Abstract:
A wide-field optical microscope and method capable of resolving images down to 0.1 Å with a magnification range in excess of 250 million power includes an objective having a primary and a secondary element. A sample is held so that the area of interest is at a location that is closer to the primary element than the focal length of the primary element. The primary element collects and collimates light reflected from the sample. The secondary element then focuses the collimated light onto a pinhole aperture, which blocks all light rays that were not parallel, thus producing a non-focused reflected pattern. The non-focused reflected pattern passes through a field stop and is then magnified by one or more negative optical elements and additional field stops to produce an enlarged pattern.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    This invention relates generally to optical microscopy and in particular to reflected-light (epi-illumination) wide-field microscopy. 
         [0002]    In a conventional non-scanning (wide-field) optical microscope, radiation (typically visible light, ultraviolet light or x-rays) interacts (e.g. by reflection, refraction, or diffraction) with a specimen located at the object plane of the microscope optics, to create a pattern that is processed by the microscope to create an image that is enlarged to a size that is many times greater than the specimen itself. In a conventional reflected-light wide-field compound refractive microscope, radiation reflected from the specimen passes through an objective that focuses the radiation into an enlarged real image on an intermediate image plane. The intermediate image is then additionally magnified by an eye lens which produces an enlarged real or virtual image depending on the particular application. 
         [0003]    It is well-know that at very high magnifications optical microscopes will exhibit the characteristic that point objects are seen as fuzzy disks surrounded by diffraction rings. The resolving power of a microscope is taken as the ability of the microscope to reveal adjacent structural detail as distinct and separate. Even assuming perfect refraction by the lens system of the microscope, the minimum size of the diffraction rings, which determines the resolution (d) of the microscope is still limited function by both the wavelength of light (λ), and the numerical aperture (NA) of the objective lens as expressed by the following equation. 
         [0000]    
       
         
           
             d 
             = 
             
               λ 
               
                 2 
                  
                 NA 
               
             
           
         
       
     
         [0004]    In practice, the highest numerical aperture that can be achieved in air is about 0.95. Therefore and with green light (about 550 nm wavelength) the diffraction limit of a conventional optical microscope is about 200 nm (the Abbe Limit). 
         [0005]    Numerous methods have been successfully employed over the years in order to gain ever increasing magnification and resolution, including use of ultraviolet light and x-rays (shorter wavelength) and oil immersion (increased numerical aperture). These methods, however, are still limited by the Abbe limit of the optical system. A technique for increasing resolution beyond the Abbe limit of an optical system is laser scanning confocal microscopy. In a laser confocal microscope, a laser beam passes through a light source pinhole aperture and beam splitter and is focused by an objective lens onto the specimen. The light reflected from the specimen is focused through the objective lens onto the beam splitter, which reflects the light onto a photo-detector through a pinhole aperture. The pinhole aperture blocks any reflected light that is not emanating from the focal point of the objective (which would be out of focus). Laser confocal microscopes operate above the Abbe limit of the optical system but do not produce a wide-field image. Consequently the image must be constructed by scanning the sample point by point. 
       SUMMARY OF THE INVENTION 
       [0006]    What is needed therefore and what the prior art lacks is an apparatus and method for producing a wide-field image that is capable of magnification and resolution far in excess of the Abbe limit of the optical system. The present invention satisfies the foregoing need by providing a wide-field optical microscope and method capable of resolving images down to 0.1 Å with a magnification range in excess of 250 million power. According to an illustrative embodiment of the invention, the microscope includes an objective having a primary and a secondary element. A sample is held so that the area of interest is at a location that is closer to the primary element than the focal length of the primary element. The primary element collects and collimates light reflected from the sample. The secondary element then focuses the collimated light onto a pinhole aperture, which blocks all light rays that were not parallel, thus producing a non-focused reflected pattern. The non-focused reflected pattern passes through a field stop and is then magnified by one or more negative optical elements and additional field stops to produce an enlarged pattern which can be displayed on a screen or subjected to additional processing to produce a positive image of the sample. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0007]    The present invention will be better understood from a reading of the following detailed description, taken in conjunction with the accompanying drawing figures in which like references designate like elements and, in which: 
           [0008]      FIG. 1  is a schematic view of a microscope incorporating features of the present invention; 
           [0009]      FIG. 2  is an enlarged view of a portion of the microscope of  FIG. 1 ; 
           [0010]      FIG. 3  is an enlarged view of another portion of the microscope of  FIG. 1 ; 
           [0011]      FIG. 4  is an enlarged view of yet another portion of the microscope of  FIG. 1 ; 
           [0012]      FIG. 5  is a schematic view of an alternative embodiment of a microscope incorporating features of the present invention; 
           [0013]      FIG. 6  is a schematic view of another alternative embodiment of a microscope incorporating features of the present invention; and 
           [0014]      FIG. 7  is a schematic view of yet another alternative embodiment of a microscope incorporating features of the present invention; 
           [0015]      FIG. 8  is a schematic view of a prototype embodiment of a microscope incorporating features of the present invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0016]    The drawing figures are intended to illustrate the general manner of construction and are not necessarily to scale. In the detailed description and in the drawing figures, specific illustrative examples are shown and herein described in detail. It should be understood, however, that the drawing figures and detailed description are not intended to limit the invention to the particular form disclosed, but are merely illustrative and intended to teach one of ordinary skill how to make and/or use the invention claimed herein and for setting forth the best mode for carrying out the invention. 
         [0017]    With reference to the figures and in particular to  FIG. 1 , a microscope  10  incorporating features of the present invention comprises a primary magnification stage  12  and a secondary magnification stage  14 . Primary magnification stage  12  comprises a first optical element comprising a first lens  16  and a second optical element comprising a second lens  18 . As discussed more fully hereinafter, although the illustrative embodiment of  FIG. 1  incorporates refractive elements, equivalent reflective elements may be incorporated into the apparatus without departing from the scope of the invention. Accordingly, as used herein, optical elements means and refers both to reflective and refractive optical elements. 
         [0018]    First optical element  16  has a focal point  20 , which is the single point at which collimated light entering first optical element  16  is focused. Microscope  10  further includes means for supporting a specimen  22 . As shown in  FIG. 2 , means for supporting specimen  22  is configured so that the specimen is located at a distance “D” which is less than the distance from first optical element  16  to focal point  20 . Means for supporting specimen  22  may be any conventional microscope specimen support, platten or other stable movable support and therefore the invention should not be considered as limited to any particular support. 
         [0019]    Primary magnification stage  12  further comprises a primary stage field stop  24  which comprises a pinhole aperture  26  located at the focal point  28  of second optical element  18 . As can be determined from the foregoing, light from a light source  30  is reflected off of a specimen located on specimen support  22 . That portion of the reflected light which is parallel to a corresponding ray emanating from focal point  20  is collimated by first optical element  16 . The collimated light then passes through secondary optical element  18  which focuses the light onto primary stage field stop  24 . Pinhole aperture  26  blocks all light that is not is parallel to the optical axis between the primary optical element  16  and primary optical element  18 . Light source  30  can be any source of illumination in the electromagnetic spectrum from infrared, to visible, to ultraviolet, to x-rays and may be coherent or non-coherent, polarized or non-polarized from any number of sources including LEDs, lasers, arc lamps, incandescent lamps or any other source of electromagnetic radiation. 
         [0020]    As can be determined from the foregoing, since the sample surface is always closer to the first optical element  16  than the distance to its focal point  20  (i.e. the sample is always within the focal cone of first optical element  16 ), the reflected image is not a focused image in the classical sense but is a reflected pattern of the sample surface. The distance from the sample to the first optical element  16  determines the magnification. The closer to the first optical element  16  the sample is, the lower the magnification and the closer to the focal point of the first optical element  16  the higher the magnification. The larger the physical dimension (diameter) of the first optical element  16 , and the smaller the focal spot size, the higher the magnification (as opposed to a conventional microscope in which magnification is determined by the half angle of the focal cone). In practice, magnification of the primary magnification stage of the present invention can be up to 100,000× power. 
         [0021]    With additional reference to  FIG. 4 , in the illustrative embodiment, secondary magnification section  14  comprises a first secondary stage field stop  32 , a second secondary stage field stop  34  and a third secondary field stop  36 . Light passing through pinhole aperture  26  of primary stage field stop  24  impinges first secondary stage field stop  32  where all but all but that portion of the light passing through field stop aperture  38  is blocked. The portion of light that passes through field stop aperture  38  then passes through third optical element  40 , which in the illustrative embodiment is a double concave lens or similar negative optical element, which causes the light to diverge until it impinges second secondary stage field stop  34 . Second secondary field stop  34  blocks all but that portion of light passing through field stop aperture  42 . 
         [0022]    That portion of light passing through field stop aperture  42  then passes through fourth optical element  44 , which in the illustrative embodiment is a double concave lens or similar negative optical element, which causes the light to diverge until it impinges third secondary stage field stop  36 . Third secondary field stop  36  blocks all but that portion of light passing through field stop aperture  46 . Light exiting through field stop aperture  46  then forms a pattern on display means  50  which in the illustrative embodiment of  FIG. 1  is a view screen. Although in the illustrative embodiment, display means  50  is a view screen, any conventional apparatus for viewing a light pattern including a CCD camera, or conventional microscope eyepiece may be incorporated without departing from the scope of the present invention and therefore the invention should not be considered as limited to any particular display means. 
         [0023]    With additional reference to  FIG. 5 , in an alternative embodiment microscope  510  comprises a primary magnification stage  512  and a secondary magnification stage  514 . Primary magnification stage  512  comprises a first optical element comprising a first lens  516  and a second optical element comprising a second lens  518 . First optical element  516  has a focal point  520 , which is the single point at which collimated light entering first optical element  516  is focused. Microscope  510  further includes means for supporting a specimen  522  so that the specimen is located at a distance “D” which is less than the distance from first optical element  516  to focal point  520 . 
         [0024]    Primary magnification stage  512  further comprises light focusing elements  410  and  412 , a non-polarizing beam splitter  414 , a light absorber  416 , and a primary stage field stop  524  which comprises a pinhole aperture  526  located at the focal point  528  of second optical element  518 . As can be determined from the foregoing, light from a light source  530  passes through light focusing elements  410  and  412  which collimate the light from light source  530  and direct the light onto non-polarizing beam splitter  414 . A portion of the light passing through non-polarizing beam splitter  414  is focused on to specimen  522  which is positioned within the focal cone of first optical element  516 . That portion of the light reflected off of specimen  522  which is parallel to a corresponding ray emanating from focal point  520  is collimated by first optical element  516 . The collimated light then passes through non-polarizing beam splitter  414  and a portion is directed onto secondary optical element  518 . Secondary optical element  518  focuses the light onto primary stage field stop  524 . Pinhole aperture  526  blocks all light that is not is parallel to the optical axis between the primary optical element  516  and primary optical element  518 . The portion of the collimated light passing through non-polarizing beam splitter  414  that is not directed onto secondary optical element  518  is absorbed by light absorber  416 . 
         [0025]    In the illustrative embodiment of  FIG. 5 , secondary magnification section  514  comprises a first secondary stage field stop  532 , a second secondary stage field stop  534  and a third secondary stage field stop  536 . Light passing through pinhole aperture  526  of primary stage field stop  524  impinges first secondary stage field stop  532  where all but all but that portion of the light passing through field stop aperture  538  is blocked. The portion of light that passes through field stop aperture  538  then passes through third optical element  540 , which in the illustrative embodiment is a double concave lens or similar negative optical element, which causes the light to diverge until it impinges second secondary stage field stop  534 . Second secondary field stop  534  blocks all but that portion of light passing through field stop aperture  542 . That portion of light passing through field stop aperture  542  then passes through fourth optical element  544 , which in the illustrative embodiment is a double concave lens or similar negative optical element, which causes the light to diverge. Light exiting through field stop aperture  546  then forms a pattern on display means  550 . 
         [0026]    As noted hereinbefore, although the illustrative embodiment of  FIG. 1  incorporated refractive elements, equivalent reflective elements may be incorporated into the apparatus without departing from the scope of the invention. In the illustrative embodiment of  FIG. 6 , a microscope  610  incorporating features of the present invention comprises a primary magnification stage  612  and a secondary magnification stage  614 . Primary magnification stage  612  comprises a first optical element comprising a first off-axis parabolic mirror  616  and a second optical element comprising a second off-axis parabolic mirror  618 . First optical element  616  has a focal point  620 , which is the single point at which collimated light reflected by first optical element  616  is focused. Microscope  610  further includes means for supporting a specimen  622  so that it is located at a distance “D” which is less than the distance from first optical element  616  to focal point  620 . 
         [0027]    Primary magnification stage  612  further comprises a primary stage field stop  624  which comprises a pinhole aperture  626  located at the focal point  628  of second optical element  618 . As can be determined from the foregoing, light from a light source  630  is reflected off of a specimen  622 . That portion of the reflected light which is parallel to a corresponding ray emanating from focal point  620  is collimated by first optical element  616 . The collimated light is then reflected by secondary optical element  618  which focuses the light onto primary stage field stop  624 . Pinhole aperture  626  blocks all light that is not is parallel to the optical axis between the primary optical element  616  and primary optical element  618 . 
         [0028]    In the illustrative embodiment of  FIG. 6 , secondary magnification section  614  comprises a first secondary stage field stop  632  and a second secondary stage field stop  634 . Light passing through pinhole aperture  626  of primary stage field stop  624  impinges first secondary stage field stop  632  where all but all but that portion of the light passing through field stop aperture  638  is blocked. The portion of light that passes through field stop aperture  638  is then reflected by third optical element  640 , which in the illustrative embodiment is a convex off-axis parabolic mirror or similar negative optical element, which causes the light to diverge until it impinges second secondary stage field stop  634 . Second secondary field stop  634  blocks all but that portion of light passing through field stop aperture  642 . That portion of light passing through field stop aperture  642  is then reflected by fourth optical element  644 , which in the illustrative embodiment is a convex off-axis parabolic mirror or similar negative optical element, which causes the light to diverge until it impinges third secondary stage field stop  636 . Third secondary field stop  636  blocks all but that portion of light passing through field stop aperture  646 . Light exiting through field stop aperture  646  then forms a pattern on display means  650 . 
         [0029]    With additional reference to  FIG. 7 , in an alternative embodiment microscope  710  comprises a primary magnification stage  712  and a secondary magnification stage  714 . Primary magnification stage  712  comprises a first optical element comprising a first concave off-axis parabolic mirror  716  and a second optical element comprising a second concave off-axis parabolic mirror  718 . First optical element  716  has a focal point  720 , which is the single point at which collimated light reflected by first optical element  716  is focused. Microscope  710  further includes means for supporting a specimen  722  so that the specimen is located at a distance “D” which is less than the distance from first optical element  716  to focal point  720 . 
         [0030]    Primary magnification stage  712  further comprises light focusing elements  420  and  422 , a non-polarizing beam splitter  424 , a light absorber  426 , and a primary stage field stop  724  which comprises a pinhole aperture  726  located at the focal point  728  of second optical element  718 . As can be determined from the foregoing, light from a light source  730  passes through light focusing elements  420  and  422  which collimate the light from light source  730  and direct the light onto non-polarizing beam splitter  424 . A portion of the light passing through non-polarizing beam splitter  424  is focused on to specimen  722  which is positioned within the focal cone of first optical element  716 . That portion of the light reflected off of specimen  722  which is parallel to a corresponding ray emanating from focal point  720  is collimated by first optical element  716 . The collimated light then passes through non-polarizing beam splitter  424  and a portion is directed onto secondary optical element  718 . Secondary optical element  718  focuses the light onto primary stage field stop  724 . Pinhole aperture  726  blocks all light that is not is parallel to the optical axis between the primary optical element  716  and primary optical element  718 . The portion of the collimated light passing through non-polarizing beam splitter  424  that is not directed onto secondary optical element  718  is absorbed by light absorber  426 . 
         [0031]    In the illustrative embodiment of  FIG. 7 , secondary magnification section  714  comprises a first secondary stage field stop  732  and a second secondary stage field stop  734 . Light passing through pinhole aperture  726  of primary stage field stop  724  impinges first secondary stage field stop  732  where all but all but that portion of the light passing through field stop aperture  738  is blocked. The portion of light that passes through field stop aperture  738  is then reflected by third optical element  740 , which in the illustrative embodiment is a convex off-axis parabolic mirror or similar negative optical element, which causes the light to diverge until it impinges second secondary stage field stop  734 . Second secondary field stop  734  blocks all but that portion of light passing through field stop aperture  742 . That portion of light passing through field stop aperture  742  is then reflected by fourth optical element  744 , which in the illustrative embodiment is a convex off-axis parabolic mirror similar negative optical element, which causes the light to diverge until it impinges third secondary stage field stop  736 . Third secondary field stop  736  blocks all but that portion of light passing through field stop aperture  746 . Light exiting through field stop aperture  746  then forms a pattern on display means  750 . 
         [0032]    According to an inventive method, specimen  22 ,  422 ,  522 ,  622 ,  722  is moved away from first optical element  16 ,  516 ,  616 ,  716  in order to increase magnification of the reflected image. Alternatively, the focal length of first optical element  16 ,  516 ,  616 ,  716  is decreased (although still remaining greater than “D”) to increase magnification of the reflected image. 
         [0033]    A prototype microscope was constructed in accordance with the basic dimensions shown on  FIG. 8 . As can be determined from an inspection of  FIG. 8 , the first optical element comprised a positive  200  mm by convex lens with a focal length of 220 mm. The sample was placed at a location 219.9 mm from the first optical element. Light from a 488 nanometer 25 milliwatt argon laser was focused onto the specimen through a 25 mm non-polarized cubic beam splitter. Light reflected off the specimen passed through a second optical element which comprised a 68 mm by convex lens located 25 mm from the beam splitter. The primary stage field stop comprised a pinhole aperture 0.030 inches in diameter located 60 mm from the second optical element. The secondary magnification stage consisted of a −20 mm biconcave lens and a −25 mm biconcave lens with associated field stops which projected the final image onto a 0.3 micron lapping film view screen. The actual magnification of the apparatus shown in  FIG. 8  was 5 million power. 
         [0034]    Although certain illustrative embodiments and methods have been disclosed herein, it will be apparent from the foregoing disclosure to those skilled in the art that variations and modifications of such embodiments and methods may be made without departing from the invention. Accordingly, it is intended that the invention should be limited only to the extent required by the appended claims and the rules and principles of applicable law. Additionally, as used herein, references to direction such as “up” or “down” are intend to be exemplary and are not considered as limiting the invention and, unless otherwise specifically defined, the terms “generally,” “substantially,” or “approximately” when used with mathematical concepts or measurements mean within ±10 degrees of angle or within 10 percent of the measurement, whichever is greater.