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.
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.
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 θ1 relative to the second surface, where the angle θ1 is in the range of 0°<θ1<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 θ1 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 θ1 relative to a horizontal axis that is orthogonal to a vertical axis. The angle θ1 is in the range of 0<θ1<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 θ1 relative to a horizontal axis that is orthogonal to a vertical axis, where the angle θ1 is in the range of 0<θ1<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 θ1 relative to a horizontal axis that is orthogonal to a vertical axis, where the angle θ1 is in a range of 0<θ1<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.
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:
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
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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 θ1 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 θ1 relative to a horizontal axis that is orthogonal to a vertical axis, in the range of 0<θ1<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, θ1 relative to a horizontal axis that is orthogonal to a vertical axis, in the range of 0<θ1<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.
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.
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.
Number | Date | Country | |
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60860755 | Nov 2006 | US |
Number | Date | Country | |
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Parent | 11943222 | Nov 2007 | US |
Child | 12788186 | US |