Patent Publication Number: US-2023152206-A1

Title: Observation device and observation method

Description:
TECHNICAL FIELD 
     The present disclosure relates to an observation apparatus and an observation method. 
     BACKGROUND ART 
     It is difficult to observe a transparent observation object such as, for example, a cell with a normal microscope, and therefore, an observation apparatus capable of acquiring a phase image of the observation object such as a quantitative phase microscope is used in general. Several implementation methods of the above observation apparatus are known. 
     One of conventional arts (hereinafter referred to as “conventional art 1”) uses a Michelson interferometer or a Mach-Zehnder interferometer in which splitting and combining of two light beams are performed, and places an observation object on an optical path of one of the two light beams. Further, in the conventional art 1, an optical path difference (phase difference) between the two light beams is sequentially set to each value by moving a mirror or the like on the optical path, a plurality of interference images of the observation object are acquired, and a phase image is generated based on the plurality of interference images. 
     An observation apparatus described in Patent Document 1 (hereinafter referred to as “conventional art 2”) is an application of a differential interference contrast microscope. In the conventional art 2, linearly polarized light output from a light source and passed through a first polarizer is split by a first prism into two linearly polarized light beams having oscillation planes orthogonal to each other and slightly shifted by an optical path, and the two linearly polarized light beams passed through an observation object are combined by a second prism and passed through a second polarizer to interfere with each other, thereby acquiring an interference image. A phase difference between the two light beams is sequentially set to each value by a spatial light modulator interposed between the second prism and the second polarizer to acquire a plurality of interference images. Further, a phase differential image is generated based on the plurality of interference images, and a phase image is generated based on the phase differential image. 
     An observation apparatus described in Non Patent Document 1 (hereinafter referred to as “conventional art 3”) is also an application of the differential interference contrast microscope. In the conventional art 3, a polarization camera is attached to a camera port of a commercially available differential interference contrast microscope, and a plurality of interference images are simultaneously acquired by the polarization camera. Further, a phase differential image is generated based on the plurality of interference images, and a phase image is generated based on the phase differential image. 
     Further, in the above observation apparatuses, an amplitude image of the observation object can be acquired in addition to the phase image, and further, a complex amplitude image can be acquired. 
     CITATION LIST 
     Patent Literature 
     
         
         Patent Document 1: US Patent Publication No. 10132609 
       
    
     Non Patent Literature 
     
         
         Non Patent Document 1: S. Shibata et al., “Video-rate quantitative phase analysis by a DIC microscope using a polarization camera”, Biomed. Opt. Express Vol. 10, pp. 1273-1281, 2019 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     The conventional art 1 can obtain a phase image excellent in quantitativeness. However, since the conventional art 1 uses the Michelson interferometer or the Mach-Zehnder interferometer in which splitting and combining of the two light beams are performed, adjustment of the optical system is not easy and vibration resistance is poor. Further, since the conventional art 1 acquires the plurality of interference images by sequentially setting the optical path difference (phase difference) between the two light beams to each value, it takes time for acquiring the plurality of interference images. 
     The conventional art 2 may solve the problems of optical system adjustment and vibration resistance. However, in the conventional art 2, since the plurality of interference images are acquired by sequentially setting the optical path difference (phase difference) between the two light beams to each value, it takes time for acquiring the plurality of interference images. 
     The conventional art 3 may solve the problems of optical system adjustment and vibration resistance, and may also solve the problem of time for acquiring the interference images. However, in the conventional art 3, an accurate phase differential image cannot be obtained, and therefore, the phase image is generated by performing necessary processing based on the phase differential image under an approximation condition of an assumption that a phase change is small. Therefore, the obtained phase image is poor in quantitativeness. 
     An object of an embodiment is to provide an observation apparatus and an observation method capable of easily adjusting an optical system and obtaining a complex amplitude image with improved quantitativeness in a short time. 
     Solution to Problem 
     An embodiment is an observation apparatus. The observation apparatus includes (1) a light source for outputting spatially incoherent light; (2) an irradiation optical system for focusing light output from the light source and irradiating an observation object with the light; (3) a polarizer provided on an optical path of the irradiation optical system, and for inputting the light output from the light source and outputting linearly polarized light; (4) a first prism provided on the optical path of the irradiation optical system and between the polarizer and the observation object, and for inputting light output from the polarizer and outputting two linearly polarized light beams orthogonal to each other; (5) an imaging optical system for forming an image by inputting light generated in the observation object in response to irradiation of the observation object with the light by the irradiation optical system; (6) a second prism provided on an optical path of the imaging optical system, and for combining two light beams output from the observation object and outputting light; (7) a polarization conversion element provided on the optical path of the imaging optical system and at a subsequent stage of the second prism, and for inputting the light output from the second prism and outputting two circularly polarized light beams having different rotation directions; (8) a polarization camera having an imaging plane disposed at a position where the image is formed by the imaging optical system, and for inputting the two circularly polarized light beams having different rotation directions output from the polarization conversion element and acquiring an interference image on the imaging plane for each of three or more polarization components; and (9) an analysis unit for generating a complex amplitude image of the observation object based on the interference image for each of the three or more polarization components acquired by the polarization camera. 
     An embodiment is an optical module. The optical module is an optical module being detachably attached to a camera port of a differential interference contrast microscope, and includes (1) a relay optical system constituting a part of an imaging optical system for forming an image by inputting light generated in an observation object placed in the differential interference contrast microscope, and for inputting light output from a second prism of the differential interference contrast microscope through the camera port; (2) a polarization conversion element provided on an optical path of the relay optical system, and for inputting the light and outputting two circularly polarized light beams having different rotation directions; and (3) a polarization camera having an imaging plane disposed at a position where the image is formed by the relay optical system, and for inputting the two circularly polarized light beams having different rotation directions output from the polarization conversion element and acquiring an interference image on the imaging plane for each of three or more polarization components. 
     An embodiment is an observation method. The observation method includes (1) outputting spatially incoherent light from a light source; (2) by a polarizer provided on an optical path of an irradiation optical system for focusing light output from the light source and irradiating an observation object with the light, inputting the light output from the light source and outputting linearly polarized light; (3) by a first prism provided on the optical path of the irradiation optical system and between the polarizer and the observation object, inputting light output from the polarizer and outputting two linearly polarized light beams orthogonal to each other; (4) by a second prism provided on an optical path of an imaging optical system for forming an image by inputting light generated in the observation object in response to irradiation of the observation object with the light by the irradiation optical system, combining two light beams output from the observation object and outputting light; (5) by a polarization conversion element provided on the optical path of the imaging optical system and at a subsequent stage of the second prism, inputting the light output from the second prism, converting the light into two circularly polarized light beams having different rotation directions and outputting the light beams; (6) by a polarization camera having an imaging plane disposed at a position where the image is formed by the imaging optical system, inputting the two circularly polarized light beams having different rotation directions output from the polarization conversion element and acquiring an interference image on the imaging plane for each of three or more polarization components; and (7) generating a complex amplitude image of the observation object based on the interference image for each of the three or more polarization components acquired by the polarization camera. 
     Advantageous Effects of Invention 
     According to the observation apparatus and the observation method of the embodiments, an optical system can be easily adjusted, and a complex amplitude image can be obtained with improved quantitativeness in a short time. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    is a diagram illustrating a configuration of an observation apparatus  1 A of a first embodiment. 
         FIG.  2    is a diagram illustrating a configuration example of an image sensor of a polarization camera  26 . 
         FIG.  3    is a diagram illustrating another configuration example of the image sensor of the polarization camera  26 . 
         FIG.  4    is a diagram illustrating a configuration of an observation apparatus  1 B of a second embodiment. 
         FIG.  5    is a diagram illustrating a configuration of an observation apparatus  1 C of a third embodiment. 
         FIG.  6    is a diagram illustrating a configuration of an observation apparatus  1 D of a fourth embodiment. 
         FIG.  7    is a diagram illustrating a configuration of an observation apparatus  1 E of a fifth embodiment. 
         FIG.  8    is a diagram illustrating a configuration of an observation apparatus  1 F of a sixth embodiment. 
         FIG.  9    is a diagram for describing reduction of polarization conversion using two mirrors M 1  and M 2 . 
         FIG.  10    is a diagram illustrating a configuration of an observation apparatus  1 G of a seventh embodiment. 
         FIG.  11    is a phase differential image obtained in an example. 
         FIG.  12    is a phase image generated from the phase differential image ( FIG.  11   ). 
         FIG.  13    is a three-dimensional refractive index distribution image generated from the phase image ( FIG.  12   ) acquired at each position in a depth direction. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, embodiments of an observation apparatus and an observation method will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference signs, and redundant description will be omitted. The present invention is not limited to these examples. 
       FIG.  1    is a diagram illustrating a configuration of an observation apparatus  1 A of a first embodiment. The observation apparatus  1 A includes a light source  11 , a lens  12 , a polarizer  13 , a first prism  14 , a condenser lens  15 , an objective lens  21 , a second prism  22 , a ¼ wave plate  23 , a lens  24 , a polarization camera  26 , and an analysis unit  40 . In the observation apparatus  1 A, the light source  11 , the lens  12 , the polarizer  13 , the first prism  14 , the condenser lens  15 , the objective lens  21 , the second prism  22 , the ¼ wave plate  23 , the lens  24 , and the polarization camera  26  are optically coupled. 
     The lens  12 , the polarizer  13 , the first prism  14 , and the condenser lens  15  are sequentially provided on an optical path of an irradiation optical system for focusing light output from the light source  11  and irradiating an observation object S with the light. Further, the objective lens  21 , the second prism  22 , the ¼ wave plate  23 , and the lens  24  are sequentially provided on an optical path of an imaging optical system for forming an image on an imaging plane of the polarization camera  26  by inputting light generated in the observation object S in response to irradiation of the observation object S with the light by the irradiation optical system. 
     The light source  11  outputs spatially incoherent light. The light output from the light source  11  may be temporally coherent or temporally incoherent. The light output from the light source  11  may be linearly polarized light or unpolarized light. The light source  11  includes, for example, a halogen lamp, a light emitting diode, or a laser diode, and further, preferably includes a diffuser plate at a subsequent stage thereof. 
     The lens  12  is optically coupled to the light source  11 . The lens  12  collimates the light output from the light source  11 , and outputs the light to the polarizer  13 . 
     The polarizer  13  is optically coupled to the lens  12 . The polarizer  13  inputs the light collimated and output by the lens  12 , and outputs linearly polarized light having a polarization plane (oscillation plane) according to an orientation of an optic axis of the polarizer  13  to the first prism  14 . 
     The first prism  14  is optically coupled to the polarizer  13 . The first prism  14  inputs the linearly polarized light output from the polarizer  13 , and outputs two linearly polarized light beams orthogonal to each other in different directions. A polarization plane of each of the two linearly polarized light beams output from the first prism  14  and orthogonal to each other is inclined by 45° with respect to the polarization plane of the linearly polarized light input from the polarizer  13  to the first prism  14 . 
     The condenser lens  15  is optically coupled to the first prism  14 . The condenser lens  15  focuses the two linearly polarized light beams orthogonal to each other output from the first prism  14  and irradiates the observation object S with the light beams. The two linearly polarized light beams orthogonal to each other output from the first prism  14  travel in different directions after being output from the first prism  14 , and thus, focusing positions on the observation object S by the condenser lens  15  are slightly different. 
     The objective lens  21  inputs the two linearly polarized light beams orthogonal to each other and output from the observation object S in response to irradiation of the observation object S with the light by the irradiation optical system, collimates the light beams, and outputs the light beams to the second prism  22 . 
     The second prism  22  is optically coupled to the objective lens  21 . The second prism  22  inputs the two linearly polarized light beams orthogonal to each other and collimated and output by the objective lens  21 , combines the light beams in the optical paths shifted by the first prism  14 , and outputs the light to the ¼ wave plate  23 . 
     The ¼ wave plate  23  is a polarization conversion element being optically coupled to the second prism  22 . The ¼ wave plate  23  inputs the light output from the second prism  22 , and outputs two circularly polarized light beams having different rotation directions. When the light output from the light source  11  is temporally incoherent, the ¼ wave plate  23  is preferably an achromatic wave plate or the like having small wavelength dependency. 
     The lens  24  is optically coupled to the ¼ wave plate  23 . The lens  24  inputs the two circularly polarized light beams having different rotation directions output from the ¼ wave plate  23 , and forms an image on the imaging plane of the polarization camera  26 . 
     The polarization camera  26  is optically coupled to the lens  24 . The polarization camera  26  has the imaging plane disposed at a position where the image is formed by the imaging optical system. The polarization camera  26  inputs the two light beams being circularly polarized in different rotation directions by the ¼ wave plate  23 , and acquires an interference image on the imaging plane for each of three or more polarization components. In addition, circular polarization in the present invention is not limited to the case where a phase difference is ±π/2 [rad] and amplitudes are equal between electric field vectors in two directions orthogonal to each other, and includes elliptical polarization. 
     The analysis unit  40  generates a complex amplitude image (an amplitude image and a phase image) of the observation object S based on the interference images respectively for the three or more polarization components acquired by the polarization camera  26 . The analysis unit  40  is, for example, a computer. 
     The analysis unit  40  includes an operation unit including a CPU for performing operational processing such as generation of the complex amplitude image, a storage unit including a hard disk drive, a RAM, a ROM, and the like for storing the interference image, the complex amplitude image, and the like, a display unit including a liquid crystal display for displaying the interference image, the complex amplitude image, and the like, and an input unit including a keyboard, a mouse, and the like for receiving input of various conditions at the time of acquisition of the interference image and display of the image. The analysis unit  40  may be constituted by a smart device such as a tablet terminal including a touch panel and the like as the input unit. Further, the operation unit and the storage unit of the analysis unit  40  may be constituted by a field-programmable gate array (FPGA) or a microcomputer. 
     The configuration from the light source  11  to the polarization camera  26  corresponds to a configuration in which an analyzer provided at a subsequent stage of the second prism  22  is removed in a configuration of a normal differential interference contrast microscope and the ¼ wave plate  23  and the polarization camera  26  are provided. Each of the first prism  14  and the second prism  22  may be a Wollaston prism or a Nomarski prism used in a normal differential interference contrast microscope. 
       FIG.  2    is a diagram illustrating a configuration example of an image sensor of the polarization camera  26 . An image sensor  26 A illustrated in this diagram has a configuration in which a plurality of pixels are arranged two-dimensionally in the imaging plane, and can acquire a two-dimensional interference image. 
     The image sensor  26 A includes a photodiode array  261  formed on a semiconductor substrate, a polarizer array  262  provided on the photodiode array, and a lens array  263  provided on the polarizer array, and has a configuration in which these are stacked. In the photodiode array  261 , a plurality of photodiodes are arranged two-dimensionally. One polarizer in the polarizer array  262  is provided corresponding to each photodiode in the photodiode array  261 , and one lens in the lens array  263  is provided corresponding thereto. 
     Each polarizer of the polarizer array  262  has an optic axis in any orientation of four orientations (0°, 45°, 90°, 135°). In this diagram, an orientation of hatching in each polarizer of the polarizer array  262  indicates the orientation of the optic axis of the polarizer. 
     By using the above image sensor  26 A, two-dimensional images for linear polarizations of four orientations can be simultaneously acquired. An image sensor (Polarsens (registered trademark)) commercialized by Sony Corporation has the configuration illustrated in this diagram. Further, an image sensor (Area Scan Polarization Sensor) commercialized by Teledyne DALSA also has the configuration illustrated in this diagram. 
       FIG.  3    is a diagram illustrating another configuration example of the image sensor of the polarization camera  26 . An image sensor  26 B illustrated in this diagram has a configuration in which a plurality of pixels are arranged one-dimensionally in the imaging plane, and can acquire a one-dimensional interference image. 
     The image sensor  26 B includes photodiode arrays  264   a  to  264   d  formed on a semiconductor substrate, and polarizers  265   a  to  265   c  provided on the photodiode arrays  264   a  to  264   c . A polarizer is not provided on the photodiode array  264   d . In each of the photodiode arrays  264   a  to  264   d , a plurality of photodiodes are arranged one-dimensionally in an x direction. The photodiode arrays  264   a  to  264   d  are arranged in parallel in a y direction. 
     An orientation of an optic axis of the polarizer  265   a  on the photodiode array  264   a  is 0°, an orientation of an optic axis of the polarizer  265   b  on the photodiode array  264   b  is 135°, and an orientation of an optic axis of the polarizer  265   c  on the photodiode array  264   c  is 90°. In this diagram, an orientation of hatching in each polarizer indicates the orientation of the optic axis of the polarizer. 
     By using the above image sensor  26 B, one-dimensional images for linear polarizations of three orientations can be simultaneously acquired. Further, by relatively moving the observation object S in a direction (for example, the y direction) different from the x direction, it is possible to simultaneously acquire two-dimensional images for the linear polarizations of the three orientations. For example, the observation object S may be a cell which moves together with a fluid in a transparent tube as in flow cytometry. An image sensor (Line Scan Polarization Sensor) commercialized by Teledyne DALSA has the configuration illustrated in this diagram. 
     Next, a method of acquiring the complex amplitude image of the observation object S using the observation apparatus  1 A will be described. 
     Spatially incoherent light output from the light source  11  is collimated by the lens  12 , made into linearly polarized light having a polarization plane according to the orientation of the optic axis of the polarizer  13 , and input to the first prism  14 . When the linearly polarized light is input from the polarizer  13  to the first prism  14 , two linearly polarized light beams orthogonal to each other are output from the first prism  14  in different directions, and are focused on and applied to the observation object S by the condenser lens  15 . 
     Two linearly polarized light beams orthogonal to each other output from the observation object S in response to irradiation of the observation object S with the light are collimated by the objective lens  21  and input to the second prism  22 . The second prism  22  combines the optical paths shifted by the first prism  14 , and the ¼ wave plate  23  converts the light into two circularly polarized light beams having different rotation directions. The two circularly polarized light beams having different rotation directions output from the ¼ wave plate  23  form an image on the imaging plane of the polarization camera  26  by the lens  24 . 
     When the two light beams being circularly polarized in different rotation directions by the ¼ wave plate  23  are input, the polarization camera  26  acquires an interference image on the imaging plane for each of three or more polarization components (that is, three or more interference images having different phase differences). Further, the analysis unit  40  generates a complex amplitude image of the observation object S based on the three or more interference images acquired by the polarization camera  26 . 
     Next, capability of acquiring the complex amplitude image of the observation object S by the observation apparatus  1 A or the observation method of the present embodiment will be described in detail. In the following description, for ease of explanation, it is assumed that the light source  11  outputs spatially coherent light. Further, it is assumed that the polarization camera  26  can acquire four interference images whose phase differences are different by 90° as illustrated in  FIG.  2   . 
     In respective polarization states of light, a vector representing a horizontal polarization is set to e H , a vector representing a vertical polarization is set to e V , a vector representing a 45° polarization is set to e D , a vector representing a  1350  polarization is set to e A , a vector representing a right-handed circular polarization is set to e R , and a vector representing a left-handed circular polarization is set to e L . There are relationships of the following Formulas (1) to (4) between the above vectors. i is an imaginary unit. 
     
       
         
           
             
               
                 
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     When circularly polarized light is input to a polarizer, a phase of light output from the polarizer is represented by an inner product of the vector of the polarization state of the output light (any of e H , e V , e D , and e A ) and the vector of the polarization state of the input light (any of e R  and e L ). For the inner products between the two vectors, there are relationships of the following Formulas (5) to (12). By using these relationships, an interference image corresponding to each phase shift amount can be acquired. 
     
       
         
           
             
               
                 
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     Since e H ·e R  and e H ·e L  have the same phase, when two circularly polarized light beams having different rotation directions are projected onto the horizontal polarization by the polarizer, two light beams output from the polarizer have the same phase. Since e V ·e R  and e V ·e L  have opposite phases, when two circularly polarized light beams having different rotation directions are projected onto the vertical polarization by the polarizer, a phase difference between two light beams output from the polarizer is 180°. 
     Since phases of e D ·e R  and e D ·e L  are different by 90°, when two circularly polarized light beams having different rotation directions are projected onto the 45° polarization by the polarizer, a phase difference between two light beams output from the polarizer is 90°. Since phases of e A ·e R  and e A ·e L  are different by 270°, when two circularly polarized light beams having different rotation directions are projected onto the 135° polarization by the polarizer, a phase difference between two light beams output from the polarizer is 270°. 
     When an electric field of light to be measured is set to U(r), a wavefront of light input to the polarization camera  26  is represented by the following Formula (13). r is a variable indicating a position. The variable r in a first term of this Formula and the variable r+δr in a second term are due to the fact that focusing regions of two linearly polarized light beams orthogonal to each other are slightly different when focused irradiation is performed on the observation object S by the condenser lens  15 . 
       [Formula 13] 
         U ( r )· e   R   +U ( r+δr )· e   L   (13)
 
     From the above Formulas, an interference image I θn  acquired by the polarization camera  26  is represented by the following Formulas (14) and (15). θ n  indicates an orientation of an optic axis of four types of polarizers of the polarization camera  26 , and is a value of 0°, 45°, 90°, or 135°. ϕ n  indicates a phase difference after two circularly polarized light beams having different rotation directions pass through the polarizer, and is a value of 0, π/2, π, or 3π/2. There is a one-to-one correspondence between θ n  and ϕ 2 . From Formula (14), the following Formula (16) is obtained. 
     
       
         
           
             
               
                 
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     An absolute value of Formula (16) represents an amplitude image. A phase of Formula (16) represents a phase differential image obtained by spatially phase-differentiating a wavefront. For reconstructing a phase image from the phase differential image, basically, integration may be performed in a differential direction. A complex amplitude image can be obtained from the amplitude image and the phase image. 
     As described above, in the present embodiment, the two light beams propagate coaxially, and thus, adjustment of an optical system is easy, and a mechanism for stabilization of an optical path length and the like is not necessary, and vibration resistance is excellent. In the present embodiment, since the interference images of the observation object in which the phase difference between the two light beams is set to respective values can be acquired simultaneously (in a single shot), a time required for acquiring the plurality of interference images is short, and thus, it is superior to the conventional art 2. Further, compared to the conventional art 3, quantitativeness of the phase image obtained in the present embodiment is excellent. 
     Next, configurations of other embodiments will be described. In addition, the same elements as those in the configuration of the observation apparatus  1 A illustrated in  FIG.  1    are denoted by the same reference signs, and redundant description will be omitted. These have the same effects as the observation apparatus  1 A of the first embodiment illustrated in  FIG.  1   . 
       FIG.  4    is a diagram illustrating a configuration of an observation apparatus  1 B of a second embodiment. As compared with the observation apparatus  1 A ( FIG.  1   ) of the first embodiment, the observation apparatus  1 B ( FIG.  4   ) of the second embodiment is different in the position where the ¼ wave plate  23  is provided. In the present embodiment, the ¼ wave plate  23  is provided between the lens  24  and the polarization camera  26 . The ¼ wave plate  23  may be provided at any position between the second prism  22  and the polarization camera  26 , however, for reducing influence of aberration, the configuration of the observation apparatus  1 A of the first embodiment in which the ¼ wave plate is provided at a position where the light is collimated is more preferable. 
       FIG.  5    is a diagram illustrating a configuration of an observation apparatus  1 C of a third embodiment. As compared with the observation apparatus  1 A ( FIG.  1   ) of the first embodiment, the observation apparatus  1 C ( FIG.  5   ) of the third embodiment is different in that a wave plate  25  is provided between the second prism  22  and the ¼ wave plate  23 . 
       FIG.  6    is a diagram illustrating a configuration of an observation apparatus  1 D of a fourth embodiment. As compared with the observation apparatus  1 B ( FIG.  4   ) of the second embodiment, the observation apparatus  1 D ( FIG.  6   ) of the fourth embodiment is different in that the wave plate  25  is provided between the second prism  22  and the ¼ wave plate  23 . 
     The wave plate  25  provided in each of the observation apparatus  1 C of the third embodiment ( FIG.  5   ) and the observation apparatus  1 D of the fourth embodiment ( FIG.  6   ) is a polarization conversion compensation optical element for reducing polarization conversion occurring in optical elements other than the second prism  22  and the ¼ wave plate  23  in the imaging optical system. The wave plate  25  as the polarization conversion compensation optical element is, for example, a configuration in which a ¼ wave plate and a ½ wave plate are combined, a Babinet-Soleil compensator, a Berek compensator, or the like. By providing the polarization conversion compensation optical element, a more accurate complex amplitude image can be obtained. 
       FIG.  7    is a diagram illustrating a configuration of an observation apparatus  1 E of a fifth embodiment. As compared with the observation apparatus  1 A ( FIG.  1   ) of the first embodiment, the observation apparatus  1 E ( FIG.  7   ) of the fifth embodiment is different in the configuration of the imaging optical system from the observation object S to a polarization camera  35 . In the present embodiment, the objective lens  21 , the second prism  22 , the lens  24 , a mirror  27 , a lens  31 , a wave plate (polarization conversion compensation optical element)  32 , a ¼ wave plate (polarization conversion element)  33 , a lens  34 , and the polarization camera  35  are provided in this order on an optical path of the imaging optical system. 
     The objective lens  21  inputs the two linearly polarized light beams orthogonal to each other and output from the observation object S in response to irradiation of the observation object S with the light by the irradiation optical system, collimates the light beams, and outputs the light beams to the second prism  22 . 
     The second prism  22  is optically coupled to the objective lens  21 . The second prism  22  inputs the two linearly polarized light beams orthogonal to each other and collimated and output by the objective lens  21 , combines the light beams in the optical paths shifted by the first prism  14 , and outputs the light to the lens  24 . 
     The lens  24  is coupled to the second prism  22  and an optical system. The lens  24  inputs the light output from the second prism  22 , converges the light, and outputs the light to the mirror  27 . 
     The mirror  27  is optically coupled to the lens  24 . The mirror  27  reflects the light output from the lens  24  to the lens  31 . 
     The lens  31  is optically coupled to the mirror  27 . The lens  31  inputs the light output from the lens  24  and reflected by the mirror  27 , collimates the light, and outputs the light to the wave plate  32 . There is an image plane between the lens  24  and the lens  31 . 
     The wave plate  32  is optically coupled to the lens  31 . The wave plate  32  is the polarization conversion compensation optical element for reducing polarization conversion occurring in optical elements other than the second prism  22  and the ¼ wave plate  33  in the imaging optical system. 
     The ¼ wave plate  33  is optically coupled to the wave plate  32 , and the ¼ wave plate  33  inputs the light output from the wave plate  32  and outputs the two circularly polarized light beams having different rotation directions. 
     The lens  34  is optically coupled to the ¼ wave plate  33 . The lens  34  inputs the two circularly polarized light beams having different rotation directions output from the ¼ wave plate  33 , and forms an image on the imaging plane of the polarization camera  35 . The lens  31  and the lens  34  constitute a relay optical system. 
     The polarization camera  35  is optically coupled to the lens  34 . The polarization camera  35  has the imaging plane disposed at a position where the image is formed by the imaging optical system. The polarization camera  35  inputs the two light beams being circularly polarized in different rotation directions by the ¼ wave plate  33 , and acquires the interference image on the imaging plane for each of the three or more polarization components. The polarization camera  35  may have the same configuration as the configuration of the polarization camera  26  described with reference to  FIG.  2    and  FIG.  3   . 
     In the configuration of the present embodiment, a commercially available differential interference contrast microscope can be used for the configuration from the light source  11  to the mirror  27 . Further, the lens  31 , the wave plate  32 , the ¼ wave plate  33 , the lens  34 , and the polarization camera  35  can be modularized into an optical module  30 E being detachably attached to a camera port of the differential interference contrast microscope. By attaching the optical module  30 E to the camera port of the commercially available differential interference contrast microscope, the observation apparatus  1 E of the present embodiment can be configured at low cost. 
       FIG.  8    is a diagram illustrating a configuration of an observation apparatus  1 F of a sixth embodiment. As compared with the observation apparatus  1 E ( FIG.  7   ) of the fifth embodiment, the observation apparatus  1 F ( FIG.  8   ) of the sixth embodiment is different in that an optical module  30 F is provided instead of the optical module  30 E. As compared with the optical module  30 E in the fifth embodiment, the optical module  30 F in the sixth embodiment is different in that the wave plate (polarization conversion compensation optical element)  32  is not provided and in that mirrors  36  to  38  are provided. 
     The mirror  36  and the mirror  37  are provided between the lens  31  and the ¼ wave plate  33 . The mirror  36  reflects the light arriving from the lens  31  to the mirror  37 . The mirror  37  reflects the light arriving from the mirror  36  to the ¼ wave plate  33 . The mirror  38  is provided between the lens  34  and the polarization camera  35 . The mirror  38  reflects the light arriving from the lens  34  to the imaging plane of the polarization camera  35 . 
     The mirrors  36  to  38  may be provided simply for reflecting the light, and further, may be preferably provided as the polarization conversion compensation optical element for reducing polarization conversion occurring in optical elements other than the second prism  22  and the ¼ wave plate  33  in the imaging optical system. 
     A dielectric multilayer mirror, which is often used, generates polarization conversion when reflecting the light. However, as illustrated in  FIG.  9   , two dielectric multilayer mirrors M 1  and M 2  having a property of compensating for the polarization conversion (for example, the same reflection property) are used and incident angles of the light to the mirrors M 1  and M 2  are equalized, so that a p-polarized component of the light incident on the mirror M 1  becomes s-polarized light when incident on the mirror M 2 , and a s-polarized component of the light incident on the mirror M 1  becomes p-polarized light when incident on the mirror M 2 . 
     As a result, polarization conversion occurring when each of the two mirrors M 1  and M 2  reflects the light is canceled out, and polarization conversion can be reduced as a whole. In the present embodiment, when the four mirrors  27 ,  36  to  38  have the above relationship, polarization conversion can be reduced as a whole. 
       FIG.  10    is a diagram illustrating a configuration of an observation apparatus  1 G of a seventh embodiment. As compared with the observation apparatus  1 F ( FIG.  8   ) of the sixth embodiment, the observation apparatus  1 G ( FIG.  10   ) of the seventh embodiment is different in that a moving unit  28  is further provided. The polarization camera  35  acquires the interference image at each position of movement by the moving unit  28 . The analysis unit  40  generates a three-dimensional complex amplitude image of the observation object S based on the interference images acquired by the polarization camera  35  at the respective positions. 
     The moving unit  28  moves an optical element included in the imaging optical system or the observation object in a direction parallel to the optical axis of the imaging optical system, and preferably moves the objective lens  21  in the optical axis direction. The moving unit  28  may be a linear stage, a piezoelectric actuator, or a combination of a linear stage and a piezoelectric actuator. 
     The position conjugate to the imaging plane of the polarization camera  35  is scanned in the depth direction in the observation object S by the movement of the objective lens  21  by the moving unit  28 , and thus, a three-dimensional phase differential image, a phase image, an amplitude image, and a complex amplitude image of the observation object S can be generated. In addition, a three-dimensional refractive index distribution image of the observation object S can also be reconstructed by performing processing such as deconvolution on the three-dimensional phase differential image or the phase image of the observation object S. 
     Next, an example will be described. In the example, the observation apparatus  1 G ( FIG.  10   ) of the seventh embodiment is used to observe a cell as the observation object. 
       FIG.  11    is a phase differential image. A shear direction by the first prism  14  (splitting direction of the two linearly polarized light beams orthogonal to each other) is a horizontal direction in this diagram.  FIG.  12    is a phase image generated from the phase differential image ( FIG.  11   ).  FIG.  13    is a three-dimensional refractive index distribution image generated from the phase image ( FIG.  12   ) acquired at each position in the depth direction. As illustrated in this diagram, a three-dimensional shape of the cell being the observation object can be visualized. 
     The observation apparatus and the observation method are not limited to the embodiments and configuration examples described above, and various other modifications are possible. 
     The observation apparatus of the above embodiment includes (1) a light source for outputting spatially incoherent light; (2) an irradiation optical system for focusing light output from the light source and irradiating an observation object with the light; (3) a polarizer provided on an optical path of the irradiation optical system, and for inputting the light output from the light source and outputting linearly polarized light; (4) a first prism provided on the optical path of the irradiation optical system and between the polarizer and the observation object, and for inputting light output from the polarizer and outputting two linearly polarized light beams orthogonal to each other; (5) an imaging optical system for forming an image by inputting light generated in the observation object in response to irradiation of the observation object with the light by the irradiation optical system; (6) a second prism provided on an optical path of the imaging optical system, and for combining two light beams output from the observation object and outputting light; (7) a polarization conversion element provided on the optical path of the imaging optical system and at a subsequent stage of the second prism, and for inputting the light output from the second prism and outputting two circularly polarized light beams having different rotation directions; (8) a polarization camera having an imaging plane disposed at a position where the image is formed by the imaging optical system, and for inputting the two circularly polarized light beams having different rotation directions output from the polarization conversion element and acquiring an interference image on the imaging plane for each of three or more polarization components; and (9) an analysis unit for generating a complex amplitude image of the observation object based on the interference image for each of the three or more polarization components acquired by the polarization camera. 
     In the above observation apparatus, the polarization conversion element may be a ¼ wave plate. Further, the above observation apparatus may further include a polarization conversion compensation optical element for reducing polarization conversion occurring in an optical element other than the second prism and the polarization conversion element in the imaging optical system. 
     In the above observation apparatus, the polarization conversion compensation optical element may be a mirror for compensating for a reflection property of a dielectric multilayer mirror provided in the imaging optical system, or a wave plate. Further, in the above observation apparatus, the imaging optical system may include a relay optical system having a mirror, and the polarization conversion compensation optical element may be the mirror included in the relay optical system. 
     In the above observation apparatus, the imaging optical system may include a relay optical system, and the polarization conversion element may be provided on an optical path of the relay optical system. 
     In the above observation apparatus, the polarization camera may have a configuration in which a plurality of pixels are arranged two-dimensionally in the imaging plane, and may acquire a two-dimensional interference image. 
     In the above observation apparatus, the polarization camera may have a configuration in which a plurality of pixels are arranged one-dimensionally in the imaging plane, and may acquire a one-dimensional interference image. Further, in this case, a two-dimensional interference image of the observation object may be acquired by relatively moving the observation object in a direction different from an arrangement direction of the plurality of pixels in the imaging plane of the polarization camera. 
     The above observation apparatus may further include a moving unit for moving an optical element included in the imaging optical system or the observation object in a direction parallel to an optical axis of the imaging optical system, and the polarization camera may acquire the interference image at each position of movement by the moving unit, and the analysis unit may generate a three-dimensional complex amplitude image of the observation object based on the interference image acquired by the polarization camera at each position. 
     The optical module of the above embodiment is an optical module being detachably attached to a camera port of a differential interference contrast microscope, and includes (1) a relay optical system constituting a part of an imaging optical system for forming an image by inputting light generated in an observation object placed in the differential interference contrast microscope, and for inputting light output from a second prism of the differential interference contrast microscope through the camera port; (2) a polarization conversion element provided on an optical path of the relay optical system, and for inputting the light and outputting two circularly polarized light beams having different rotation directions; and (3) a polarization camera having an imaging plane disposed at a position where the image is formed by the relay optical system, and for inputting the two circularly polarized light beams having different rotation directions output from the polarization conversion element and acquiring an interference image on the imaging plane for each of three or more polarization components. 
     In the above optical module, the polarization conversion element may be a ¼ wave plate. Further, the above optical module may further include a polarization conversion compensation optical element for reducing polarization conversion occurring in an optical element other than the second prism and the polarization conversion element in the imaging optical system. 
     In the above optical module, the polarization conversion compensation optical element may be a mirror for compensating for a reflection property of a dielectric multilayer mirror provided in the imaging optical system, or a wave plate. 
     In the above optical module, the polarization camera may have a configuration in which a plurality of pixels are arranged two-dimensionally in the imaging plane, and may acquire a two-dimensional interference image. 
     In the above optical module, the polarization camera may have a configuration in which a plurality of pixels are arranged one-dimensionally in the imaging plane, and may acquire a one-dimensional interference image. 
     The observation method of the above embodiment includes (1) outputting spatially incoherent light from a light source; (2) by a polarizer provided on an optical path of an irradiation optical system for focusing light output from the light source and irradiating an observation object with the light, inputting the light output from the light source and outputting linearly polarized light; (3) by a first prism provided on the optical path of the irradiation optical system and between the polarizer and the observation object, inputting light output from the polarizer and outputting two linearly polarized light beams orthogonal to each other; (4) by a second prism provided on an optical path of an imaging optical system for forming an image by inputting light generated in the observation object in response to irradiation of the observation object with the light by the irradiation optical system, combining two light beams output from the observation object and outputting light; (5) by a polarization conversion element provided on the optical path of the imaging optical system and at a subsequent stage of the second prism, inputting the light output from the second prism, converting the light into two circularly polarized light beams having different rotation directions and outputting the light beams; (6) by a polarization camera having an imaging plane disposed at a position where the image is formed by the imaging optical system, inputting the two circularly polarized light beams having different rotation directions output from the polarization conversion element and acquiring an interference image on the imaging plane for each of three or more polarization components; and (7) generating a complex amplitude image of the observation object based on the interference image for each of the three or more polarization components acquired by the polarization camera. 
     In the above observation method, the polarization conversion element may be a ¼ wave plate. Further, the above observation method may further include, by a polarization conversion compensation optical element, reducing polarization conversion occurring in an optical element other than the second prism and the polarization conversion element in the imaging optical system. 
     In the above observation method, the polarization conversion compensation optical element may be a mirror for compensating for a reflection property of a dielectric multilayer mirror provided in the imaging optical system, or a wave plate. Further, in the above observation method, the imaging optical system may include a relay optical system having a mirror, and the polarization conversion compensation optical element may be the mirror included in the relay optical system. 
     In the above observation method, the imaging optical system may include a relay optical system, and the polarization conversion element may be provided on an optical path of the relay optical system. 
     In the above observation method, the polarization camera may have a configuration in which a plurality of pixels are arranged two-dimensionally in the imaging plane, and may acquire a two-dimensional interference image. 
     In the above observation method, the polarization camera may have a configuration in which a plurality of pixels are arranged one-dimensionally in the imaging plane, and may acquire a one-dimensional interference image. Further, in this case, a two-dimensional interference image of the observation object may be acquired by relatively moving the observation object in a direction different from an arrangement direction of the plurality of pixels in the imaging plane of the polarization camera. 
     The above observation method may further include moving an optical element included in the imaging optical system or the observation object in a direction parallel to an optical axis of the imaging optical system; acquiring the interference image at each position by the polarization camera; and generating a three-dimensional complex amplitude image of the observation object based on the interference image acquired by the polarization camera at each position. 
     INDUSTRIAL APPLICABILITY 
     The embodiments can be used as an observation apparatus and an observation method capable of easily adjusting an optical system and obtaining a complex amplitude image with improved quantitativeness in a short time. 
     REFERENCE SIGNS LIST 
       1 A- 1 G—observation apparatus,  11 —light source,  12 —lens,  13 —polarizer,  14 —first prism,  15 —condenser lens,  21 —objective lens,  22 —second prism,  23 —¼ wave plate (polarization conversion element),  24 —lens,  25 —wave plate (polarization conversion compensation optical element),  26 —polarization camera,  27 —mirror,  28 —moving unit,  30 E,  30 F—optical module,  31 —lens,  32 —wave plate (polarization conversion compensation optical element),  33 —¼ wave plate (polarization conversion element),  34 —lens,  35 —polarization camera,  36 - 38 —mirror,  40 —analysis unit.