Abstract:
A phase contrast observation device for observing a phase object (O), and phase apertures for same. The device comprises, in order along an optical axis (AX), a light source (LS) capable of providing light (L), an illumination optical system (G 2  and G 3 ) for condensing the light and illuminating the object, an aperture stop (AP) having an aperture (AO) therein, arranged in the illumination optical system, an objective lens system (G 2  and G 3 ) for converging light from the illuminated object and forming an image of the object. The device also includes one of a number of novel phase apertures (Ph 1- Ph 4 ) arranged at a position inside said objective lens conjugate to the aperture stop. The phase apertures of the present invention allow for high-contrast and low-contrast imaging regardless of the phase content of the object.

Description:
FIELD OF THE INVENTION 
     The present invention pertains to a phase contrast observation device, more particularly, to a phase contrast microscope that allows for observing a transparent object, such as biological specimen, without the need to stain the object. 
     BACKGROUND OF THE INVENTION 
     In phase contrast microscopy, an object is illuminated by an illumination optical system defined by an aperture stop located at the pupil position of the illumination optical system. A phase plate, providing a phase modulation, is arranged inside an objective lens at a location conjugate with the pupil of the illumination optical system. Then, the phase difference of light introduced by the object (phase object) is converted into a difference in light intensity. Therefore, the phase difference of the object is visualized as light and shade of the image (i.e., contrast), and can be observed. The phase contrast microscope was invented by Fritz Zernike in 1935, and is described in chapter 8.6 of the textbook “Principles of Optics (Sixth Edition),” by M. Born and E. Wolf, Pergamon Press, 1980. 
     The principle of the phase contrast microscope will be explained. With reference to FIG. 1, prior art phase contrast microscope  10  comprises, in order along an optical axis AX, a light source LS capable of emitting light L, an aperture stop AP arranged at a pupil plane P 1  and having an annular opening (aperture) AO (FIG.  2 ), a condenser lens G 1  having a front focal point F positioned on-axis at pupil plane P 1 , an object stage OS for supporting an object O to be observed, a first objective lens G 2  having a rear focal point F′ positioned on-axis, a phase plate Ph 0  arranged at rear focal point F′ and optically conjugate to aperture AP (FIG.  3 ), a second objective lens G 3 , and an image plane IP. First and second objective lenses G 2  and G 3  constitute an objective optical system. Phase plate Ph 0  has an opening (aperture) AO 0  similar to aperture opening AO, and also has a phase film PP thereon covering annular opening AO 0 . Phase film PP provides a phase difference of a quarter wavelength to light transmitted therethrough. Also, phase plate Ph 0  has the same shape as phase film PP, and has an absorbing film which reduces the amount of transmitted light. 
     The operation of phase contrast microscope  10  is now explained. Illumination light L of wavelength λ is emitted from light source LS and passes through annular opening AO in ring aperture AP. The latter determines the amount and nature of llumination of object O. Objective lenses G 2  and G 3  collect the light transmitted through object O and form an image of the object on object plane IP. 
     Light L is diffracted upon passing through object O and is thereby separated into a direct (undiffracted) light beam L 1  and a ±1 st  order diffracted light beam L 2 . Light beams L 1  and L 2  then pass through phase plate Ph 0 . Phase film PP covers aperture opening AOD, which changes the phase of light beam L 1  by a quarter of the wavelength (λ/4) relative to diffracted light beam L 2 . Light beam L 1 , with its phase advanced by (λ/4) interferes destructively with diffracted light beam L 2  at image plane IP. On the other hand, light L having passed through a part of object O having no phase-altering properties does not produce diffracted light, and takes part in the background of direct light beam L 1 . Therefore, the phase difference of object O can be observed as light and shade in the image. Moreover, the amplitude of ±1 st  order diffracted light L 2  can be expressed as a Bessel function J 1 (B). According to the intensity J 1 (B) 2  of the diffracted light varying in accordance with the amount of the phase difference, a transmittance-modulation film, such as a neutral density film ND reducing the amount of transmitted light, is applied to the phase film PP. If the amplitude of direct light beam L 1  is made equal to that of the diffracted light beam L 2  with the help of neutral density transmittance-modulation film ND, the phase object can be observed with maximum intensity contrast against the background. 
     According to prior art contrast microscope  10  described above, if the amount of the phase difference of the object is small, a high detection sensitivity to the amount of phase difference, using low transmittance of direct light beam L 1 , is utilized. This is referred to as a high-contrast type microscope, or high contrast imaging. In this instance, the transmittance of the modulation film applied on phase film PP is about 0.1 to 0.25. In the case of a high-contrast imaging, an object having a small amount of phase difference can be easily observed. However, when an object having large amount of phase difference is observed, the ratio of the amplitude of the direct light to that of the diffracted light is reversed, and a reverse contrast image is formed. Therefor, a fringe-shaped blurring of light in accordance with the phase difference or structure of the object is formed around the image of the object. The phenomenon is called “halo.” If halo is produced, good observation of the object is disturbed. Moreover, there is good possibility of misidentification of structure in the object. 
     When an object having a large amount of phase difference is observed, a low-contrast type phase contrast microscope having high transmittance of the direct (undiffracted) light beam is utilized to avoid producing halo. In such a microscope, it is desirable that the transmittance of transmittance-modulation film ND applied on phase film PP is on the order of 0.25 to 0.50. In a low-contrast type phase contrast microscope, instead of producing halo, other problems occurs. For example, when observing an object having small amount of phase difference, it is difficult to get a good image because of low image contrast. 
     In addition to the problems mentioned above, when an object is observed with white light using a phase contrast microscope, further problems arise. For example, with reference to FIG. 4, consider a phase object  11  having an interior medium width t and a refractive index n 1 , surrounded by a medium of refractive index n 2 . When phase object  11  is observed with a phase-contrast microscope, although refractive index n 1  varies in accordance with wavelength, the refractive index is considered to be approximately constant within a normal extent of dispersion. The optical path length inside of phase object  11  can be expressed as n 1 ×t. The amount of phase difference is expressed as (n 1 −n 2 )×t. Since the amount of phase difference is expressed in units of wavelength, the amount of phase difference produced in the same object is approximately inversely proportional to the wavelength. For example, an object having the amount of phase difference of 0.1λ at a wavelength of 550 nm will produce the amount of phase difference of 0.14λ at a wavelength of 400 nm. Therefore, there is a problem that even an object producing no halo at a given wavelength, such as 550 nm, produces halo at a different wavelength, such as 400 nm. 
     To solve this problem, the transmittance T 1  of transmittance-modulation film ND is varied in accordance with the wavelength, from about 0.1 to about 0.4. For example, with reference also to FIG. 5, the transmittance T ND  of the transmittance-modulation film ND at short wavelengths is made higher than that at long wavelengths. This allows the phase contrast microscope to work as low-contrast type at short-wavelengths and as high-contrast type at long wavelengths. Therefore, it is possible to mitigate the generation of a halo likely to be produced at the short-wavelength side. However, if the transmittance of the transmittance-modulation film ND is set high at short wavelengths, the spectral transmittance of the background of the image is changed, so that the background has color. For example, if the transmittance is high at short wavelengths, as shown in FIG. 5, the background becomes blue. 
     SUMMARY OF THE INVENTION 
     The present invention pertains to a phase contrast observation device, more particularly, to a phase contrast microscope that allows for observing a transparent object, such as biological specimen, without the need to stain the object. 
     The present invention has for its object, in the light of above-mentioned problems, to provide a phase contrast observation device always capable of making use of good contrast image despite an amount of phase difference inherent in an object. 
     Accordingly, a first aspect of the present invention is a phase contrast microscope for imaging an object having phase features. The microscope comprises, in order along an optical axis, a light source capable of providing light, an illumination optical system for condensing the light from said light source and illuminating the object, an aperture stop having an aperture therein, arranged in said illumination optical system, and an objective lens for converging light from the illuminated object and forming an image of the object. The microscope also includes one of a number of phase apertures according to the present invention, arranged at a position inside the objective lens at a position conjugate to the aperture stop. 
     A second aspect invention is a phase aperture suitable for use in the phase contrast microscope, described above. The phase aperture comprises, radially outwardly from a center, a first circular portion having a transmittance of 1, a second annular portion having a first transmittance, a third annular portion having a second transmittance, a fourth annular portion having said first transmittance, and a fifth annular portion having a transmittance of 1. 
     A third aspect of the invention is a phase aperture suitable for use in the phase contrast microscope, described above. The phase aperture comprises, radially outwardly from a center, a first circular portion having a transmittance of 1, a first annular portion having a first transmittance, a second annular portion having a second transmittance, a third annular portion having a third transmittance, a fourth annular portion having the second transmittance, a fifth annular portion having the first transmittance, and a sixth annular portion having a transmittance of 1. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic optical diagram of a prior art phase contrast microscope; 
     FIG. 2 is a plan view of a prior art aperture for use in combination with the phase aperture of FIG. 3 in the microscope of FIG. 1; 
     FIG. 3 is a plan view of a prior art phase filter for use with the microscope of FIG. 1; 
     FIG. 4 is an exemplary phase object of the kind that may be viewed with the microscope of FIG. 1, or with the microscope of the present invention; 
     FIG. 5 is an exemplary plot of the transmission T ND  vs. wavelength λ (nm) for the prior art phase aperture of FIG. 3; 
     FIG. 6 is a schematic optical diagram of a phase contrast microscope according to the present invention; 
     FIG. 7 is a plan view of a phase aperture according to a first embodiment of the present invention, suitable for use in the phase contrast microscope of FIG. 6; 
     FIG. 8 is a plan view of a phase aperture according to a second embodiment of the present invention, suitable for use in the phase contrast microscope of FIG. 6; 
     FIG. 9 is a plan view of a phase aperture according to a third embodiment of the present invention, suitable for use in the phase contrast microscope of FIG. 6; 
     FIG. 10 is a plan view of a phase aperture according to a fourth embodiment of the present invention, suitable for use in the phase contrast microcope of FIG. 6; 
     FIG. 11 illustrates the transmittance profile (T vs. x) of the phase aperture of FIG. 10; 
     FIG. 12 is a plot of the spectral transmittance (T vs. λ (nm)) of the phase aperture of FIG. 11; 
     FIG. 13 illustrates a construction of a phase contrast microscope according to a sixth embodiment of the present invention; and 
     FIG. 14 is a schematic optical diagram of an objective lens of the phase contrast microscope shown in FIG.  13 . 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention pertains to a phase contrast observation device, more particularly, to a phase contrast microscope that allows for observing a transparent object, such as biological specimen, without the need to stain the object. 
     Referring to FIG. 6, a phase contrast microscope  20  in accordance with the present invention is described. Those elements making up phase microscope  20  that are common with phase microscope  10  are given the same reference numbers and symbols. In addition, only the differences between the microscopes are described. 
     The operation of the microscope  20  similar to that of the microscope  10 . That is, illumination light L having a wavelength λ is emitted from light source LS and passes through an annular opening AO in a ring aperture AP. By using objective lenses G 2  and G 3 , the light transmitted through object O is concentrated at image plane IP, where an image of the object is formed. A phase aperture Phn is arranged at the rear focal point F′ of the objective lens G 2 . The phase aperture Phn is conjugate with ring aperture AP. The phase aperture Phn has an opening similar to aperture opening AO of ring aperture AP (FIG.  2 ), which may be annular, as shown, or some other desired shape. The phase aperture Phn also has a phase-modulation means, such as phase plate PP, which provides a phase difference of a quarter wavelength to the transmitted light. A neutral-density filter ND may also be included on phase plate PP, as discussed above in connection with aperture plate PhO. Described below are phase apertures Phn (i.e., Ph 1 -Ph 4 ) of the present invention suitable for use with the microscope  20 . 
     First Embodiment 
     With reference now to FIG. 7, phase aperture Ph 1  has transmittance-modulation annular portions A 1  and B 1  having respective widths of W A1  and W B1 , and composed of neutral density film. Surrounds B 1  surronds both, the inner- and outer-peripheries A 1 i and A 1 o of portion A 1 . Portions A 1  and B 1  correspond to first and second transmittance-modulation portions respectively. The phase plate Ph 1  has a no-modulation portion C 1  surrounding B 1  on both the inner- and outer-peripheries B 1 i and B 1 o of portion B 1 . 
     Portion A 1  has a similar shape, at a given magnification, to aperture opening AO of ring aperture AP. It is desirable that the given magnification of microscope  20  be set to be a combined magnification of a condenser lens G 1  and an objective lens G 2  such that ring aperture AP can be relayed by lenses G 1  and G 2  to the phase aperture Ph 1 . It is more desirable that the width W A1  of portion A 1  be set slightly wider than the width determined by the combined magnification with respect to aperture opening AO, to facilitate adjustment of the position of phase aperture Ph 1 . Portion B 1  is provided at inner- and outer-peripheries A 1 i and A 1 o of portion A 1  such that portion B 1  surrounds the periphery of portion A 1 . It is desirable that width W B1  of portion B 1  is the same, or slightly wider, than width W A1 , but the invention is not limited to this construction. Moreover, no-modulation portion C 1  surrounds portion B 1 . Portions A 1  and B 1  preferably have an annular shape in either case. 
     Use of a neutral density film controls the transmittance such that the transmittance TA 1  of portion A 1  is about 0.2 and the transmittance TB 1  of the portion B 1  is about 0.5. Portion C 1  of the phase aperture Ph 1  does not have a transmittance-modulation film, and thus has a transmittance TC 1  of 1. 
     In other words, phase aperture Ph 1  comprises, radially outwardly from its center, a first circular portion C 1  having a transmittance of 1, a second annular portion B 1  having a first transmittance, a third annular portion A 1  having a second transmittance, a fourth annular portion, also B 1 , having the first transmittance, and a fifth annular portion, also C 1 , having a transmittance of 1. 
     Referring to FIG. 6, the principle of the phase contrast observation using phase aperture Ph 1  in phase contrast microscope  20  is now explained. The explanation below also applies in a general sense to the phase apertures of the present invention as set forth below. Diffraction angle θ of diffracted light L 2  produced by the structure (not shown) of object O can be expressed in the following equation, where the wavelength is λ, the refractive index of the medium is n, and the period of the structure of the object is t: 
     
       
         θ=sin −1 (0.61λ /n·t ),  (1) 
       
     
     wherein θ is limited to the range 0≦θ≦π/4. 
     Where object O comprises a biological specimen, the amount of phase difference of the object is generally approximately proportional to the dimension of the structure of the object, so that an object having large amount of phase difference usually has a large structure. In other words, it is characteristic that t in the equation (1) immediately above is large. In equation (1), if n and λ are constant, θ becomes small. 
     Accordingly, the distance P perpendicular to axis AX between diffracted light L 2  and direct light L 1  can be expressed as: 
     
       
           P=f   2 ·sin θ,  (2) 
       
     
     wherein f 2  is the focal length of objective lens G 2 , and θ is the diffraction angle. 
     When an object having a large amount of phase difference (i.e., a large structured object) is observed, diffraction angle θ is small, and the intensity of the diffracted light is relatively large. Therefore, distance P between direct light L 1  and diffracted light L 2  becomes small, so that the direct light L 1  and the ±1 st  order diffracted light L 2  are passed through portions A 1  and B 1 , respectively. Accordingly, the ratio of the transmittance TA 1 =0.2 of portion A 1  to the transmittance TB 1 =0.5 of portion B 1  is the essential amount of the modulation of transmittance between direct light L 1  and diffracted light L 2 . Thus, low-contrast type imaging results. 
     On the other hand, when an object having small amount of phase difference (i.e., a small structured object) is observed, diffraction angle θ is large, and the intensity of the diffracted light is relatively weak. Therefore, direct light L 1  and the ±1 st  order diffracted light L 2  are passed through portions A 1  and C 1 , respectively. Accordingly, the ratio of the transmittance TA 1 =0.2 of portion A 1  to the transmittance TC 1 =1 of portion C 1  becomes substantially the value of the modulation of transmittance between direct light L 1  and diffracted light L 2 . So, the effect that the amplitude of only the direct light L 1  can be decreased. Accordingly, high-contrast type imaging results. 
     In phase aperture Ph 1 , the transmittance varies step by step, from B 1  to C 1 , relative to portion A 1 . So, the ratio of the amplitude (and consequently, the ratio of the light intensity) of direct light L 1  relative to that of diffracted light L 2  can be moderately adjusted. Preferably, this can be adjusted to be almost the same. Then, good image contrast can be observed. 
     Second Embodiment 
     With reference now to FIG. 8, a phase aperture Ph 2  in accordance with to a second embodiment of the present invention is described. The phase aperture Ph 2  comprises a transmittance-modulation portion A 2  with a transmittance TA 2 =0.2 having a ring shape, a transmittance-modulation portion B 2  with a transmittance TB 2 =0.5 located symmetrically with portion A 2 , a transmittance-modulation portion C 2  with a transmittance TC 2 =0.8, and no-modulation portion D 2  with a transmittance of 1. As can be seen from FIG. 8, the phase aperture Ph 2  is constructed such that portion B 2  surrounds portion A 2 , portion C 2  surrounds portion B 2  and portion D 2  surrounds portion C 2 . The central section of portion D 2  is circular. Portions A 2 , B 2 , and C 2  are all annular shaped. Portion A 2  corresponds to a first transmittance-modulation portion and portions B 2  and C 2  correspond to a second transmittance-modulation portion. 
     In other words, the phase aperture Ph 2  comprises, radially outwardly from its center, a first circular portion D 2  having a transmittance of 1, a first annular portion C 2  having a first transmittance, a second annular portion B 2  having a second transmittance, a third annular portion A 2  having a third transmittance, a fourth annular portion, also B 2 , having the second transmittance, a fifth annular portion, also C 2 , having the first transmittance, and a seventh annular portion, also D 2 , having a transmittance of 1. According to this construction, the transmittance of portions B 2  and C 2  relative to portion A 2  is radially graded with finer steps than that of phase aperture Ph 1 , so that an image having better contrast can be observed. 
     Third Embodiment 
     With reference to FIG. 9, phase aperture Ph 3  representing a third embodiment of the present invention is described. Aperture AP is used with a phase aperture Ph 3  in microscope  20  and has a circular aperture opening AO instead of an annular aperture. Phase aperture Ph 3  comprises a circular transmittance-modulation portion A 3  with a transmittance TA 3 =0.2, an annular transmittance-modulation portion B 3  with a transmittance TB=0.5, an annular transmittance-modulation portion C 3  with a transmittance=0.8, and an annular no -modulation portion D 3  with a transmittance of 1. Portion A 3  corresponds to a first transmittance-modulation portion and portions B 3  and C 3  correspond to a second transmittance-modulation portion. As can be seen from FIG. 9, phase aperture Ph 3  is constructed such that portion B 3  surrounds portion A 3 , portion C 3  surrounds portion B 3  and portion D 3  surrounds portion C 3 . 
     In other words, phase aperture Ph 3  comprises, radially outwardly from its center, a first circular portion A 3  having a first transmittance&lt;1, a first annular portion B 3  having a second transmittance, a third annular portion C 3  having a third transmittance, and a fourth annular portion D 3  having a transmittance of 1. 
     Fourth Embodiment 
     With reference now to FIGS. 10 and 11, a phase aperture Ph 4  in accordance with to a fourth embodiment of the present invention is described. The phase aperture Ph 4  has transmittence-modulation regions A 4  and B 4 . The transmittance of these regions are described by the transmittance variation curve (i.e., transmission profile T vs. x) of FIG.  11 . In FIG. 11, the horizontal X axis is the distance from optical axis AX, normalized to an outer-most periphery PR=1. The vertical axis is the transmittance. The transmittance of portion B 4  of phase aperture Ph 4  decreases continuously from center CE outwardly to portion A 4  and then increases continuously relative to portion A 4  from portion A 4  outwardly to periphery PR. The transmittance-modulation portions A 4  and B 4  correspond to a first and a second transmittance-modulation portions, respectively. 
     Preferably, it is desirable that the transmittance-variation curve satisfies with the following equation: 
     
       
           TA   4 +(1 −TA   4 )× d /D   4 &lt; TB   4 &lt;1  (3) 
       
     
     where TA 4  is the transmittance of portion A 4 , TB 4  is the transmittance of portion B 4 , D 4  is the width of portion B 4 , and d 4  is the distance toward periphery PR from portion A 4 . An image having better contrast can be obtained by satisfying the equation. 
     Moreover, it is desirable for this embodiment to satisfy following equation: 
     
       
           TA   4 &lt;0.3  (4) 
       
     
     By satisfying equation (4), an image of an object even having small phase difference can be obtained with better contrast. 
     Fifth Embodiment 
     Referring to FIG.  7  and phase aperture Ph 1 , a fifth embodiment of the present invention is described. 
     In phase aperture Ph 1 , the transmittance TA 1  of portion A 1  is set to be about 0.25 over the range of the visible wavelength, 400 nm through 700 nm. Use of a neutral density film or the like controls transmittance TB 1  of portion B 1  such that the transmittance TB 1   400  at wavelength of 400 nm is 0.3, the transmittance TB 1   550  at a wavelength of 550 nm is 0.4, and the transmittance TB 1   700  at a wavelength of 700 nm is 0.5. Referring to FIG. 12, the transmittance T 1  within the scope of the wavelength 400 nm through 700 nm almost varies along the straight line passing through the points TB 1   400 , TB 1   550 , and TB 1   700 . 
     Sixth Embodiment 
     FIG. 13 illustrates a construction of a phase contrast microscope  30  in accordance with a sixth embodiment of the present invention. An objective lens  40  is fitted to a revolver assembly  44  and is disposed in an observation optical path along an optical axis AX 1 . The revolver assembly  44  is detachably fitted to a base  48 , which serves as the main body of microscope  30 . A body tube  52  is mounted on the base  48 . An eyepiece holder  56  for storing an eyepiece through which an image of a specimen is visually observed, is fitted on body tube  52 . Inside of body tube  52 , as shown by dotted line, is contained an imaging lens  60 , which converges the passing through objective lens  40 , and forms the image of the specimen. Also inside body tube  52  is a prism  64  and a mirror M, which leads the light passing through imaging lens  60  to eyepiece holder  56 . A stage  68 , on which a specimen (object) O is placed, and an illumination unit  74  providing light to illuminate the specimen, are fitted to base  48 . The light provided by illumination unit  74  illuminates specimen O placed on stage  68  through a condenser lens  80 , thereby providing a transmitted illumination. A phase aperture Phn (e.g., one of phase apertures Ph 1 -Ph 4 ) is arranged inside of objective lens  40 . Ring aperture AP is placed at a position conjugate with phase aperture Phn. 
     Lens specifications of objective lens  40  are set forth in Table 1, below. In Table 1, a cover glass CG, imaging lens  60 , and prism  64  are included. Also, the numerical aperture of objective lens  40  at the specimen O side is NA, the focal length of objective lens  40  is F, the curvature of radius of the respective lens surface is r, the distance between adjacent lens surfaces is d, and the refractive index and the Abbe number with respect to d-line light (587.6 nm) of the lens are n d  and ν d , respectively. Also, the first column lists lens surfaces S (from the specimen side) and the focal length of imaging lens  60  is fI. Phase aperture Phn is formed at the eleventh surface of objective lens  40  by vacuum evaporation. The characteristic of phase aperture Phn is that of the third embodiment, i.e., phase aperture Ph 3 . 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 S 
                 r (mm) 
                 d (mm) 
                 n d   
                 ν d   
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 cover glass 
                   
                   
                   
                   
               
               
                   
                  1 
                 ∞  
                 0.17 
                 1.5222 
                 58.80 
               
               
                   
                  2 
                 ∞   
                 4.20 
               
               
                   
                 objective lens 40 
               
               
                   
                  3 
                 −10.059 
                 13.29 
                 1.8041 
                 46.54 
               
               
                   
                  4 
                 −13.053 
                 0.30 
               
               
                   
                  5 
                 23.466 
                 4.50 
                 1.4978 
                 82.52 
               
               
                   
                  6 
                 −19.900 
                 0.20 
               
               
                   
                  7 
                 15.451 
                 4.27 
                 1.4339 
                 95.57 
               
               
                   
                  8 
                 −16.087 
                 7.25 
                 1.6727 
                 32.17 
               
               
                   
                 9 
                 11.863 
                 4.70 
               
               
                   
                 10 
                 −7.017 
                 1.65 
                 1.6127 
                 44.41 
               
               
                   
                 11 
                 94.422 
                 7.00 
                 1.5932 
                 67.87 
               
               
                   
                 12 
                 −12.305 
                 0.20 
               
               
                   
                 13 
                 209.577 
                 4.20 
                 1.7408 
                 27.63 
               
               
                   
                 14 
                 −24.512 
                 0.50 
               
               
                   
                 15 
                 −49.165 
                 2.00 
                 1.7440 
                 45.00 
               
               
                   
                 16 
                 22.005 
                 5.00 
                 1.4978 
                 82.52 
               
               
                   
                 17 
                 −50.715 
                 100.00 
               
               
                   
                 imaging lens 60 
               
               
                   
                 18 
                 75.043 
                 5.10 
                 1.6228 
                 57.03 
               
               
                   
                 19 
                 −75.043 
                 2.00 
                 1.7495 
                 35.19 
               
               
                   
                 20 
                 1600.580 
                 7.50 
               
               
                   
                 21 
                 50.256 
                 5.10 
                 1.6676 
                 41.96 
               
               
                   
                 22 
                 −84.541 
                 1.80 
                 1.6127 
                 44.41 
               
               
                   
                 23 
                 36.911 
                 10.00 
               
               
                   
                 prism 64 
               
               
                   
                 24 
                 ∞ 
                 30.00 
                 1.5688 
                 56.04 
               
               
                   
                 25 
                 ∞ 
                 139.31 
               
               
                   
                   
               
             
          
         
       
     
     NA=0.45, F=20.0, fI=200 
     When the phase difference is set between the direct light and the diffracted light in the phase contrast observation device of the present invention, either the bright contrast or dark contrast method can be applied. In the dark contrast method, the phase difference of the direct light is advanced by a quarter of the wavelength such that the object having a higher refractive index than surrounding medium is seen darker. In, the bright contrast method, the phase difference of the direct light is delayed by a quarter of the wavelength such that the object having a higher refractive index than surrounding medium is seen brighter. 
     According to the phase contrast observation device of the present invention explained above, when an object having a large amount of phase difference is observed, it is possible to obtain a low-halo image, without the help of an electrical-contrast-modulation device. Moreover, when an object having a small amount of phase difference is observed, it is possible to obtain a high-contrast image. Therefore, it is always possible to obtain a good-contrast image, regardless of the amount of phase difference produced by the object. 
     While the present invention has been described in connection with working examples and various embodiments, it will be understood that it is not so limited. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined in the appended claims.