Abstract:
In accordance with one embodiment of the present invention an apparatus for a low numerical aperture exclusion imaging apparatus is provided. The apparatus may include an electromagnetic illumination source for illuminating a portion of a specimen; and for collecting an image created by the electromagnetic radiation an objective lens optically coupled to the electromagnetic illuminated portion of the specimen. The apparatus also includes an optical blocking plate disposed between the objective lens and a focusing lens. The optical blocking plate is positioned to substantially block undesired electromagnetic radiation from image sources distally aligned in the same optical axis as the specimen. This invention is enhances narrow depth of field characteristics in imaging. It also enhances discreet imaging in a narrow focus field by eliminating some or most of the light which contributes to wide depth of field focus. This is useful for optical sectioning ranging from microscopy to photography. Optical sectioning provides the information necessary for 3D image reconstructions and other X Axis spatial measurements.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is related to, claims the earliest available effective filing date(s) from (e.g., claims earliest available priority dates for other than provisional patent applications; claims benefits under 35 USC §119(e) for provisional patent applications), and incorporates by reference in its entirety all subject matter of the following listed application(s) (the “Related Applications”) to the extent such subject matter is not inconsistent herewith; the present application also claims the earliest available effective filing date(s) from, and also incorporates by reference in its entirety all subject matter of any and all parent, grandparent, great-grandparent, etc. applications of the Related Application(s) to the extent such subject matter is not inconsistent herewith.
       1. U.S. patent application Ser. No. 12/994,264 entitled “LOW NUMERICAL APERTURE EXCLUSION IMAGING”, naming Guy G. Kennedy as inventor, filed Nov. 23, 2010.       
 
     
    
     BACKGROUND 
       [0003]    1. Field of Use 
         [0004]    These teachings relate generally to a system and method for microscopy imaging in general and more particularly to a low numerical aperture exclusion. 
         [0005]    2. Description of Prior Art (Background) 
         [0006]    Microscopes have been known for some time in the existing art. Very generally, in upright and inverted light microscopes, focusing of the specimen image is accomplished by way of a corresponding positioning of the specimen relative to the objective, specifically in such a way that a specimen region to be detected is arranged in the focal plane of the objective. This can be achieved on the one hand by the fact that the objective, optionally together with the objective turret receiving the objective, is positioned along the optical axis relative to the specimen. In this case the specimen, for example mounted on a conventional specimen slide, is clamped in a corresponding holder on the microscope stage, this microscope stage then not being moved in the direction of the optical axis of the microscope objective. This type of focusing is usually utilized with inverted light microscopes. 
         [0007]    On the other hand, the microscope stage can be arranged movably relative to the microscope stand, and positioned in the direction of the optical axis for focusing. In this case the objective does not perform a motion in the direction of its optical axis relative to the microscope stand. The latter type of focusing is usually utilized with upright light microscopes. 
         [0008]    Focusing with the aid of the microscope stage also exists, for purposes of the present invention, when the microscope stage comprises a mechanism with which a specimen slide performs a positioning relative to the objective with the aid of a linear or pivoting motion controlled by a galvanometer, as is the case, for example, with some confocal laser scanning microscopes. 
         [0009]    Imaging for scientific research has been evolving since the invention of the microscope. Demand has driven the directions in which these innovations have evolved. In recent years, science has brought the demand for high Z axis resolution to satisfy the need for 3D spatial information in molecular studies. Several technologies have been developed to satisfy this need. Notably, Confocal Microscopy, Two Photon Microscopy, TIRF Microscopy, and most recently STED Microscopy have come to the forefront. All of these techniques have high resolution imaging in a narrow field of focus. 
         [0010]    A narrow field of focus facilitates stacking layers of images called “optical sections” to create high resolution 3D composites of thicker sections. Confocal Microscopy and Two Photon Microscopy are very expensive, but fairly versatile. TIRF Microscopy costs less, has the most discrete Z axis imaging, but is only useful within 200 nanometers of the glass surface thus allowing only one optical section. STED Microscopy is also very expensive, with limited availability. 
         [0011]    All of these techniques benefit from the elimination or minimization of the out of focus light, improving the signal to noise ratio. Research has resulted in a demand for mapping trajectories of molecules within cells in 3D. This demand is coupled with the need to image with ever increasing frame rates to provide high resolution 3D position at high temporal resolution. There are several variables which limit the speed of such a system, such as the sensitivity of the detection system (camera, photo-detector, etc.), the speed in which the excitation can scan (Confocal, Two Photon), and the speed in which stage steps in the z axis between scans or images. All of these techniques are effective at excluding unwanted out of focus light. This allows improved imaging in a crowded environment. 
         [0012]    Traditional microscopy benefits from a wide depth of field. Depth of field refers to the z axis distance in which an image is in high resolution focus. This is a fundamental property of imaging optics. Cameras have an F-stop which is an adjustable aperture located next to the focusing lens. Some microscope lenses have a similar adjustable aperture. When this aperture is reduced in diameter, less light goes through the lens. More importantly, the light that remains is centered through the middle region on the lens with the exclusion of light near the edges. This condition results in a projected image which is illuminated by light rays which are proportionately more normal in angle to the object and image. Because the angles of these rays are smaller with respect to each other, the focus point is less distinct in the Z axis resulting in a wider range of acceptable focus resolution. An extreme example of this effect is a pinhole camera. In this case the rays are not bent at all by a lens, and thus everything at any distance is in focus. A general observation of this effect would to squint one&#39;s eyes in order to read in low light. We benefit in our focus ability from bright light because our pupils are small. A wide aperture passes more light, but has a narrower depth of field. A narrow aperture, passes less light, and produces a wider depth of field. With traditional camera or microscopy configurations when the aperture is open, the depth of field is at its smallest. 
       BRIEF SUMMARY 
       [0013]    The foregoing and other problems are overcome, and other advantages are realized, in accordance with the presently preferred embodiments of these teachings. 
         [0014]    In accordance with one embodiment of the present invention an apparatus for a low numerical aperture exclusion imaging apparatus is provided. The apparatus includes an electromagnetic illumination source for illuminating a portion of a specimen; and for collecting an image created by the electromagnetic radiation an objective lens optically coupled to the electromagnetic illuminated portion of the specimen. The apparatus also includes an optical blocking plate disposed between the objective lens and a focusing lens, wherein the optical blocking plate is positioned to substantially block undesired electromagnetic radiation images transmitted through a center portion of the objective lens. 
         [0015]    The invention is also directed towards a low numerical aperture exclusion imaging apparatus having an electromagnetic illumination source for examining a specimen. Wherein the low numerical aperture exclusion imaging apparatus includes an objective lens optically coupled to the electromagnetic illuminated portion of the specimen, for and a focusing lens for focusing the image onto a focus plane. The imaging apparatus also includes an optical blocking plate disposed between the objective lens and the focusing lens and is positioned to substantially block electromagnetic radiation transmitted through a center portion of the objective lens. The optical blocking plate includes an absorptive optical filter disposed substantially concentrically with the optical blocking plate; and an optically transparent window disposed contiguously around the outer edge of the absorptive optical filter. 
         [0016]    In accordance with another embodiment of the present invention an apparatus a system for low numerical aperture exclusion imaging of a specimen is included. The system includes an optical blocking plate; and an objective lens disposed between the specimen and optically coupled to the specimen, for collecting an image of the specimen. The optical blocking plate is positioned to substantially block electromagnetic radiation. In addition, the system includes multiple types of objective lenses such as, but not limited to, achromat objective lens, a plan achromat objective lens, a fluorite objective lens, a plan fluorite objective lens, and a plan apochromat objective lens. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]    The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings in which: 
           [0018]      FIG. 1  is a side view of a simple microscope in which the teachings of the present invention is implemented; 
           [0019]      FIG. 2  is a perspective view of the microscope shown in  FIG. 1 , in which several elements are removed so as to obtain a better impression of the microscope stand in accordance with the invention shown in  FIG. 1 ; 
           [0020]      FIG. 3  is a perspective view of the microscope from the front in accordance with the invention shown in  FIG. 1 ; 
           [0021]      FIG. 4  is an optical diagram of one embodiment of the low numerical aperture imaging features using a single optical blocking aperture in accordance with the invention shown in  FIG. 1 ; 
           [0022]      FIG. 5  is an optical diagram of an alternate embodiment of the low numerical aperture imaging features using a plurality of optical blocking apertures in accordance with the invention shown in  FIG. 1 ; 
           [0023]      FIG. 6  is an optical diagram of another embodiment of the low numerical aperture imaging features using a second plurality of optical blocking apertures in accordance with the invention shown in  FIG. 5 ; 
           [0024]      FIG. 7  is an optical diagram of another embodiment of the low numerical aperture imaging features using a third plurality of optical blocking apertures in accordance with the invention shown in  FIG. 5 ; and 
           [0025]      FIG. 8  is an optical diagram of another embodiment of the low numerical aperture imaging features using a fourth plurality of optical blocking apertures in accordance with the invention shown in  FIG. 5 . 
       
    
    
     DETAILED DESCRIPTION 
       [0026]    Referring now to  FIG. 1  there is shown a side view of a simple microscope in which the teachings of the present invention is implemented. It will be understood that any suitable microscope or microscopy system may be used in accordance with the present invention. 
         [0027]    Still referring to  FIG. 1 , microscope  1  encompasses a microscope stand  2 . Microscope  1  stands on a support surface  10 . Also provided on microscope stand  2  is a turret  3  that carries at least one objective  4 . Objective  4  can be pivoted by means of turret  3  into a working position. Objective  4  possesses an optical axis  5  that, in the working position of objective  4 , runs perpendicular to a microscope stage  6 . A specimen  7  to be investigated can be placed on microscope stage  6 . 
         [0028]    Microscope  1  also includes a focusing device  20  serving to focus specimen  7 , focusing device  20  being installed in the interior of microscope stand  2 . With this focusing device  20 , microscope stage  6  is positionable relative to microscope stand  2  in the direction of optical axis  5  of objective  4 . Focusing device  20  carries two operating elements  8  (only one operating element  8  is depicted in the side view of  FIG. 1 ). Operating elements  8  are provided on the two side walls  2   a  and  2   b  of microscope stand  2 . With these operating elements  8 , a user can enable the displacement of microscope stage  6  in the direction of optical axis  5 . The displacement of microscope stage  6  results in a focusing of specimen  7  present on microscope stage  6 . 
         [0029]    Still referring to  FIG. 1 , a displacement element  9  for microscope stage  6  is positioned directly in front of operating element  8 . Displacement element  9  is connected to microscope stage  6  and enables a displacement of microscope stage  6  perpendicular to optical axis  5 , by which means specimen  7  can be positioned in the image field of objective  4 . Displacement element  9  encompasses an X element  9   a  that enables displacement of microscope stage  6  in the X direction. Displacement element  9   a  further encompasses a Y element  9   b  that enables displacement of microscope stage  6  in the Y direction. 
         [0030]    Still referring to  FIG. 1 , microscope  1  includes low numerical aperture (NA) exclusion imaging aperture plates  44  and  44 A which will be discussed in more detail herein. 
         [0031]      FIG. 2  is a perspective view of microscope  1  in which some elements are removed in order to obtain a better impression of microscope stand  2  and its low NA exclusion imaging aperture  44  configuration. As described herein, Low NA Exclusion Microscopy does not require X-Y axis scans for its excitation or detection when applied in a wide field configuration. The focus simply needs to be shifted between image acquisitions. These discrete focal plane images can be used as optical sections for accurate three dimensional image reconstructions. Because the light gathered is specific to the narrow depth of field component of the light, out of focus portions of the image are excluded improving the discreetness of the focal plane. 
         [0032]    Microscope stand  2  possesses a flange  11 , e.g. for attaching a binocular eyepiece (not depicted). This is not, however, to be construed in any way as a limitation. Microscope stand  2  further comprises a holding element  12  for microscope stage  6  (see  FIG. 1 ). Holding element  12  is movable by focus device  20  parallel to optical axis  5  of objective  4  that is located in the working position. Focusing device  20  installed in the interior of microscope stand  2  possesses a first end  14   a  and a second end  14   b  (not shown). First end  14   a  and second end  14   b  engage through an opening  15  on first and on second side wall  2   a  and  2   b , respectively, of microscope stand  2 . Illumination source  21  may be any suitable illumination source such as used in bright field microscopy. As already explained in the description relating to  FIG. 1 , an operating element  8  can be attached respectively onto first and second ends  14   a  and  14   b  of focusing device  20 . 
         [0033]    In the exemplary embodiment depicted in  FIG. 2 , opening  15  possesses the shape of a curved elongated hole  13  that is embodied on the oppositely located side walls  2   a  and  2   b  of microscope stand  2 . A focusing device (not shown) installed in the interior of microscope stand  2  likewise engages with first end  14   a  and with second end  14   b  (not shown) through opening  15  on first and on second side wall  2   a  and  2   b , respectively, of microscope stand  2 . In this embodiment, opening  15  is configured as longitudinal opening in first and in second side wall  2   a  and  2   b.    
         [0034]    Referring also to  FIG. 3  there is shown a perspective view of microscope  1  from the front. In the interior, microscope  2  is constructed in part from multiple struts  16 . Several installation positions  17  are likewise configured in the interior of holding element  12  of microscope stage  2 . Each of installation positions  17  comprises a first stop surface  17   a  and a second stop surface  17   b . First and second stop surface  17   a  and  17   b  run perpendicular to one another and are configured in such a way that a component (not depicted) to be attached at that position can be attached with screws in the position without further alignment. In  FIG. 3 , second end  14   b  of focusing device  20  provided in the interior of microscope stand  2  is visible on second side wall  2   b . A rotation axis  18  of focusing device  20  is likewise accessible via first and second side wall  2   a  and  2   b.    
         [0035]    Referring also to  FIG. 4 , there is shown an optical diagram of one embodiment of the low numerical aperture imaging features using a single optical blocking aperture  44  in accordance with the invention shown in  FIG. 1 . Objective lens  4  may be any suitable objective lens type. For example, the objective lens type may be achromat, plan achromat, fluorite, plan fluorite, or plan apochromat. It will be further appreciated that the present invention may be used with any suitable imaging lens or assembly in addition to, or in place of, the objective lens. 
         [0036]    Still referring to  FIG. 4 , optical blocking aperture  44  may be any suitable optical blocking aperture with a suitable reflection and absorption coefficient to prevent or minimize undesired light from reflecting into objective lens  4 . It will also be understood that aperture  44  may also be any suitable wavelength specific blocking aperture. Aperture  44  is suitably positioned to block or eliminate undesired light  49 A (or other portions of the electromagnetic spectrum) emanating from object  46  which is distally aligned with particle  48  in the same optical axis  5 . It will be appreciated that this arrangement allows desired light depicted by dashed lines  49 B, 49 C (or other portions of the electromagnetic spectrum) emanating or reflecting from particle of interest  48  through objective  4 , to be detected directly by the eye, imaged on a photographic plate or captured digitally. It will be understood that particle  48  may be illuminated by any suitable light source. It will be further understood that particle  48  may be a fluorescent particle emitting light when excited by a suitable excitation source. It will also be further appreciated that the light depicted by  49 B,  49 C which travels through the outer diameter of the objective lens  4  contributes to a narrow depth of field component of an image of the particle of interest  48 , because the rays travel at greater angles. 
         [0037]    It is also understood that aperture  44  excluding the light traveling through the near center of objective lens  4 , that light which produces the widest depth of field, is eliminated. It will be appreciated that this creates a specificity of light, selecting the portion which is derived within the image plane. The result is a narrower depth of focus than could be achieved with conventional optical microscopy or photographic configurations. 
         [0038]    Referring also to  FIG. 5 , there is shown an optical diagram of an alternate embodiment of the low numerical aperture imaging features using a plurality of optical blocking apertures in accordance with the invention shown in  FIG. 1 . As described earlier, optical blocking apertures  54 A,  54 B,  54 C,  54  may by any suitable optical blocking apertures. In addition, it will be understood that each of the blocking apertures  54 A,  54 B,  54 C and  54  may be different types of blocking apertures. For example, blocking apertures  54 A,  54 B, and  54 C may each be different wavelength filters. Still referring to  FIG. 5 , particle  56 A lying before the object plane emanates light, depicted by dashed lines  59 A, which is blocked by blocking aperture  54 A. Particle  58 A emanates or reflects light, depicted by dashed lines  59 B,  59 C which passes by blocking aperture  54  to focusing lens  52  to be focused as image  58 B on image plane  51 . It will be understood that in alternate embodiments blocking aperture  54  may also include optically transparent windows  542 ,  541 . It will be appreciated that optically transparent windows  542 ,  541  may be any suitable optical material such as optical glass. It will also be appreciated that optically transparent windows  542 ,  541  may be wavelength specific filters allowing only selected wavelengths from particle  58 A to be focused on focus plane  58 B. This overall configuration advantageously blocks defocusing light from particle  56 A while simultaneously selecting specific wavelengths of interest. 
         [0039]    Referring also to  FIG. 6 , there is shown an optical diagram of another embodiment of the low numerical aperture imaging features using a second plurality of optical blocking apertures in accordance with the invention shown in  FIG. 5 . Light rays emanating from particle  68 A on the object plane  41  passes through objective lens  4  and focusing lens  52  to be imaged  68 B on image plane  51 . Light rays emanating from particle  66 , lying in the Z direction beyond the objective plane are blocked by blocking apertures  64 A and  64 B. 
         [0040]    Referring also to  FIG. 7  there shown is an optical diagram of another embodiment of the low numerical aperture imaging features using a third plurality of optical blocking apertures in accordance with the invention shown in  FIG. 5 . As shown in  FIG. 7 , unwanted optical rays from particle  7 Z, depicted as dashed lines  79 A,  79 B are blocked by blocking aperture  74 B. Likewise, although not shown in  FIG. 7 , unwanted light rays are also blocked by blocking aperture  74 A located after focusing lens  52 . It will be understood that blocking apertures  74 A and  74 B prevent light from particle  7 Z from defocusing, or otherwise interfering with light from particle  78 A passing through objective  4  and focus lens  52  and imaging on image plane  51  as particle  78 B. 
         [0041]    Referring also to  FIG. 8 , there is shown an optical diagram of another embodiment of the low numerical aperture imaging features using a fourth plurality of optical blocking apertures in accordance with the invention shown in  FIG. 5 . Blocking apertures  84 A and  84 C prevent unwanted light rays with obtuse angles, with respect to the objective  4 , from defocusing desired light from particle  88 A. Likewise, blocking aperture  84 D prevents unwanted light from passing through center region of objective  4 . Blocking aperture  84 B prevents unwanted focused light from reaching the image plane and distorting the focused light from particle  88 A and imaged as particle  88 B on the image plane. 
         [0042]    As shown by  FIG. 4  through  FIG. 8 , it will be appreciated that the location and method of the light exclusion blocking apertures is variable depending upon the specific application. It will also be appreciated, as noted earlier, that the light exclusion blocking apertures can be wavelength specific. An example of this would be use of an optical filter medium which would filter certain wavelengths, while allowing other wavelengths to pass. 
         [0043]    It should be understood that the foregoing description is only illustrative of the invention. Thus, various alternatives and modifications can be devised by those skilled in the art without departing from the invention. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variances that fall within the scope of the appended claims. For example, Low NA Exclusion Objective microscopy features, as described herein, can be applied to most forms of light microscopy. The benefit of detecting and or exciting with the High NA component of light is achievable in both Brightfield and Fluorescence microscopy. For example, In Confocal Microscopy, the benefit from a Low NA Exclusion Objective lens arrangement as described herein would also apply to fluorescence excitation and fluorescence emission. The Z axis of the Confocal spot, is longer than the XY axis. If only the High NA component of excitation light is utilized to produce the Confocal spot, The Z axis illumination will be smaller resulting in a thinner Z axis scan. 
         [0044]    It will also be appreciated that there is an ever increasing demand for higher resolution light microscopy. New developments continue to provide ever increasing resolution, particularly in the XY axis which is beyond what was believed possible only 10 years ago. Recently major microscopy manufactures have introduced High Numerical Aperture Objective lenses. These lenses were created to satisfy the demand for “Through the Lens” TIRF Microscopy. These lenses are a natural choice when considering Low NA Exclusion Microscopy features as described herein. Low NA Exclusion Microscopy features as described herein can be a modification made to older modest microscopy systems, or as a feature to new manufactured microscopes, including TIRF microscopes. It can be a very inexpensive addition that would add important capability. There are numerous configurations for this invention that dovetail into existing microscopy systems. 
         [0045]    Likewise, the inventive features described herein can also be used as enhancement for Photo-Activated Localization Microscopy, also known as PALM Microscopy. This new form of high resolution light microscopy can use the narrower field of light provided by the invention described herein to make the photo-activated regions more specific in the Z axis. Similarly, Stimulated Emission Depletion Microscopy, or STEAD, is another apparatus that can use the narrower field of light provided by the invention described herein to result in higher Z axis resolution.