Abstract:
A focusing apparatus for use with an optical system having a high NA objective lens includes an image forming and capturing mechanism for forming an image in an intermediate image zone and for capturing an image by receiving and refocusing light from a selected focal plane within the intermediate image zone, and a focus adjusting mechanism for adjusting the position of the selected focal plane within the intermediate image zone. The image forming and capturing mechanism includes at least one high NA lens. In use, spherical aberration introduced by the high NA objective lens is reduced.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 13/296,975, filed Nov. 15, 2011, which is a division of U.S. patent application Ser. No. 12/519,517, filed Aug. 17, 2009, now U.S. Pat. No. 8,144,395, which is a U.S. National Phase filing under 35 U.S.C. §371 of PCT/GB2007/004916, filed Dec. 20, 2007, which claims priority to Great Britain Patent Application No. 0625775.2, filed Dec. 22, 2006. The disclosures of all these prior applications are incorporated herein by reference in their entireties for all purposes. 
    
    
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present invention relates to a focusing apparatus and method and in particular, but not exclusively, to a focusing apparatus and method for use in microscopic applications. The invention may also have various non-microscopic applications. 
     The term “microscopic” as used herein refers to optical applications having a resolution in the range of approximately 10 −4  to 10 −7  meter. Such applications are generally characterized by the use of high numerical aperture (NA) objective optics, typically with a NA in the range 0.5 to 0.95 in air, or 0.7 to 1.4 in immersion oil. 
     The invention may be used either in applications where light is collected from an object in order to form an image or detect certain properties of the object (for example wide field or scanning microscopy, fluorescence microscopy and so on), or in applications where light is focused onto an object in order to illuminate or affect the object in some way (for example micro-fabrication or laser surgery). It should be understood that any references herein to focusing and image formation apply equally to both types of application. The invention may also be used in applications where light is focused onto an object and an image of the object is then detected. 
     Description of the Related Art 
     In conventional microscopy, light from a specimen is collected by an objective lens and focused either by an ocular for viewing by eye or by an imaging lens onto a detector, for example a charge-coupled device (CCD). A typical arrangement is shown in  FIG. 1 . This includes an objective lens  2 , an imaging lens  4  and a CCD detector  6 . Light from a specimen  8  is collected by the objective lens  2  and focused by the imaging lens  4  onto the CCD detector  6 . The image recorded by the CCD will represent a thin section  10  of the specimen. Light from all other parts of the specimen will be blurred out. 
     In a conventional arrangement, the image represents a two-dimensional plane (the X-Y plane) that is perpendicular to the optical axis  12  (the Z-axis) of the objective  2 . Sometimes, however, it may be desirable to obtain a three-dimensional image or an image from a plane that is not perpendicular to the optical axis  12  of the objective  2 . In either of these cases it is necessary to adjust the focal plane of the objective, so that it collects light from different regions of the specimen. A number of images obtained from different focal planes can then be combined to obtain either a 3D image or a 2D image in a non-perpendicular plane (for example the X-Z plane). 
     SUMMARY OF THE INVENTION 
     There are two ways in which the microscope can be adjusted in order to obtain images from different planes in the specimen. The first method simply involves adjusting the distance between the objective and the specimen, by moving either the objective or the specimen in the direction of the optical axis. However, there are two major drawbacks to this approach: 
     (i) The speed at which mechanical adjustments can be carried out is limited by the mass of the object being moved (the objective or the specimen), which typically leads to slow response times. 
     (ii) Mechanical movements can sometimes disturb the specimen, thereby altering the properties that are of interest to the user. 
     The second method involves moving the detector relative to the objective, so as to obtain images from different planes of the specimen. However, this method is not employed commercially as it has the following drawbacks: 
     (i) Owing to the high magnification of most such systems, a large translation of the detector is required for even a small shift of the imaging plane in the specimen; 
     (ii) The discrepancy between the large numerical aperture (NA) of the objective lens and the small NA of the imaging optics that focus the image on the detector introduces a large amount of spherical aberration when the detector is not located at its optimum designed position relative to the objective. As a result, the image quality at the shifted focal plane is seriously reduced, the loss of quality becoming progressively worse as the detector displacement increases. There is therefore a large reduction in the signal to noise ratio when the detector is displaced from its optimum position. This causes real problems in most applications and especially in non-linear microscopy. 
     (iii) In any sectioning technique such as confocal microscopy, Nipkow disc microscopy or structured illumination microscopy, the presence of spherical aberration leads to an apparent loss of image sectioning. This is very detrimental to such imaging modes as they all rely heavily on consistent sectioning to be of practical value. 
     The ability to refocus a microscope so as to interrogate different focal planes is of great importance. Similarly, there is a need in certain manufacturing and other systems for the ability to refocus an illumination system quickly and accurately. Current technology allows a line scan in a single focal plane to be obtained quickly, for example at a frequency of about 5 kHz. However, mechanical limitations restrict axial scans to a much lower frequency, typically about 15 Hz. There is a need therefore for a system that allows axial scanning at a much higher frequency, without moving the specimen or encountering the problem of spherical aberration. 
     It is an object of the present invention to provide a focusing apparatus and method that mitigates at least some of the problems described above. 
     According to one aspect of the present invention there is provided a focusing apparatus for use with an optical system having a high NA objective lens, the apparatus including an image forming and capturing means for forming an image in an intermediate image zone, and for capturing an image by receiving and refocusing light from a selected focal plane within said intermediate image zone, and a focus adjusting means for adjusting the position of the selected focal plane within the intermediate image zone, wherein said image forming and capturing means includes at least one high NA lens whereby, in use, spherical aberration introduced by the high NA objective lens is reduced. 
     The invention makes it possible to refocus the optical system rapidly at different distances from the objective lens without introducing spherical aberration. Therefore, in the case of a microscopic imaging system, an image can be obtained from different planes within the specimen, so allowing axial scanning without moving either the specimen or the objective lens. In the case of an illuminating system, an image (that is, a light pattern) can be focused onto an object at different distances from the objective, again without introducing spherical aberration. 
     In one preferred embodiment, the image forming and capturing means includes a first high NA lens for forming an image in the intermediate image zone and a second high NA lens for receiving and refocusing light from the intermediate image zone, and the focus adjusting means includes means for adjusting the axial position of the second high NA lens relative to the first high NA lens. In this embodiment the components of the system can be set out in a co-axial arrangement, which provides for compact packaging. There is also no significant loss of light as it passes through the apparatus, apart from normal transmission losses. 
     Advantageously, the first and second high NA lenses are opposed objective lenses. Preferably, the first and second high NA lenses are identical. 
     In another preferred embodiment, the image forming and capturing means includes a high NA lens and a reflective element for reflecting the image formed by the high NA lens back into the same high NA lens so as to capture an image from a selected focal plane within said intermediate image zone, and wherein the focus adjusting means includes means for adjusting the axial position of the reflective element. This form of the system allows for very rapid refocusing, since only the reflective element has to be moved. As the reflective element may be a very small and light mirror, rapid adjustment is possible, for example using a piezo transducer. 
     In this second form of the system, the focusing apparatus preferably includes a beam splitter for dividing the reflected light from the light entering the focusing apparatus. Advantageously, the apparatus includes a polarizing beam splitter and a polarization adjuster located between the polarizing beam splitter and the intermediate image zone for adjusting the polarization of the reflected light. The polarization adjuster may be a quarter wave plate that is positioned between the beam splitter and the mirror. 
     Advantageously, the polarizing beam splitter is constructed and arranged to transmit incident light having a first polarization in the direction of a first axis and to reflect light having a second polarization in the direction of a second axis, the apparatus including a first image forming and capturing means including a first high NA lens and a first reflective element for forming and capturing an image in a first intermediate image zone on said first axis, and a second image forming and capturing means including a second high NA lens and a second reflective element for forming and capturing an image in a second intermediate image zone on said second axis, and means for recombining said first and second captured images. This arrangement provides the advantage that all the light passing through the beam splitter is captured, resulting in a brighter image. 
     Alternatively, the apparatus may include a discriminator for dividing the reflected light from the light entering the focusing apparatus. The discriminator may for example be a Glan-Thompson prism, which divides the reflected light from the light entering the focusing apparatus by shifting it at an angle away from the axis of the input beam. 
     Advantageously, the at least one high NA lens of the image forming and capturing means has a numerical aperture in air of at least 0.5, preferably at least 0.7, more preferably at least 0.9. For example, the high NA lens may have a numerical aperture in air of about 0.95. 
     According to another aspect of the invention there is provided an optical system including a high NA objective lens and a focusing apparatus according to any one of the preceding statements of invention for adjusting the focus of the high NA objective lens. 
     Advantageously, the at least one high NA lens of the focusing apparatus (the “focusing lens”) is optically matched to the high NA objective lens. By “optically matched”, we mean that a ray of light entering or leaving the objective lens in the object space at an angle γ relative to the optical axis of the objective lens will be mapped by the system to a ray that enters or leaves the focusing lens in the intermediate image space at the same angle γ relative to the optical axis of the focusing lens. This arrangement ensures that any spherical aberration introduced by the objective lens is cancelled by the focusing lens. In the simplest case, the objective lens and the focusing lens are identical. However, this is not essential: different lens may be used providing that they are optically matched. Thus, an oil immersion objective lens may for example be optically matched to a dry, high NA focusing lens. 
     The angular aperture of the at least one high NA lens of the focusing apparatus (the “focusing lens”) is preferably at least as great as the angular aperture of the high NA objective lens. This ensures that the resolution of the system is not adversely affected by the focusing lens. For example, an oil immersion objective lens with a numerical aperture of 1.4 and an angular aperture of 67° may be used in combination with a dry, high NA focusing lens with a numerical aperture of 0.95 and an angular aperture of 71.8°. 
     The optical system may comprise a light detection system including a detector and means for focusing the captured image onto the detector. Alternatively, the optical system may comprise an illumination system that includes a light source for illuminating an object through the high NA objective lens. In another alternative, the optical system includes both an illumination system and a light detection system, as for example in a scanning microscope. 
     In another alternative form, the optical system comprises a microscope system having a large NA microscope objective lens. 
     According to another aspect of the invention there is provided a microscope objective lens assembly that includes a high NA objective lens and a focusing apparatus comprising a first high NA lens for forming an image in an intermediate image zone, a second high NA lens for receiving and refocusing light from the intermediate image zone, and focus adjusting means for adjusting the axial position of the second high NA lens relative to the first high NA lens. This objective lens assembly may be fitted to an existing microscope, allowing the microscope to be refocused without adjusting the position of either the specimen or the microscope objective. Advantageously, the high NA objective lens, the first high NA lens and the second high NA lens are mounted coaxially within a housing. 
     According to another aspect of the invention there is provided a method of focusing an optical system having a high NA objective lens, the method including forming an image in an intermediate image zone using at least one high NA lens, capturing an image by receiving and refocusing light from a selected focal plane within said intermediate image zone, and adjusting the position of the selected focal plane within the intermediate image zone, whereby spherical aberration introduced by the high NA objective lens is reduced. 
     According to another aspect of the invention there is provided an imaging method including gathering light from an object using an optical system having a high NA objective lens, adjusting the focus of the system by a method according to the previous statement of invention, and focusing the light onto a detector to obtain an image. 
     According to yet another aspect of the invention there is provided an illumination method that includes generating light from a light source, adjusting the focus of the light by a method according to the preceding statement of invention, and directing the light onto an object using an optical system having a high NA objective lens. 
     Certain embodiments of the invention will now be described by way of example with reference to the accompanying drawings, in which: 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram of a conventional prior art microscope system; 
         FIG. 2  is a diagram of a microscope system according to a first embodiment of the invention; 
         FIG. 3  is a diagram of a microscope system according to a second embodiment of the invention; 
         FIG. 4  is a diagram of a microscope system according to a third embodiment of the invention; 
         FIG. 5  is a diagram of a microscope objective lens, comprising a fourth embodiment of the invention; 
         FIG. 6  is a diagram of a plug-in assembly for the camera port of a microscope, according to a fifth embodiment of the invention; 
         FIG. 7  is a diagram of an alternative plug-in assembly for the camera port of a microscope, according to a sixth embodiment of the invention; 
         FIG. 8  is a diagram of a slit scanning confocal microscope according to a seventh embodiment of the invention; 
         FIG. 9  is a diagram of a microscope system similar to that shown in  FIG. 3 ; 
         FIG. 10  is a diagram of a microscope system according to an eighth embodiment of the invention; 
         FIG. 11  is a diagram of a microscope system according to a ninth embodiment of the invention, and 
         FIGS. 12 and 13  are diagrams of a discriminator forming part of the microscope system shown in  FIG. 11 . 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     In the first embodiment of the invention shown in  FIG. 2 , the microscope system includes an objective lens  20 , a mapping lens system  22 , beam splitter  24 , a reference lens  26  and a reference mirror  28 , all spaced along a first optical axis  30 . The objective lens  20  and the reference lens  26  are identical high NA lenses. An imaging lens  32  (or “tube” lens) and a CCD detector  34  are spaced along a second optical axis  36  that intersects the first optical axis  30  at right angles where it passes through the beam splitter  24 . 
     The mapping lens system  22  is a 4f system comprising two lenses  22   a , 22   b , each of which has a focal length f and is spaced from the other one of the two lenses by a distance 2f and from the pupil plane of the adjacent one of the objective lens  20  and the reference lens  26  by a distance f. The mapping lens system  22  maps the wavefront appearing at the pupil plane of the objective lens  20  onto the pupil plane of the reference lens  26 . 
     The reference mirror  28  is mounted on a piezo transducer (not shown), which allows its position relative to the reference lens  26  to be adjusted rapidly in the direction of the first optical axis  30 , as indicated by arrow  38 . 
     Light from a specimen  42  in an object space  40  is collected by the objective lens  20  and mapped by the mapping lens system  22  onto the pupil plane of the reference lens  26 , which focuses the light to form an image of the specimen  42  in an intermediate image space  44  in front of the reference lens  26  and adjacent the reference mirror  28 . Because the objective lens and the reference lens  26  are matched and designed to the same mathematical condition (known as the “sine condition”), the image of the specimen  42  is reconstructed in the intermediate image space  44  without any spherical aberration. The reference mirror  28  reflects this reconstructed image back into the reference lens  26 . If the reference mirror is located in the normal focal plane of the reference lens  26 , the wavefront reconstructed at the pupil plane of the reference lens  26  will be identical to that entering the reference lens from the mapping lens system  22 . However, if the reference mirror  28  is displaced axially, it will reflect light from a different plane within the intermediate image zone and the reconstructed wavefront will then contain information representing a different plane in the object space. The CCD detector  34  observes only the information in the focal plane of the reference lens  26 . This makes it possible to select the plane of the reconstructed image that is refocused by the reference lens. 
     The reflected light emerging from the reference lens  26  is reflected by the beam splitter  24  along the second optical axis  36  and is focused by the imaging lens  32  onto the CCD detector  34 . The CCD detector observes only the information in the focal plane of the reference lens  26 . Thus, by shifting the reference mirror  28  it is possible to select different planes of the image for observation. As the reference lens  26  is matched to the high NA objective lens  20 , these different planes are imaged without introducing spherical aberration. 
     As the mirror  28  can be very small and light, and there is no contact between the mirror and the reference lens, its position can be adjusted very quickly using the piezo transducer, allowing for axial scans at a high frequency, for example typically 100-1000 Hz, which is many times faster than the frequency of about 15 Hz available with existing systems. 
     In the example shown in  FIG. 2  the objective lens  20  and the reference lens  26  are identical. It is also possible to use two different high NA lenses, providing that the mapping lens system  22  is modified to provide the correct magnification. For example, it is possible to use a 0.95 NA dry lens as the reference lens together with any other lens as the objective lens without affecting the resolution set by the objective lens, since the 0.95 NA dry lens has the largest angular aperture of all commercially available lenses. It is therefore possible to use a dry lens for the reference lens and an immersion lens for the objective. In this case, the reference mirror would not need to be brought into contact with the reference lens via an immersion medium. This provides the advantage that the reference mirror and the reference lens could be placed in a sealed chamber, since the user would not need access to those components. A modified form of the system is depicted in  FIG. 3 . In this system, the beam splitter  24  is replaced by a polarizing beam splitter  24 ′, and a quarter wave plate  45  is located between the beam splitter  24 ′ and the reference lens  26 . The other components of the system are the same as those of the first system described above. When used with polarized light, this system provides the advantage that there is no attenuation of the light reaching the CCD detector  34 . 
     A third embodiment of the invention is shown in  FIG. 4 . In this embodiment, the microscope system includes an objective lens  120 , a mapping lens system  122   a,b , a first reference lens  124 , a second reference lens  126 , an imaging lens  132  and a CCD detector  134 , all spaced along an optical axis  136 . The mapping lens system  122   a,b  maps the wavefront appearing at the pupil plane of the objective lens  120  onto the pupil plane of the first reference lens  124 . The objective lens  120  and the first and second reference lenses  124 , 126  are identical or matched high NA lenses. 
     The second reference lens  126  is mounted so as to be moveable relative to the first reference lens  124  along the optical axis  136 , and is attached to a piezo transducer (not shown) that allows its position relative to the first reference lens  124  to be adjusted rapidly as indicated by arrow  137 . 
     Light from a specimen is collected by the objective lens  120  and mapped by the mapping lens system  122   a,b  onto the pupil plane of the first reference lens  124 , which focuses the light to form an image in an intermediate image space  138  between the first reference lens  124  and the second reference lens  126 . As the objective lens  120  and the first reference lens  124  are matched and designed to the same mathematical condition, an image of the specimen is reconstructed without aberration in the intermediate image space  138 . The second reference lens  126  collects and focuses the light emerging from this image space. The wavefront emerging from the second reference lens  126  is focused by the imaging lens  132  onto the CCD detector  134 . 
     If the focal plane of the second reference lens  126  is located in the focal plane of the first reference lens  124 , the wavefront reconstructed at the pupil plane of the second reference lens  126  will be identical to that entering the first reference lens  124  from the mapping lens system  122 . However, if the second reference lens  126  is displaced axially, it will gather light from a different plane within the intermediate image zone and the reconstructed wavefront will then contain information representing a different plane in the object space. The CCD detector  134  observes only the information in the focal plane of the second reference lens  126 . Thus, by shifting the second reference lens  126 , it is possible to select different planes of the image for observation, without introducing spherical aberration. As with the previous system, the position of the second reference lens  126  can be adjusted very quickly, for example using a suitable transducer, allowing for axial scans at a high frequency. 
     The system is designed to map a ray leaving a specimen in the object space  140  at an angle γ to a conjugate ray entering an intermediate image space  138  also with an angle γ. If this is the case then the light distribution in the object space is re-imaged without aberration in the intermediate image space  138 . The detector assembly  132 , 134  images a single plane of intermediate image space. Moving the second reference lens  126  along the axis selects different planes for imaging and the resulting images formed on the detector  134  are all free from aberration. In this embodiment it may be preferable for packaging reasons that the objective lens  120  and the first reference lens  124  are identical. However, this is not essential, providing that the objective lens  120  and the first reference lens  124  are optically matched. 
     The arrangement shown in  FIG. 4  has the advantage that there is no significant light loss with either polarized or unpolarized light, since a beam splitter is not used. The disadvantage is that refocusing may be slower than with a mirror, owing to the greater mass of the second reference lens  126 . However, it should still be considerably faster than conventional systems. 
     The systems described above and shown in  FIGS. 2 to 4  are all intended for use in microscopic imaging systems, in which an image of a specimen is focused onto a detector. Such applications include wide field microscopy, confocal microscopy, non-linear microscopy, stereo microscopy, scanning microscopy and slit scanning microscopy, using techniques such as reflection, transmission, fluorescence, phase contrast and polarization imaging. 
     The invention is however also applicable to illumination systems in which light from a source is focused onto a specimen. Such applications include micro-fabrication (e.g. micro-machining through optical ablation and photo-induced polymerization), laser surgery, photoporation of cells (the selective creation of a hole in a cell wall for transfection), optical trapping, atom trapping, data storage (e.g. the writing of data in 3-dimensional multilayer optical storage media), and non-linear microscopy (e.g. multi-photon microscopy, second harmonic generation microscopy and third harmonic generation microscopy). 
     The layout of the illumination system may be virtually identical to any one of the imaging systems shown in  FIGS. 2 to 4 , except that in each case the CCD detector is replaced by a suitable light source and, if required, a suitably-scaled mask or equivalent device for creating an illumination pattern that is to be focused onto the specimen. The axial position of the illumination pattern within the specimen can be adjusted by moving the reference mirror or the second reference lens as appropriate, thereby refocusing the system. Again no movement of either the objective lens or the specimen is required and spherical aberration is avoided. 
     The invention is also applicable to applications that involve both illuminating a specimen and collecting light emerging from the specimen, for example scanning confocal microscopy (by reflection or fluorescence), non-linear microscopy with a detection pinhole and data storage (e.g. with confocal read-out of 3D, multilayer storage media). The invention may be used to eliminate spherical aberration both in the illuminating radiation and in the light collected from the specimen, thus allowing high quality images to be captured in axially-spaced planes. 
     A practical embodiment of the invention in the form of a microscope objective assembly  200  is shown in  FIG. 5 . The objective assembly  200  is designed for use on a conventional microscope, as a replacement for an existing objective assembly. 
     This objective assembly  200  is based on the arrangement shown in  FIG. 4  and includes an objective lens  220 , a first reference lens  224  and a second reference lens  226 , all mounted within a housing  228  and spaced along a common optical axis  230 . The microscope also includes an imaging lens (or tube lens)  232  and a CCD detector  234 . The objective lens  220  and the first reference lens  224  are mounted back-to-back so that their pupil planes coincide. The wavefront appearing at the pupil plane of the objective lens  220  in thus mapped onto the pupil plane of the first reference lens  224 . The objective lens  220  and the first and second reference lenses  224 , 226  are identical high NA lenses. 
     The second reference lens  226  is mounted within the housing  228  so as to be moveable along the optical axis  230  relative to the first reference lens  224 , and is attached to a suitable transducer (not shown) that allows its position relative to the first reference lens  224  to be adjusted rapidly as indicated by arrow  238 . Light emerging from the pupil plane of the second reference lens  226  is focused by the imaging lens  232  onto the CCD detector  234 . 
     In use, light from a specimen in the object space  240  is collected by the objective lens  220  and focused by the first reference lens  224  in an intermediate image space  242  between the first reference lens  224  and the second reference lens  226 . As the objective lens  220  and the first reference lens  224  are identical, an image of the specimen is reconstructed without aberration in the intermediate image space  242 . The second reference lens  226  collects and focuses the light emerging from this image space  242 . The wavefront emerging from the second reference lens  226  is focused by the imaging lens  232  onto the CCD detector  234 . 
     If the focal plane of the second reference lens  226  is located in the focal plane of the first reference lens  224 , the wavefront reconstructed at the pupil plane of the second reference lens  226  will be identical to that entering the first reference lens  224  from the objective lens  220 . However, if the second reference lens  226  is displaced axially, it will gather light from a different plane within the intermediate image zone and the reconstructed wavefront will then contain information representing a different plane in the object space. The CCD detector  234  observes only the information in the focal plane of the second reference lens  226 . Thus, by shifting the second reference lens  226 , it is possible to select different planes of the image for observation without introducing spherical aberration. As with the previous system, the position of the second reference lens can be adjusted very quickly, allowing for axial scans at a high frequency. 
     Another practical embodiment of the invention in the form of a plug-in assembly  300  for the camera port of an existing microscope is shown in  FIG. 6 . The assembly  300  is designed to plug in between the microscope objective lens  310  and an existing camera or CCD detector  312 . Alternatively, the CCD detector  312  may be part of the plug-in assembly  300 . The microscope also includes a tube lens  314  that focuses light from the pupil plane of the objective lens  310  onto an entry window  316  of the assembly  300 . 
     The plug-in assembly  300  is again based on the arrangement shown in  FIG. 4  and includes a housing  318 , a mapping lens  322 , a first reference lens  324 , a second reference lens  326 , and an imaging lens  328 , all spaced along a common optical axis  330 . The tube lens  314  and the mapping lens  322  together map the wavefront appearing at the pupil plane of the objective lens  310  onto the pupil plane of the first reference lens  324 . The objective lens  310  and the first reference lens  324  are identical or matched high NA lenses. 
     The second reference lens  326  is mounted within the housing  318  so as to be moveable along the optical axis  330  relative to the first reference lens  324 , and is attached to a transducer (not shown) that allows its position relative to the first reference lens  324  to be adjusted rapidly as indicated by arrow  332 . Light emerging from the pupil plane of the second reference lens  326  is focused by the imaging lens  328  through an exit window  334  onto the CCD detector  312 . 
     In use, light from a specimen in the object space  340  is collected by the objective lens  310  and mapped by the tube lens  314  and the mapping lens  322  onto the pupil plane of the first reference lens  324 . This light is focused by the first reference lens  324  in an intermediate image space  342  between the first reference lens  324  and the second reference lens  326 . The objective lens  310  and the first reference lens  324  are identical or matched, and an image of the specimen is thus reconstructed without aberration in the intermediate image space  342 . The second reference lens  326  collects and focuses the light emerging from this image space. The wavefront emerging from the second reference lens  326  is focused by the imaging lens  328  through an exit window  334  onto the CCD detector  312 . 
     When the focal plane of the second reference lens  326  is located in the focal plane of the first reference lens  324 , the wavefront reconstructed at the pupil plane of the second reference lens  326  will be identical to that entering the first reference lens  324  from the objective lens  310 . However, if the second reference lens  326  is displaced axially, it will gather light from a different plane within the intermediate image zone and the reconstructed wavefront will then contain information representing a different plane in the object space. The CCD detector  312  observes only the information in the focal plane of the second reference lens  326 . Thus, by shifting the second reference lens  326 , it is possible to select different planes of the image for observation without introducing spherical aberration. The position of the second reference lens can be adjusted very quickly, allowing axial scans to be performed at a high frequency. 
     An alternative plug-in assembly  400  for the camera port of an existing microscope is shown in  FIG. 7 . The assembly  400  is designed to plug in between the microscope objective lens  410  and the existing camera or CCD detector  412 . The microscope also includes a tube lens  414  that focuses light from the pupil plane of the objective lens  410  onto an entry window  416  of the assembly  400 . 
     The plug-in assembly  400  is based on the arrangement shown in  FIG. 3  and includes a housing  418 , a mapping lens  422 , beam splitter  424 , a quarter wave plate  425 , a reference lens  426  and a reference mirror  428 , all spaced along a first optical axis  430 . The objective lens  410  and the reference lens  426  are identical high NA lenses. An imaging lens (or “tube” lens)  432  and the CCD detector  412  are spaced along a second optical axis  436  that intersects the first optical axis  430  at right angles where it passes through the beam splitter  424 . 
     The tube lens  414  and the mapping lens  422  map the wavefront appearing at the pupil plane of the objective lens  410  onto the pupil plane of the reference lens  426 . The objective lens  410  and the reference lens  426  are identical or matched high NA lenses. 
     The mirror  428  is mounted within the housing  418  so as to be moveable along the optical axis  430  relative to the reference lens  426 , and is attached to a piezo transducer (not shown) that allows its position relative to the reference lens  426  to be adjusted rapidly as indicated by arrow  438 . Light emerging from the pupil plane of the reference lens  426  is focused by the imaging lens  432  through an exit window  434  onto the CCD detector  412 . 
     In use, light from a specimen in the object space  440  is collected by the objective lens  410  and focused by the reference lens  426  in an intermediate image space  442  adjacent the mirror  428 . As the objective lens  410  and the reference lens  426  are identical or matched, an image of the specimen is reconstructed without aberration in the intermediate image space  442 . This light is reflected by the mirror  428  back towards the reference lens  426 , which collects and focuses the reflected light. The wavefront emerging from the reference lens  426  is then focused by the imaging lens  432  onto the CCD detector  412 . 
     When the mirror  428  is located in the focal plane of the reference lens  426 , the wavefront reconstructed at the pupil plane of the reference lens  426  will be identical to that entering the lens from the objective lens  410 . However, if the mirror  428  is displaced axially, it will reflect light from a different plane within the intermediate image zone and the reconstructed wavefront will then contain information representing a different plane in the object space. The CCD detector  412  observes only the information in the focal plane of the reference lens  426 . Thus, by shifting the mirror  428 , it is possible to select different planes of the image for observation without introducing spherical aberration. The position of the mirror can be adjusted very quickly using the piezo transducer, allowing axial scans to be performed at a high frequency. 
       FIG. 8  illustrates a slit scanning confocal microscope according to a seventh embodiment of the invention. The microscope includes a laser light source  510 , a beam expander  512 , a cylindrical lens  514  and a narrow slit  516 , which together form a flat beam of light, which is focused by a lens  518  into a reference lens  520 . This light is focused by the reference lens  520  into an intermediate image space  521  adjacent a reference mirror  522 , which is mounted for movement along the optical axis  524  of the aforesaid components. The light is reflected back into the reference lens  520  by the mirror  522  and then reflected along a second optical axis  526  by a beam splitter  528 . This light is focused through an intermediate lens system  530  into an objective lens  532 , which focuses the flat light beam onto a specimen  534 . 
     The axial position at which the light beam is focused within the specimen  534  can be adjusted by moving the reference mirror  522 . The objective lens  532  and the reference lens  520  are matched to eliminate any spherical aberration from the illuminating beam. 
     The light reflected by the specimen is collected by the objective lens  532  and focused through the intermediate lens system  530 , the beam splitter  528  and the reference lens  520  into the intermediate image space  521  adjacent the mirror  522 . The light is then reflected by the mirror  522  back into the reference lens  520  and along the first optical axis  524 . This light is reflected by a second beam splitter  536 , a pair of lenses  538 ,  540  and a rotatable mirror  542  onto a CCD detector  544 . Rotating the mirror  542  allows a line image to be scanned across the detector  544 . 
     Moving the reference mirror  522  in the direction of the first optical axis  524  adjusts simultaneously both the position of the focused illuminating beam within the specimen  534 , and the image plane focused onto the detector  544 . The image plane can thus be scanned through the specimen in the direction of the optical axis  526  (the Z axis) of the objective lens  532 . By simultaneously adjusting the axial position of the reference mirror  522  and the angular position of the rotatable mirror  542 , an image representing an XZ plane of the specimen can be captured in real time, without requiring any subsequent processing. 
       FIG. 9  shows a microscope system similar to that shown in  FIG. 3 , including a polarizing beam splitter  24 ′ and a quarter wave plate  45  located between the beam splitter  24 ′ and the reference lens  26 . The other components of the system are the same as those of the system described above. 
     When used with polarized light, there is no attenuation of the light reaching the image plane  34 ′. However, when the system is used with unpolarized light, the light  130  entering the polarizing beam splitter  24 ′ is divided into two parts, the first part  130   a  passing through the beam splitter while the second part  130   b  is reflected from it. As this second part is not captured, the light reaching the image plane  34 ′ is attenuated by 50%. 
       FIG. 10  shows a modified version of the microscope system shown in  FIG. 9 , in which this problem is addressed. The microscope system includes an objective lens  620   a,b , a mapping lens system  622 , a polarizing beam splitter  624  and a first image forming and capturing system  625 , all located on a first optical axis  630 . The first image forming and capturing system  625  is similar to that shown in  FIG. 9 , comprising a reference lens  626 , a reference mirror  628  and a quarter wave plate  645 . A second image forming and capturing system  625 ′ is located on a second optical axis  636  that intersects the first optical axis  630  at a right angle where it passes through the beam splitter  624 . The second image forming and capturing system  625 ′ comprises a reference lens  626 ′, a reference mirror  628 ′ and a quarter wave plate  645 ′. An imaging lens  632  and a detector  634  are also located on the second optical axis  636 , on the opposite side of the beam splitter  624 . 
     Light from a specimen  642  is collected by the objective lens  620 , split into two parts by the beam splitter  624  and mapped onto the pupil planes of the reference lenses  626 , 626 ′. Each reference lens  626 , 626 ′ focuses the light to form an image of the specimen in an intermediate image space adjacent the respective reference mirror  628 , 628 ′, which reflects the reconstructed image back into the respective reference lens  626 , 626 ′. The reflected light is recombined by the beam splitter  624  and focused by the imaging lens  632  onto the detector  634 . The two mirrors are adjusted synchronously to ensure that the reflected images are identical. Both parts of the light emerging from the beam splitter  624  are used, thereby avoiding undesirable attenuation. 
     An alternative arrangement is shown in  FIGS. 11-13 , in which the microscope system includes an objective lens  720 , a mapping lens system  722 , a discriminator  724  and an image forming and capturing system  725 , all located on a first optical axis  730 . The image forming and capturing system  725  comprises a reference lens  726 , a reference mirror  728  and a quarter-wave plate  745 . An imaging lens  732  and a detector  734  are located on a perpendicular second optical axis  736 . 
     The discriminator  724 , which is shown in more detail in  FIGS. 12 and 13 , comprises a polarizing prism (in this case a Glan-Thompson prism) made of two birefringent calcite crystals  746  that are joined by a layer of cement  748 . As shown in  FIG. 12 , the prism splits the unpolarized light beam  750  entering the prism into an s-polarized extraordinary ray  752  that is transmitted without significant deviation through the prism and an p-polarized ordinary ray  754  that is reflected internally from the calcite/cement interface at an angle to the extraordinary ray  752 . Both rays pass through the quarter-wave plate  745  and the reference lens  726  and are then reflected by the mirror  728  back through the lens and the plate. This rotates the polarization of both rays through 90°. The rays then re-enter the discriminator as shown in  FIG. 13 , which recombines the rays to form a single unpolarized beam  756  that leaves the prism at a small angle relative to the original beam  750 . 
     The recombined beam  756  is then reflected by an angled mirror  758  along the second optical axis  736 , so that it passes through the imaging lens  732  into the detector  734 . Because the recombined beam  756  is shifted through a small angle relative to the original beam, the mirror  758  can be displaced to one side of the first optical axis, so that it does not impede the input beam. 
     In this arrangement, all the light from a specimen  742  is collected and focused onto the detector  734 , thus avoiding attenuation. Only a single refocusing mirror  728  is required and this can be adjusted at high speed to image different planes of the sample.