Patent Publication Number: US-2011058252-A1

Title: Bottomless micro-mirror well for 3d imaging for an object of interest

Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATION 
     This application is a continuation-in-part of U.S. patent application Ser. No. 11/943,222, filed Nov. 20, 2007, entitled “PHOTOLITHOGRAPHED MICRO-MIRROR WELL FOR 3D TOMOGRAM IMAGING OF INDIVIDUAL CELLS,” by Kevin Truett Seale, Ron Reiserer, and John Wikswo, which is incorporated herein by reference in its entirety and which itself claims the benefit, pursuant to 35 U.S.C. §119(e), of U.S. provisional patent application Ser. No. 60/860,755, filed Nov. 22, 2006, entitled “PHOTOLITHOGRAPHED MICRO-MIRROR WELL FOR 3D TOMOGRAM IMAGING OF INDIVIDUAL CELLS,” by Kevin Truett Seale, Ron Reiserer and John Wikswo, which is incorporated herein by reference in its entirety. 
     Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference. 
    
    
     STATEMENT OF FEDERALLY-SPONSORED RESEARCH 
     The present invention was made with Government support awarded by the Air Force Office of Scientific Research (AFOSR) under Contract No. FA9550-05-1-0349. The United States Government has certain rights to this invention pursuant to this grant. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to a bottomless micro-mirrored well, and more particularly to a bottomless micro-mirrored pyramidal well, its applications in multi-perspective three-dimensional (3D) microscopy to simultaneously collect images of an object of interest from multiple vantage points and a method of manufacturing same. 
     BACKGROUND OF THE INVENTION 
     One of the burgeoning areas of the development of modern microscopy is three-dimensional (3D) microscopy, which acquires a three-dimensional image with every image plane sharply in focus. This is in contrast to conventional microscopy where the image of the in-focus plane is superposed with a blurred image of out-of-focus planes. Several developments of 3D microscopy have been reported. These techniques have been gaining popularity in the scientific and industrial communities. Typical applications include life sciences and semiconductor inspection. 
     An inverted microscope is a microscope with its light source and condenser on the top above the stage pointing down, and the objectives and turret are below the stage pointing up. Inverted microscopes are useful for observing living cells or organisms at the bottom of a container of fluid (e.g., a well-plate with a thin bottom, or a tissue culture flask) under more natural conditions than on a glass slide, with an objective some distance above the sample, as is the case with a conventional microscope. Some inverted microscopes are also capable of epi-illumination, in which light is passed through the microscope objective lens to illuminate the sample from the same side that it is observed. This is particularly useful for fluorescence imaging, wherein the excitation and emission light are at different wavelengths and can be separated with optical filters. 
     In confocal scanning microscopy (CSM), the out-of-focus signal is spatially filtered out by confocal aperturing of the object illumination and the detector points. The 3D image is constructed by pixel-by-pixel mechanical scanning of the entire object volume, which places a fundamental limit on the image acquisition speed. 
     A catadioptric system that uses a curved mirror to map a panoramic view onto a single sensor is able to obtain multi-perspective 3D images of an object, but has limited sensor resolution. Furthermore, the resolution varies significantly with the viewing direction across the field of view (FOV). 
     Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies. 
     SUMMARY OF THE INVENTION 
     In one aspect, the present invention relates to a bottomless micro-mirror well. In one embodiment, the bottomless micro-mirror well includes a substrate having a first surface and an opposite, second surface and defining a body portion between the first surface and the second surface, where the body portion defines an inverted pyramidal well having at least three side surfaces extending to each other and defining an opening between a first sidewall and a second sidewall, and where each of the at least three side surfaces is configured to reflect light emitting from an object of interest. The horizontal cross-sectional shape of the inverted pyramidal well is a polygon or a circle. The vertical cross-sectional shape of the inverted pyramidal well is a trapezoid if the well has an even number of sides or is circular, and an irregular quadralateral if the well has an odd number of sides. The object of interest is a biological analyte that includes cells and proteins. Also, each of the side surfaces includes a dichroic mirror. 
     In another embodiment, the bottomless micro-mirror well enables transillumination of an object within or adjacent to the well, such that light scattered or emitted fluorescently from the object is collected by the mirrors on the sides of the well. 
     In one embodiment, the bottomless micro-mirror well enables epi-illumination of the object through the microscope objective but does not produce a direct reflection of this illumination light back into the objective because the well does not have a mirrored bottom as would occur in the wells described in the earlier patent application. 
     In one embodiment, the bottomless micro-mirror well enables the use of a post or platform passing through the smaller opening of the well to allow mechanical positioning of the object within the well, for example to adjust the position of the object within the well to the point where the images are at a chosen depth of focus for the objective. 
     In another embodiment, the bottomless micro-mirror well allows the flow of fluids through the opening of the well to deliver drugs or toxins to affect the object being observed or provide nutrients and remove metabolic products of a living cell or organism being maintained within the well. 
     In one embodiment, the substrate includes a silicon wafer, and the object of interest includes a biological analyte with cells and proteins. Each of the at least three side surfaces defines an angle θ 1  relative to the second surface, where the angle θ 1  is in the range of 0°&lt;θ 1 &lt;90°. For a point object, the inverted pyramidal well produces images that are equidistant from all of the side surfaces, and the position where the object should be located to accomplish this is termed the focus of the well, is inside the inverted pyramidal well and equidistant from all side surfaces. In operation, the inverted pyramidal well is positioned in relation to the object of interest such that the object of interest is located inside of the inverted pyramidal well. 
     In another embodiment, the position of the focus is outside the inverted pyramidal well. In this embodiment, in operation, the inverted pyramidal well is positioned in relation to the object of interest such that the object of interest is located outside of the inverted pyramidal well. 
     In another aspect, the present invention relates to a process of fabricating a bottomless micro-mirror well. In one embodiment, the process includes the steps of providing a silicon substrate and etching off the silicon substrate to form a bottomless inverted pyramidal well in the silicon substrate, where the bottomless inverted pyramidal well has a first end, an opposite, second end, and a plurality of side surfaces extending to each other and defining an opening at the second end. The process also includes the step of performing photolithographically masking and evaporating processes on the plurality of side surfaces, to form a mirrored pyramidal well. The bottomless inverted pyramidal well has a central axis running through the center of the well, from a first end to a second end, as well as a planar axis that is perpendicular to the central axis, where each of the plurality of side surfaces is formed to define an angle θ 1  relative to the planar axis. The etching step is performed with a potassium hydroxide (KOH) etching process. 
     In one embodiment, the process further includes the step of fabricating a master mold from the mirrored pyramidal well, for replication of at least one additional mirrored pyramidal well. The master mold is fabricated through hot embossing, injection molding, casting, and/or another method of mass fabrication of objects. 
     In yet another aspect, the present invention relates to a three-dimensional microscope. In one embodiment, the three-dimensional microscope includes a microscope objective lens adapted for focusing light from a plurality of mirrors configured to simultaneously collect images of an object of interest from multiple vantage points. The object of interest includes a biological analyte including cells and proteins. The plurality of mirrors forms one or more bottomless mirrored pyramidal wells, where each of the plurality of mirrors has an angle θ 1  relative to a horizontal axis that is orthogonal to a vertical axis. The angle θ 1  is in the range of 0&lt;θ 1 &lt;90°. 
     In one embodiment, the three-dimensional microscope also includes a microfluidic structure in communication with the one or more bottomless mirrored pyramidal wells. The one or more bottomless pyramidal wells is/are made from the smooth angled surfaces of anisotropically etched silicon. Each of the plurality of mirrors includes a dichroic mirror capable of reflecting specific wavelength ranges into a collection cone of the microscope objective lens. The plurality of mirrors is affixed such that the perimeter of the field of view of the microscope objective lens contains reflected images of the object of interest. 
     In one embodiment, the plurality of mirrors is affixed opposite the object of interest from the microscope objective lens, for collecting reflected images of the object of interest. 
     In yet another aspect, the present invention relates to a three-dimensional microscope. In one embodiment, the three-dimensional microscope includes a microscope objective that is adapted for focusing the light produced by a plurality of mirrors that are configured to simultaneously collect images of an object of interest, from multiple vantage points. Each of the plurality of mirrors forms one or more bottomless mirrored pyramidal wells. At least one of the bottomless pyramidal wells that are formed is made from the smooth angled surfaces of anisotropically etched silicon. The object of interest includes a biological analyte with cells and proteins, and a microfluidic structure that is in communication with the bottomless mirrored pyramidal well. 
     In one embodiment, each of the plurality of mirrors defines an angle θ 1  relative to a horizontal axis that is orthogonal to a vertical axis, where the angle θ 1  is in the range of 0&lt;θ 1 &lt;90°. Each of the plurality of mirrors comprises a dichroic mirror that is capable of reflecting specific wavelength ranges into a collection cone of the microscope objective lens. Also, the plurality of mirrors is affixed such that the perimeter of the field of view of the microscope objective lens contains reflected images of the object of interest. 
     In another embodiment, the plurality of mirrors is affixed opposite an object of interest from the microscope objective lens, for collecting reflected images of the object of interest. 
     In yet another aspect, the present invention relates to a method for reconstruction of simultaneous, multi-vantage point images into three-dimensional structures of an object of interest. In one embodiment, the method includes the steps of simultaneously collecting images of the object of interest from multiple vantage points surrounding the object of interest, and mapping the collected images of the object of interest to form a three-dimensional image displaying the three-dimensional structures of the object of interest. The step of simultaneously collecting images of the object of interest is performed with a bottomless mirrored pyramidal well that has a plurality of side mirrored surfaces extending to each other and that define a first opening and a second opening. Each of the plurality of side mirrored surfaces has a first end and a second end such that the first opening is formed by the respective first ends of the side mirrored surfaces and the second opening is formed by the respective second ends of the side mirrored surfaces. Each of the plurality of side mirrored surfaces has an angle θ 1  relative to a horizontal axis that is orthogonal to a vertical axis, where the angle θ 1  is in a range of 0&lt;θ 1 &lt;90°. The diameter of the first opening is greater than the diameter of the second opening. 
     These and other aspects of the present invention will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment, and wherein: 
         FIG. 1  shows schematically a bottomless micro-mirror well according to one embodiment of the present invention: (A) a top view, and (B) a vertical cross-sectional view; 
         FIG. 2  shows an upright three-dimensional microscope with a bottomless inverted pyramidal well having introverted wells for collecting images of an object of interest within the well, according to one embodiment of the present invention; 
         FIG. 3  shows an upright three-dimensional microscope with a bottomless inverted pyramidal well having extroverted wells for collecting images of an object of interest outside the well, according to one embodiment of the present invention; 
         FIG. 4  shows an inverted three-dimensional microscope with a bottomless pyramidal well having introverted wells for collecting images of an object of interest within the well, according to one embodiment of the present invention; and 
         FIG. 5  shows an inverted three-dimensional microscope with a bottomless pyramidal well having extroverted wells for collecting images of an object of interest, according to one embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like parts throughout the views. As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. 
     The description will be made as to the embodiments of the present invention in conjunction with the accompanying drawings of  FIGS. 1-5 . In accordance with the purposes of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a bottomless micro-mirror well. Wells can be etched through the full thickness of a silicon wafer, to form a mirrored well with sides but an open bottom, or they can be molded or cast out of plastic and then coated with a reflective metallic coating to form mirrors. 
     In one embodiment, now referring to  FIGS. 1A and 1B , the bottomless micro-mirror well  100  includes a substrate  110  having a first surface  111  and an opposite, second surface  112  defining a body portion  113  between the first surface  111  and the second surface  112 . The body portion  113  has a thickness, H. The body portion  113  defines a bottomless inverted pyramidal well  120  having at least three side surfaces  121 ,  122 ,  123  and  124  extending to each other and defining a first opening  130  and a second opening  132 , between a first sidewall  126  and a second sidewall  127 . Each of the side surfaces  121 - 124  has a first end ( 121   a ,  122   a ,  123   a , or  124   a ) and a second end ( 121   b ,  122   b ,  123   b , or  124   b ) such that the first opening  130  is formed by the respective first ends  121   a ,  122   a ,  123   a ,  124   a  of the side surfaces  121 - 124  and the second opening  132  is formed by the respective second ends  121   b ,  122   b ,  123   b ,  124   b  of the side surfaces  121 - 124 . Each of the at least three side surfaces  121 - 124  is configured to reflect light emitting from an object of interest (see e.g.  FIGS. 2 ,  250 ), and each has an angle, θ 1  relative to the second surface  112 , in the range of 0&lt;θ 1 &lt;90°. That is, each of the at least three side surfaces  121 - 124  defines an angle θ 1  relative to a horizontal axis  125  that is orthogonal to a vertical axis  128  such that 0&lt;θ 1 &lt;90°. As shown, the diameter d 1  of the first opening  130  is greater than the diameter d 2  of the second opening  132 . The angles defined between the side surfaces  121 - 124  and the second surface  112  can be the same, or different. Each of the at least three side surfaces  121 - 124  may comprise a dichroic mirror, and the substrate  110  may include a silicon wafer. The horizontal cross-sectional shape of the inverted pyramidal well  120  shown in  FIG. 1  is a square, but alternatively the cross-sectional shape can be another type of polygon, a circle, or an elongated circle. A microfluidic structure may be provided that is in communication with the bottomless mirrored pyramidal well on either or both sides. 
     Now referring to  FIG. 2 , a partial, cross-sectional view of an inverted three-dimensional microscope  200  is shown, with a bottomless inverted pyramidal well  220  having introverted wells for collecting images of an object of interest  250 , according to one embodiment of the present invention. The bottomless inverted pyramidal well  220  includes a substrate  210  having a first surface  211  and an opposite, second surface  212  defining a body portion  213  between the first surface  211  and the second surface  212 . The body portion  213  defines the bottomless inverted pyramidal well  220  having side surfaces  221  (not shown),  222 ,  223  (not shown), and  224  that extend to each other and define an opening  232  between a first sidewall  226  and a second sidewall  227  of the body portion  213 . Each of the side surfaces  221 - 224  is configured to reflect light emitting from the object of interest  250  and each defines an angle θ 2  relative to the second surface  212 , in the range of 45°&lt;θ 2 &lt;90°. As shown, the objective  240  is disposed above the transparent glass or plastic coverslip  260  holding the object of interest  250  and the object of interest  250  is disposed in the opening  232  defined by the bottomless inverted pyramidal well  220 . The inverted pyramidal well  220  has a focus (not shown) being equidistant from all of the at least three side surfaces  221 - 224 , and the position of the focus is inside the inverted pyramidal well  220 . In operation, the inverted pyramidal well  220  is positioned in relation to the object of interest  250  such that the object of interest  250  is located inside of the inverted pyramidal well  220 . 
     Now referring to  FIG. 3 , a partial, cross-sectional view of an upright three-dimensional microscope  300  is shown, with a bottomless inverted pyramidal well  320  having extroverted wells for collecting images of an object of interest  350 , according to one embodiment of the present invention. The bottomless inverted pyramidal well  320  includes a substrate  310  and a body portion  313 . The body portion  313  defines the bottomless inverted pyramidal well  320 , which has side surfaces  321  (not shown),  322 ,  323  (not shown), and  324  that extend to each other and define an opening  332  between a first sidewall  326  and a second sidewall  327  of the body portion  313 . Each of the side surfaces  321 - 324  is configured to reflect light emitted from the object of interest  350  and each defines an angle θ 3  relative to a surface  312 , where 0&lt;θ 3 &lt;45°. As shown, the objective  340  is disposed above the transparent glass or plastic coverslip  360  holding the object of interest  350  and the bottomless inverted pyramidal well  320  is disposed beneath the coverslip  360  and the object of interest  350 . The inverted pyramidal well  320  has a focus (not shown) and the position of the focus is outside the inverted pyramid well  320 . In operation, the inverted pyramidal well  320  is positioned in relation to the object of interest  350  such that the object of interest  350  is located outside of the inverted pyramidal well  320 . 
     Now referring to  FIG. 4 , a partial, cross-sectional view of an inverted three-dimensional microscope  400  is shown, with a bottomless pyramidal well  420  having introverted wells for collecting images of an object of interest  450  within the well, according to one embodiment of the present invention. The bottomless pyramidal well  420  includes a substrate  410  having a first surface  411  and an opposite, second surface  412  defining a body portion  413  between the first surface  411  and the second surface  412 . The body portion  413  defines side surfaces  421  (not shown),  422 ,  423  (not shown), and  424 , which extend to each other and define an opening  432  between a first sidewall  426  and a second sidewall  427  of the body portion  413 . Each of the side surfaces  421 - 424  is configured to reflect light emitted from the object of interest  450  and each defines an angle θ 2  relative to the second surface  412 , in the range of 45&lt;θ 2 &lt;90°. As shown, the objective  440  is disposed beneath the coverslip  460  holding the object of interest  450  and the bottomless pyramidal well  420  is disposed on the coverslip  460  such that the object of interest  450  is located inside the bottomless pyramidal well  420 . 
     Now referring to  FIG. 5 , a partial, cross-sectional view of an inverted three-dimensional microscope  500  is shown, with a bottomless pyramidal well  520  having extroverted wells for collecting images of an object of interest  550 , according to one embodiment of the present invention. The bottomless pyramidal well  520  includes a substrate  510  having a first surface  511  and an opposite, second surface  512  defining a body portion  513  between the first surface  511  and the second surface  512 . The bottomless pyramidal well  520  has side surfaces  521  (not shown),  522 ,  523  (not shown), and  524 , which extend to each other and define an opening  532  between a first sidewall  526  and a second sidewall  527  of the body portion  513 . Each of the side surfaces  521 - 524  is configured to reflect light from the object of interest  550  and each defines an angle θ 3  relative to the second surface  512 , in a range of 0°&lt;θ 3 &lt;45°. As shown, the objective  540  is disposed beneath the coverslip  560 , which holds the object of interest  550 . 
     In another aspect, the present invention relates to a process of fabricating a bottomless micro-mirror well. In one embodiment, the process includes the steps of providing a silicon substrate and etching off the silicon substrate to form a bottomless inverted pyramidal well in the silicon substrate, where the bottomless inverted pyramidal well has a first end, an opposite, second end, and a plurality of side surfaces extending to each other and defining an opening at the second end. The process also includes the step of performing photolithographically masking and evaporating processes on the plurality of side surfaces so as to form the bottomless mirrored pyramidal well. 
     The etching step is performed with a potassium hydroxide (KOH) etching process. The bottomless inverted pyramidal well further includes a central axis running through the center of the well from a first end to a second end and a planar axis perpendicular to the central axis, and wherein each of the plurality of side surfaces is formed to define an angle θ 1  relative to the planar axis. 
     In another aspect, the present invention relates to a three-dimensional microscope. In one embodiment, the three-dimensional microscope includes an objective lens that focuses the light from a plurality of mirrors configured to simultaneously collect images of an object of interest from multiple vantage points, where the plurality of mirrors forms at least one bottomless mirrored pyramidal well, and where each of the plurality of mirrors has an angle θ 1  relative to a horizontal axis that is orthogonal to a vertical axis, in the range of 0&lt;θ 1 &lt;90°. The bottomless pyramidal well is made from the smooth angled surfaces of anisotropically etched silicon. In one embodiment, a microfluidic structure is in communication with the at least one bottomless mirrored pyramidal well, and the object of interest includes a biological analyte including cells and proteins. Each of the plurality of mirrors has a dichroic mirror capable of reflecting specific wavelength ranges into a collection cone of the microscope objective lens. In one embodiment, the plurality of mirrors is affixed such that the perimeter of the field of view of the microscope objective lens contains reflected images of an object of interest. 
     In another embodiment, the plurality of mirrors is affixed opposite an object of interest from the microscope objective lens for collecting reflected images of the object of interest. 
     In yet another aspect, the present invention relates to a method for reconstruction of simultaneous, multi-vantage point images into three-dimensional structures of an object of interest. In one embodiment, the method includes the steps of simultaneously collecting images of the object of interest from multiple vantage points surrounding the object of interest and mapping the collected images of the object of interest to form a three-dimensional image displaying the three-dimensional structures of the object of interest. The step of simultaneously collecting images of the object of interest is performed with a bottomless mirrored pyramidal well having a plurality of side mirrored surfaces extending to each other and defining a first opening and a second opening, where each of the plurality of side mirrored surfaces has a first end and a second end such that the first opening is formed by the respective plurality of first ends of the side mirrored surfaces and the second opening is formed by the respective second ends of the side mirrored surfaces, such that each of the plurality of side mirrored surfaces has an angle, θ 1  relative to a horizontal axis that is orthogonal to a vertical axis, in the range of 0&lt;θ 1 &lt;90°, and where the diameter of the first opening is greater than the diameter of the second opening. The step of simultaneously collecting images of the object of interest includes the step of collecting light from simultaneously emitting fluorophores of the object of interest. 
     According to one or more embodiments of the present invention as set forth above, bottomless, mirrored pyramidal wells are provided which can be physically attached to the objective of an upright microscope and lowered down over a small specimen that is attached to a slide. Bottomless wells can also be incorporated into a microfluidic device where the flow is through the open bottom, allowing the possibility of three-dimensional tracking or even dynamic trapping, as cells or particles move through the system. 
     Also, according to one or more embodiments of the present invention as set forth above, an in-vivo image of a cell (wide-field and confocal, bright-field and fluorescent) can be acquired by means of an introverted well that is polished to remove material from the back surface of the substrate into which the well is formed such that the bottom of the well no longer exists. The bottomless introverted well provides a declinated perspective of in-vivo tissue when placed (without compression) directly on the tissue in question. In addition to the ordinary (XY) microscope or confocal plane, the bottomless mirrored pyramidal well provides four planes which are nearly orthogonal to the XY plane, and thus gives access to planes which may contain the entire junction between adjacent and connected cells, for instance, the junction between epithelial cells. 
     The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. 
     The embodiments were chosen and described in order to explain the principles of the invention and their practical application so as to activate others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.