser beam L 2  is slightly changed by using the motorized holder  19 , and the process thereafter is repeated. Consequently, the second fluorescence intensity distribution with respect to a relative irradiation position can be acquired. 
     By subtracting the acquired second fluorescence intensity distribution from the acquired first fluorescence intensity distribution, a third fluorescence intensity distribution from which an offset component is removed is obtained (step S 29 ). The obtained third fluorescence intensity distribution is rotated around the axis extending through the peak so that an image indicating an autocorrelation waveform is acquired (calculating step S 30 ). The acquired image undergoes Fourier transformation (step S 31 ), is raised to the power of ½ (step S 32 ), undergoes inverse Fourier transformation (step S 33 ), and is further raised to the power of 2 (step S 34 ). Consequently, the two-photon-excitation PSF shape of the laser beams at the focal points within the sample X can be determined. 
     Furthermore, if a signal value when the focal points P 1  and P 2  have a relative distance therebetween and thus do not overlap in  FIG. 3  is to be set as an offset value, as described above, or if half of the signal peak value is to be set as an offset value, step S 25  to step S 28  can be omitted. After calculating an offset value in such a process, the offset value may be subtracted in step S 29 . 
     Although it is desirable that the position on the sample X where the PSF shape is to be measured and the position where an image of the sample X is to be acquired be the same, the positions may be different from each other if the PSF shape does not change. For example, the PSF-shape measurement and the image acquisition may be performed at different positions but at the same depth of the sample X. 
     In a case where an image of the sample X is to be three-dimensionally acquired while continuously changing the depth, for example, the PSF-shape measurement may be performed at several intermittent depth positions, and the PSF shape for regions between the positions may be calculated by interpolating the acquired PSF shape. Accordingly, an image with high spatial resolution can be effectively acquired in accordance with deconvolution while reducing the number of times the PSF shape is measured. 
       FIG. 8  illustrates the flow in a case where the PSF measurement position and the image acquisition position of the sample X are different from each other. In  FIG. 8 , a PSF-shape measurement position is set (step S 6 ), and the PSF shape is measured at that position (step S 2 ). It is determined whether or not there is another PSF-shape measurement position (step S 7 ). If there is another measurement position, the process returns to step S 6  to set the PSF measurement position again (step S 6 ), and the PSF shape is measured (step S 2 ). When the PSF measurement is completed, an image acquisition position is set (step S 1 ), image acquisition is performed (step S 3 ), and image reconfiguration is performed (step s 4 ). The image acquisition is repeated until images are acquired for the entire field of view (step S 5 ). 
     The flow of the process is not limited to the flow in  FIG. 8 . A process for calculating the PSF at positions other than the measurement positions by interpolating the PSF may be added, or the image reconfiguration may be performed collectively at the end. 
     Furthermore, a high-frequency component of the spatial frequency of the PSF may be optically accentuated by, for example, spatially modulating a laser beam to be input to the objective lens  17 . If a mask that blocks off the center of a beam is to be used as the beam shaping element  14  in  FIG. 1 , the laser beam to be output from the objective lens  17  becomes annular and has a shape in which the high-frequency component is accentuated. Normally, when performing deep observation, the high-frequency components decrease due to scattering and aberration, causing the resolution to decrease. By optically accentuating the high-frequency components, a decrease in resolution can be suppressed. 
     When the high-frequency components are accentuated as in annular illumination, the PSF has an unnatural shape having side lobes. However, by measuring the PSF shape and performing deconvolution using the obtained PSF, the effect of the side lobes can be removed, and a fluorescence image having high resolution can be acquired. Specifically, the high-frequency component is optically accentuated by the beam shaping element  14 , and the component is further accentuated by image processing in accordance with deconvolution and the effect of the side lobes is removed, so that a high-resolution image of the sample X can be obtained. In combination with the beam shaping element  14  that accentuates the high-frequency components of the PSF, the resolution of the fluorescence image can be further improved. As an alternative to a mask, the beam shaping element  14  may be a polarizing element that converts a polarized laser beam into a radially polarized beam; it is not limited so long as the beam shaping element  14  can optically accentuate the high-frequency components. 
     In order to acquire an autocorrelation waveform in this embodiment, a two-dimensional image is acquired by using the two-axis galvanometer mirror  21  while relatively changing the irradiation position of the first laser beam L 1  and the irradiation position of the second laser beam L 2 . Alternatively, since this image is intended for acquisition of an autocorrelation waveform, the image may be one-dimensional, that is, linear, instead of two-dimensional. 
     Although the irradiation positions of the first laser beam L 1  and the second laser beam L 2  are relatively moved one-dimensionally, as shown in  FIG. 2 , this is intended to create a two-dimensional, rotationally symmetric autocorrelation waveform by rotating a one-dimensional autocorrelation waveform and is based on the assumption that the PSF shape to be measured is rotationally symmetric. Therefore, this cannot be applied in a case where the PSF shape is rotationally asymmetric. 
     As shown in  FIG. 9 , if the PSF shape is rotationally asymmetric, it is preferable that the irradiation position of the first laser beam L 1  and the irradiation position of the second laser beam L 2  be relatively scanned two-dimensionally. By performing a process similar to that in the above description by using an image of a two-dimensional autocorrelation waveform acquired in this manner, an autocorrelation waveform image of two-photon-excitation fluorescence can be acquired even when the PSF shape is rotationally asymmetric. 
     When the PSF shape is measured in this manner, deconvolution is performed based on the measured PSF shape, so that the resolution of the fluorescence image can be improved. In particular, in deep observation using a two-photon-excitation microscope, the PSF distribution changes in accordance with scattering occurring within the sample X or aberrations caused by the refractive index of the sample X. The image acquisition apparatus  1  according to this embodiment is advantageous in that the distribution of the PSF shape within the sample X is measured even in a deep section of the sample X, and in that a fluorescence image with high resolution can be acquired by means of deconvolution. 
     By using an image acquired when the PSF shape is measured as a sample image for performing deconvolution, the PSF-shape measurement and the sample-image acquisition do not have to be performed separately. This is advantageous in that the overall image acquisition time can be shortened. 
     Furthermore, as an alternative to this embodiment in which the half-wave plate  8  is disposed in front of the first polarization beam splitter  9 , for example, a quarter-wave plate may be used so long as the first polarization beam splitter  9  is capable of splitting a laser beam into a first laser beam L 1  and a second laser beam L 2 . Moreover, although the quarter-wave plate  12  is disposed behind the second polarization beam splitter  11 , the quarter-wave plate  12  may be omitted so long as the polarization directions of the first laser beam L 1  and the second laser beam L 2  are orthogonal to each other when the laser beams are radiated onto the sample X. 
     However, because an autocorrelation waveform is acquired in a state where the polarization directions of the first laser beam L 1  and the second laser beam L 2  are orthogonal to each other, the cross-sectional shape of the PSF obtained with expression (7) is the same as that of circularly-polarized light, which has an intermediate shape between the shape of vertically-polarized light and the shape of horizontally-polarized light. Therefore, it is desirable that circularly-polarized light be acquired when an image of the sample X is to be acquired. Thus, it is desirable that the PSF-shape measurement be performed in a state where there is no quarter-wave plate  12  behind the second polarization beam splitter  11  and that the quarter-wave plate  12  be inserted in the optical path to obtain circularly-polarized light when an image of the sample X is to be acquired. 
     In this embodiment, the second polarization beam splitter  11 , the beam shaping element  14 , and the scanning unit  4  are disposed to have an optically conjugate positional relationship with the pupil position of the objective lens  17  by means of the two sets of relay lenses  13   a  and  13   b , the pupil projection lens  15 , and the imaging lens  16 . Alternatively, as shown in  FIG. 10 , one of the sets of relay lenses  13   a  and  13   b  may be omitted, and the beam shaping element  14  may be disposed immediately behind the second polarization beam splitter  11 . 
     Although it is preferable that the beam shaping element  14  be accurately disposed at an optically conjugate position with respect to the second polarization beam splitter  11  and the pupil position of the objective lens  17 , the beam shaping element  14  can be disposed at a substantially conjugate position by being disposed immediately behind the second polarization beam splitter  11 . Furthermore, since an angular difference between the first laser beam L 1  and the second laser beam L 2  output at different angles from the second polarization beam splitter  11  is extremely small, a variation in the positions where the first laser beam L 1  and the second laser beam L 2  pass through the beam shaping element  14  can be made small enough to be negligible even by disposing the beam shaping element  14  in this manner. 
     The configuration in  FIG. 10  is advantageous in that the configuration of the image acquisition apparatus  1  exhibiting advantages similar to those in  FIG. 1  can be simplified. 
     Furthermore, in this embodiment, a defocusing element  26  may be disposed in the optical path of the second laser beam L 2  between the first polarization beam splitter  9  and the second polarization beam splitter  11 , as shown in  FIG. 11 . The defocusing element  26  causes the second laser beam L 2  to slightly scatter or converge so as to move (defocus), in the optical-axis direction, the focal point of the second laser beam L 2  to be focused by the objective lens  17 . The defocusing element  26  used may be an active device, such as a pair of lenses or a liquid lens. 
     In the image acquisition apparatus  1  in  FIG. 1 , the PSF shape is measured by relatively scanning the two focal points in the direction orthogonal to the optical axis, making it possible to measure only the PSF shape extending in the direction orthogonal to the optical axis. However, since a PSF extends three-dimensionally in actuality, an autocorrelation waveform is acquired and calculated by relatively scanning the two focal points also in the direction parallel to the optical axis, as in an image acquisition apparatus  27  shown in  FIG. 11 , so that a three-dimensionally-extending PSF shape can also be measured. 
     Accordingly, deconvolution can be performed three-dimensionally, so that when a three-dimensional fluorescence image is acquired, the spatial resolution can be improved three-dimensionally. In this case, similar to the image acquisition apparatus  1  in  FIG. 1 , the effect can be further enhanced by using the beam shaping element  14  to accentuate the high-frequency components of the spatial frequency of the PSF. In order to create a three-dimensional image of the PSF, the three-dimensional image may be acquired in accordance with a rotating process and an interpolation process from a PSF cross-sectional profile acquired within a plane orthogonal to the optical axis and a PSF cross-sectional profile acquired in the direction parallel to the optical axis. 
     Furthermore, in this embodiment, as shown in  FIG. 12 , a spatial light modulator (light modulating unit)  28  may be disposed in place of the beam shaping element  14 . The spatial light modulator  28  is used for correcting aberration of the focal point of a laser beam focused by the objective lens  17 . First, the PSF shape is measured in a state where a laser beam is not modulated by the spatial light modulator  28 . Thus, the state of the aberration can be ascertained from the acquired PSF shape. The aberration includes aberrations caused by both the optical system and the sample X. In the state where the aberration is ascertained and a single laser beam is used, the wave front of the laser beam may be modulated such that the aberration is corrected by the spatial light modulator  28 , and a fluorescence image of the sample X may be acquired. 
     Accordingly, the PSF is corrected to have a shape with no aberration based on the measured PSF shape, whereby the resolution can be enhanced. Thus, image processing becomes unnecessary, and a fluorescence image with high spatial resolution can be acquired. 
     When ascertaining the state of aberration, simulation may be used together with the obtained PSF shape, or a PSF shape at a different depth may be compared therewith. For example, with regard to aberration occurring within the sample X, PSF shapes are measured in a shallow region and a deep region of the sample X. By comparing the shapes, aberration occurring as a result of propagation to the deep region of the sample X can be ascertained, and the laser beam is modulated by the spatial light modulator  28  such that the aberration is corrected, whereby the resolution in the deep region can be improved. 
     Furthermore, there is an advantage in that the spatial resolution can be further enhanced by measuring the PSF shape and acquiring an image of the sample X in a state where aberration is corrected by the spatial light modulator  28  and performing deconvolution by using the acquired image of the sample X and the measured PSF shape. Moreover, by optically accentuating a high-frequency component by using the beam shaping element  14 , the resolution can be further improved. 
     Furthermore, as shown in  FIG. 11 , in a state where aberration is corrected by the spatial light modulator  28 , the PSF shape may be three-dimensionally measured by using the defocusing element  26 , thereby improving the spatial resolution three-dimensionally. 
     Furthermore, an image acquisition apparatus  34  having the structure shown in  FIG. 13  may be employed. 
     The image acquisition apparatus  34  includes a first non-polarization beam splitter  29  in place of the first polarization beam splitter  9 , acousto-optic elements  30  respectively disposed in two optical paths split by the beam splitter  29 , and a second non-polarization beam splitter  31  in place of the second polarization beam splitter  11 . 
     The image acquisition apparatus  34  also includes a detector  32  that detects a laser beam passing through the second polarization beam splitter  31 , and a lock-in amplifier  33  that demodulates a signal having a predetermined frequency from a fluorescence signal detected by the photodetector  24  based on a detection signal obtained by the detector  32 . 
     The two acousto-optic elements  30  slightly change the time frequencies of laser beams L 1  and L 2  traveling along the respective optical paths to different frequencies. 
     Normally, when laser beams having different frequencies overlap, a beat having a frequency corresponding to the difference between the frequencies occurs. 
     When scanning is to be performed while changing the relative position between the first laser beam L 1  and the second laser beam L 2  having different frequencies at the position of the sample X, a frequency component corresponding to the difference in frequency between the first laser beam L 1  and the second laser beam L 2  occurs at the overlapping position. Thus, by using the lock-in amplifier  33  to demodulate a signal having the frequency difference, the signal alone can be extracted from the overlapping position. 
     Specifically, a waveform from which an offset component is removed from the fluorescence intensity distribution shown in  FIG. 3  can be acquired at one time. Therefore, by performing a process similar to that described above, a fluorescence image having high spatial resolution can be acquired by measuring the PSF shape in the image acquisition apparatus  34  in  FIG. 13 . 
     Accordingly, the image acquisition apparatus  34  shown in  FIG. 13  can directly extract a fluorescence signal from the overlapping section between the first laser beam L 1  and the second laser beam L 2  without performing an offset subtracting process, and can also be applied to a one-photon-excitation microscope not involving a nonlinear optical process. Moreover, because this embodiment is characterized in that a signal is acquired from the overlapping position between the first laser beam L 1  and the second laser beam L 2 , the spatial light modulator  28  or the defocusing element  26  described above can also be used. In a case where the embodiment is to be applied to one-photon excitation, the process for raising the acquired image to the power of 2, which corresponds to step S 34  in  FIG. 7 , is not necessary. 
     In order to cause beating of the laser beams to occur, the two polarized light beams to be multiplexed need to overlap each other. Therefore, unlike other embodiments, non-polarization beam splitters  29  and  31  are used. Although this implies that the multiplexed laser beam is output in a direction other than the direction toward the beam shaping element  14 , since this laser beam also has a beat component, this signal is detected by the detector  32  and is set as a reference signal of the lock-in amplifier  33  so as to be used without waste. 
     Although several embodiments of the present invention have been described above, the present invention is not limited to these embodiments. 
     For example, although the first laser beam L 1  and the second laser beam L 2  are generated by using a laser beam from the same light source  2 , laser beams emitted from different light sources may alternatively be used. In that case, the two laser beams L 1  and L 2  may have different wavelengths, and the PSF shape to be acquired is an average shape of the PSF shapes of the two laser beams L 1  and L 2 . Moreover, as an alternative to the use of the two-axis galvanometer mirror  21  as the scanning unit  4 , a method of acquiring an image by stage scanning may be employed. 
     Although a two-photon-excitation microscope is described in the above embodiments, the present invention may be applied to a microscope of another type, such as an SHG microscope, so long as the microscope is of a scanning type that utilizes a nonlinear optical process. 
     From the above-described embodiments, the following aspects of the present disclosure are derived. 
     An aspect of the present disclosure provides a point-spread-function measurement device comprising: a scanning unit that scans two illumination light beams emitted from a light source; an illumination optical system that radiates the two illumination light beams scanned by the scanning unit onto a sample; a relative-position adjustment unit that changes a relative irradiation position, in the sample, between the two illumination light beams radiated by the illumination optical system; a detection optical system that detects signal light generated at an overlapping position, in the sample, of the illumination light beams radiated by the illumination optical system; and a calculating unit that calculates a point spread function based on the signal light detected by the detection optical system and the relative irradiation position between the two illumination light beams when the signal light is detected. 
     According to this aspect, when the two illumination light beams emitted from the light source are scanned by the scanning unit and are radiated onto the sample by the illumination optical system, signal light is generated at the irradiation position of each illumination light beam and is detected by the detection optical system. By repeatedly scanning and detecting the two illumination light beams while actuating the relative-position adjustment unit to vary the relative irradiation position between the two illumination light beams, the point spread function is calculated by the calculating unit from the relationship between the relative irradiation position and the intensity of the detected signal light. 
     In this case, the point spread function of the illumination light beams at a freely-chosen position of the sample can be determined without applying an additional process to the sample, such as disposing a fluorescence bead in the sample as in the related art. 
     In the above aspect, the calculating unit may calculate the point spread function by determining an autocorrelation waveform. 
     Accordingly, the point spread function can be accurately calculated from the autocorrelation waveform. 
     Specifically, by varying the relative irradiation position between the two illumination light beams, the overlapping of the two illumination light beams in the sample changes. When the two illumination light beams are in a completely non-overlapped state, signal light equivalent to the sum of signal light beams generated by the two independent illumination light beams is generated. When the two illumination light beams are in a completely overlapped state, signal light with the maximum intensity is generated. By using an intensity profile of the signal light according to the relative irradiation position, the point spread function can be accurately determined. 
     Furthermore, in the above aspect, the illumination optical system may set polarization states of the two illumination light beams such that the polarization states are orthogonal to each other. 
     Accordingly, the occurrence of an excessive signal light component caused by electric-field interference between the illumination light beams when the two illumination light beams overlap can be suppressed, so that the point spread function can be accurately determined. 
     Furthermore, in the above aspect, the signal light may be generated in a nonlinear optical process in accordance with irradiation of the illumination light beams. 
     Accordingly, even when the sample is to be observed by utilizing a linear optical process, the spatial resolution can be improved in accordance with the point spread function. 
     Furthermore, in the above aspect, the illumination light beams may be ultra-short pulsed laser beams, and the signal light may be fluorescence generated in accordance with a multiphoton absorption effect. 
     Accordingly, by utilizing the multiphoton absorption effect occurring as a result of radiating an ultra-short pulsed layer beam onto the sample, the spatial resolution can be improved in accordance with the point spread function when fluorescence observation is performed. 
     Furthermore, in the above aspect, at least one signal light image having at least a one-dimensional size may be acquired by using the signal light detected by the detection optical system, and the calculating unit may calculate the autocorrelation waveform by using the signal light image. 
     Accordingly, the point spread function can be determined at any position of any kind of sample so long as signal light can be detected. 
     Furthermore, in the above aspect, the calculating unit may calculate the point spread function by performing Fourier transformation of the autocorrelation waveform. 
     Accordingly, the point spread function can be accurately determined from the autocorrelation waveform. 
     Furthermore, in the above aspect, the illumination light beams may be pulsed light beams, the illumination optical system may include a timing adjustment unit that switches the timing for radiating the two illumination light beams onto the sample between a simultaneous mode and a non-simultaneous mode, and the calculating unit may calculate the point spread function by using a difference between signal light detected in the simultaneous mode switched by the timing adjustment unit and signal light detected in the non-simultaneous mode switched by the timing adjustment unit. 
     Accordingly, when the timing adjustment unit switches the timing for radiating the two illumination light beams onto the sample to the simultaneous mode, overlapping of the two illumination light beams occurs within the sample in accordance with the relative irradiation position, and signal light that changes in intensity in accordance with the degree of overlapping can be detected. When the irradiation timing is switched to the non-simultaneous mode, overlapping of the two illumination light beams does not occur, and an offset component of signal light that does not change in response to a change in the relative irradiation position can be detected. Therefore, by calculating the difference, signal-light intensity distribution from which the offset component is removed can be calculated, and the point spread function can be accurately calculated by using this signal-light intensity distribution. 
     Furthermore, in the above aspect, the illumination optical system may include a light modulating unit that modulates a spatial distribution or polarization states of the illumination light beams so that a high-frequency component is accentuated in a spatial frequency distribution of the point spread function. 
     Accordingly, the high-frequency component can be optically accentuated when signal light is generated in the sample, and the spatial resolution can be enhanced. 
     Another aspect of the present disclosure provides a point-spread-function measurement method comprising: a scanning step for scanning two illumination light beams emitted from a light source over a sample while changing a relative irradiation position in the sample; a detecting step for detecting signal light generated at an overlapping position, in the sample, of the illumination light beams radiated in the scanning step; and a calculating step for calculating a point spread function based on the signal light detected in the detecting step and the relative irradiation position between the two illumination light beams when the signal light is detected. 
     In the above aspect, the calculating step may include calculating the point spread function by determining an autocorrelation waveform. 
     Furthermore, in the above aspect, the scanning step may include setting polarization states of the two illumination light beams such that the polarization states are orthogonal to each other and performing the scanning. 
     Furthermore, in the above aspect, the signal light may be generated in a nonlinear optical process in accordance with irradiation of the illumination light beams. 
     Furthermore, in the above aspect, the illumination light beams are ultra-short pulsed laser beams, and the signal light is fluorescence generated in accordance with a multiphoton absorption effect. 
     Furthermore, in the above aspect, the detecting step may include acquiring at least one signal light image having at least a one-dimensional size by using the detected signal light, and the calculating step may include calculating the autocorrelation waveform by using the signal light image. 
     Furthermore, in the above aspect, the calculating step may include calculating the point spread function by performing Fourier transformation of the autocorrelation waveform. 
     Furthermore, in the above aspect, the illumination light beams may be pulsed light beams, the scanning step may include performing the scanning by switching the timing for radiating the two illumination light beams onto the sample between a simultaneous mode and a non-simultaneous mode, and the calculating step may include calculating the point spread function by using a difference between signal light detected in the simultaneous mode switched in the scanning step and signal light detected in the non-simultaneous mode switched in the scanning step. 
     Furthermore, in the above aspect, the scanning step may include modulating a spatial distribution or polarization states of the illumination light beams so that a high-frequency component is accentuated in a spatial frequency distribution of the point spread function. 
     Another aspect of the present disclosure provides an image acquisition apparatus comprising: any one of the above-described point-spread-function measurement devices; and an image processor that generates an image of the sample by using the point spread function measured by the measurement device. 
     According to this aspect, the image processor generates an image of the sample by using the point spread function measured by the measurement device, so that blurriness in the sample image can be reduced. 
     In the above aspect, the image processor may generate a sample image acquired as a result of the illumination optical system radiating the illumination light beams emitted from the light source and scanned by the scanning unit onto the sample and the detection optical system detecting the signal light generated at the irradiation positions in the sample, and may reconfigure the generated sample image by using the point spread function measured by the measurement device. 
     Accordingly, by reconfiguring the sample image by using the point spread function, blurriness can be effectively reduced. 
     Furthermore, in the above aspect, the image processor may reconfigure the sample image by performing deconvolution on the sample image by using the point spread function. 
     Accordingly, by performing deconvolution, blurriness in the sample image can be effectively reduced. 
     Another aspect of the present disclosure provides image acquisition method comprising: any one of the above-described measurement methods; and an image processing step for generating a sample image by using a point spread function measured in accordance with the measurement method. 
     In the above aspect, the image processing step may include an image generating step for generating the sample image acquired as a result of scanning the illumination light beams from the light source onto the sample and detecting the signal light generated at the irradiation positions in the sample, and a reconfiguring step for reconfiguring the sample image generated in the image generating step by using the point spread function measured in accordance with the measurement method. 
     Furthermore, in the above aspect, the image processing step may include reconfiguring the sample image by performing deconvolution on the sample image by using the point spread function. 
     According to the aforementioned aspects, an advantageous effect is afforded in that a point spread function can be acquired without applying an additional process to a sample. 
     REFERENCE SIGNS LIST 
     
         
           1 ,  27 ,  34  image acquisition apparatus 
           2  light source 
           3  illumination optical system 
           4  scanning unit 
           5  detection optical system 
           10  optical-path-length adjustment optical system (timing adjustment unit) 
           14  beam shaping element (light modulating unit) 
           19  motorized holder (relative-position adjustment unit) 
           25  measurement device 
           28  spatial light modulator (light modulating unit) 
         S 3  image generating step, image processing step 
         S 4  reconfiguring step, image processing step 
         S 23 , S 27  scanning step, detecting step 
         S 30  calculating step 
         L 1 , L 2  laser beam (illumination light beam) 
         X sample