Optical system and method for illuminating a sample surface - Patents.com

Abstract

To provide a method of illuminating a sample plane.SOLUTION: The method may include providing an illumination subsystem, the illumination subsystem including an optical source and at least one lens, having an optic axis at an incident angle greater than 0° and less than 90° to a normal of the sample plane. The method may also include rotating the illumination subsystem about a pivot point between the optical source and the sample plane on the optic axis so that an adjusted illumination distribution generated by the illumination subsystem at the sample plane has greater symmetry than a reference illumination distribution generated by the illumination subsystem at the sample plane without the rotation about the pivot point.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] Various aspects of the present disclosure relate to a method for illuminating a sample plane.Various aspects of the present disclosure also relate to an optical system. [Background technology]

[0002] In certain biological applications (e.g., digital polymerase chain reaction (PCR)), an array of reaction chambers may contain a biological sample (such as deoxyribonucleic acid (DNA)) and a fluorescent dye. Adjacent reaction chambers are separated by one or more partitions. In most practical cases, it is desirable to obtain a constant fluorescent signal (or at least a nearly constant fluorescent signal) from all partitions in the array (or, in the case of more than one array, a constant fluorescent signal from all partitions in each array). By "constant signal" we mean that when illuminated by the instrument's light source, each partition will produce equal fluorescence if equal concentrations of dye are present in each partition.

[0003] From an optical system design perspective, it is necessary to achieve uniform illumination on the array of partitions in order to obtain a consistent fluorescence signal from each partition in the array of partitions. Due to the fundamental nature of light spreading from a light source to a screen, the resulting illumination distribution is often non-uniform. Figure 1A is a schematic diagram showing an illumination subsystem directly illuminating the sample surface with normal incidence (i.e., 0 degree incidence angle), resulting in a non-uniform light distribution (the center is brighter than the ends). Figure 1B is another schematic diagram showing the use of a beam splitter (commonly done in fluorescence detection) to reflect light onto the sample surface. The reflection is simply a "mirror image" of the direct illumination. Therefore, this also results in the same type of non-uniformity at the sample surface.

[0004] In the practical sense of fluorescence detection, it may be desirable to illuminate the sample face without the use of beam splitters (e.g., to eliminate stray light problems associated with beam splitters or to reduce the cost of the instrument by not having a beam splitter). In such cases, the sample face is illuminated by grazing incidence (i.e., angle of incidence >0 degrees) as shown in Figure 2. Figure 2 shows a schematic diagram of illumination by grazing incidence for fluorescence detection, which results in an asymmetric light distribution at the sample face. In this case, an asymmetric non-uniformity is created, with the end closer to the lens being brighter than the end farther from the lens. Summary of the Invention [Problem to be solved by the invention]

[0005] Various embodiments provide a method of illuminating a sample surface, the method including providing an illumination subsystem having a light source and at least one lens and having an optical axis that forms an angle of incidence with respect to a normal to the sample surface that is greater than 0° and less than 90°, the method also including rotating the illumination subsystem about a pivot point such that an adjusted illumination distribution at the sample surface produced by the illumination subsystem has greater symmetry compared to a reference illumination distribution at the sample surface produced by the illumination subsystem without the pivot about the pivot point on the optical axis between the light source and the sample surface.

[0006] Various embodiments provide an optical system comprising an illumination subsystem having a light source and at least one lens and having an optical axis with an angle of incidence greater than 0° and less than 90° relative to a normal to the sample surface, the illumination subsystem being rotated or rotatable about a pivot point such that an adjusted illumination distribution produced by the illumination subsystem on the sample surface has a higher symmetry compared to a reference illumination distribution on the sample surface produced by F without rotation about a pivot point on the optical axis between the light source and the sample surface. [Brief description of the drawings]

[0007] The invention can be better understood with reference to the detailed description, taken in conjunction with the non-limiting examples and the accompanying drawings, in which: [Figure 1A] FIG. 1A is a schematic diagram showing an illumination subsystem directly illuminating a sample surface at normal incidence (ie, 0 degree angle of incidence), resulting in a non-uniform light distribution (the center is brighter than the edges). [Figure 1B] FIG. 1B is another schematic diagram showing the use of a beam splitter to reflect light onto the sample surface, as is common in fluorescence detection. [Diagram 2] FIG. 2 is a schematic diagram showing illumination with an asymmetric light distribution on a sample surface due to oblique incidence for fluorescence detection. [Diagram 3] FIG. 3 is a general diagram of how the sample plane may be illuminated in accordance with various embodiments. [Figure 4] FIG. 4 is a general diagram of an optical system according to various embodiments. [Diagram 5] FIG. 5 is a schematic diagram of an optical system illustrating the rotation of an illumination subsystem about a pivot point or axis proximate a lens of the illumination subsystem in various embodiments. [Figure 6A] FIG. 6A is an optical design model and simulation of the optical system in various embodiments implemented on a breadboard. [Figure 6B] FIG. 6B illustrates an illumination subsystem of the optical system shown in FIG. 6A, according to various embodiments. [Figure 7A] FIG. 7A is an image showing a two-dimensional (2D) profile of a surface with asymmetric non-uniform illumination distribution. [Figure 7B] FIG. 7B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm), showing the y-dimension profile of an asymmetric non-uniform illumination distribution. [Figure 8A] FIG. 8A is a schematic diagram of the optical system of FIG. 6A with the second tilt being 2.7°, according to various embodiments. [Figure 8B] FIG. 8B is a schematic diagram of an optical illumination subsystem of the optical system shown in FIG. 8A, according to various embodiments. [Figure 9A] FIG. 9A is an image showing a two-dimensional (2D) profile of a surface of illumination with improved symmetry, according to various embodiments. [Figure 9B] FIG. 9B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm), showing the y-dimension profile of the illumination distribution with improved symmetry. [Figure 10] FIG. 10 is an image of a breadboard on which the optical system shown in FIGS. 6 and 8 is configured, according to various embodiments. [Figure 11A] FIG. 11A is an image showing a two-dimensional (2D) profile of the surface of the while paper when the second tilt angle is 0°. [Figure 11B] FIG. 11B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm) showing the y-dimension profile of white paper when the second tilt angle is 0°. [Figure 12A] FIG. 12A is an image showing a two-dimensional (2D) profile of the surface of the while paper when the second tilt angle is 2.7°. [Figure 12B] FIG. 12B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm) showing the y-dimension profile of white paper when the second tilt angle is 2.7°. [Figure 13A] FIG. 13A shows a schematic diagram of a biological entity detection system or device according to various embodiments. [Figure 13B] FIG. 13B is a schematic diagram of the optical system and camera of FIG. 13A in various embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The following detailed description refers to the accompanying drawings, which show by way of illustration specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the present invention. The various embodiments are not necessarily mutually exclusive, and some embodiments may be combined with one or more other embodiments to form new embodiments.

[0009] Embodiments described in the context of one method or optical system may be equally valid for other methods or optical systems, and similarly, embodiments described in the context of a method may be equally valid for an optical system, and vice versa.

[0010] Features described in the context of one embodiment are equally applicable to the same or similar features in other embodiments. Features described in the context of one embodiment are equally applicable to other embodiments even if not explicitly described in the other embodiments. Furthermore, additions and / or combinations and / or alternatives described with respect to features in the context of one embodiment are equally applicable to the same or similar features in the other embodiments.

[0011] In the context of various embodiments, the articles "a," "an," and "the" used in reference to a feature or element include a reference to one or more features or elements.

[0012] In the context of various embodiments, the term "about" or "approximately" applied to a numerical value encompasses the exact value and a reasonable variation.

[0013] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0014] Various embodiments attempt to address the asymmetric non-uniformity caused by oblique illumination.

[0015] The asymmetry on the sample surface shown in Figure 2 when illuminated at oblique incidence is due in part to the dependence of light intensity on 1 / r^2 (one over the square of the radius), where r is the distance from the light source to the illuminated surface (i.e., the sample surface). The shorter the distance between the light source and the illuminated surface, the higher the light intensity. Conversely, the further the light source is from the illuminated surface, the lower the light intensity. As a result, the right edge of the illumination in Figure 2 is farther from the light source (in this case, the illumination subsystem, including the lens and light source, is considered the "light source") and therefore the illumination in that area will be dimmer. Conversely, the left edge of the illumination surface in Figure 2 is closer to the light source and therefore will be brighter.

[0016] Based on the radiometric principles of optics, the intensity distribution on the illuminated surface at oblique incidence is in fact not simply a function of the distance between the light source and the surface, but can also be a function of the angle of incidence of the light rays with respect to the surface, similar to the intensity distribution of the central bright spot on the sample surface shown in Figure 1A-B. When a lens collects light rays from a uniform light source (e.g., a light-emitting diode (LED)), there is always a bright central ray among the entire collection of rays that pass from the lens to the screen. As a result, the final intensity distribution seen on the sample surface in Figure 2 depends on a combination of the distribution profile of the central bright spot, the 1 / r^2 dependence of the light intensity, and the effect of the angle of incidence of the light rays. For example, even though the left side of the illuminated surface in Figure 2 is closer to the light source, the brightest spot of the illuminance will shift slightly to the right of the left edge, which will cause a slight decrease in the brightness on the left side of the sample surface. Therefore, the brightest location on the sample surface at oblique incidence is not necessarily at the left edge of the surface. Rather, the brightest spot may be shifted slightly to the right of the left edge, but will not be in the center of the surface.

[0017] Various embodiments relate to moving the brightest point of oblique incidence illumination toward the center of the sample surface by rotating the illumination subsystem about an axis located anywhere near the lens or by performing a "second tilt" (i.e., tilting the lens and light source together).

[0018] 3 is a schematic diagram of a method for illuminating a sample surface according to various embodiments. The method includes, in step 302, providing an illumination subsystem. The illumination subsystem has a light source and at least one lens and has an optical axis that forms an angle of incidence with respect to a normal to the sample surface that is greater than 0° and less than 90°. The method also includes, in step 304, rotating the illumination subsystem about a pivot point such that an adjusted illumination distribution at the sample surface produced by the illumination subsystem has greater symmetry in comparison to a reference illumination distribution at the sample surface produced by the illumination subsystem without rotation about a pivot point on the optical axis between the light source and the sample surface.

[0019] In other words, various embodiments relate to positioning an illumination subsystem such that an optical axis of the illumination subsystem forms an angle of incidence selected from a range of greater than 0° and less than 90° with respect to a normal to the sample surface. The illumination subsystem includes a light source and at least one lens. The method further includes tilting or rotating the illumination subsystem about a pivot point located on the optical axis such that an adjusted illumination distribution produced by the illumination subsystem tilted or rotated about the pivot point is symmetrical to a reference illumination distribution (i.e., an oblique illumination distribution) produced by an illumination subsystem without tilt or rotation (i.e., a subsystem positioned such that its optical axis has an angle of incidence with respect to a normal to the sample surface without pivoting about a pivot point).

[0020] The pivoting or tilting of the illumination subsystem about the pivot point is referred to as the "second tilt."

[0021] In various embodiments, the adjusted illumination distribution produced at the sample plane by the illumination subsystem is symmetric, with the center point of the adjusted illumination distribution being the brightest. A symmetric illumination distribution can be achieved without using multiple light sources with opposing oblique incidence angles, without using normal incidence illumination, or without using a beam splitter to fold the light beam and project it onto the sample plane. Alternatively, a symmetric illumination distribution can be achieved by projecting the light beam from the light source through at least one lens.

[0022] The angle by which the illumination subsystem rotates (at the second tilt about the pivot point) depends on the angle of incidence, the light source, the at least one lens, the position of the pivot point on the optical axis, the distance between the light source and the at least one lens, and / or the distance between the light source and the sample surface.

[0023] The optical axis is referred to as the original optical axis. Tilting or rotating the illumination subsystem about the pivot point causes the illumination subsystem to align along an additional optical axis (also referred to as a new optical axis) that is different from the original optical axis. The pivot point may be located at the intersection of the optical axis and the additional optical axis. The additional optical axis has an additional angle of incidence with respect to the normal to the sample surface that is greater than 0° and less than 90°. The angle of incidence of the optical axis and the additional angle of incidence of the additional optical axis are different.

[0024] In various embodiments, before the illumination subsystem rotates about the pivot point, the light source and the at least one lens are aligned along an optical axis, and after the illumination subsystem rotates about the pivot point, the light source and the at least one lens are aligned along a further optical axis.

[0025] In various embodiments, the at least one lens comprises a single lens.

[0026] In various embodiments, the illumination subsystem has three light sources, three lenses, and five excitation and five emission filters for mixing and matching different wavelengths.

[0027] In various embodiments, the sample surface is formed by an array of reaction chambers.

[0028] Each reaction chamber of the array of reaction chambers includes a fluorescent dye and is configured to produce fluorescent light by the fluorescent dye upon illumination by the illumination subsystem.

[0029] In various embodiments, the method includes providing an excitation filter between the light source and the sample surface. The excitation filter is configured to filter light generated by the light source. The method also includes providing an imaging lens above the sample surface. The imaging lens is configured to focus fluorescence generated by the dye upon illumination by the illumination subsystem. In various embodiments, the method also includes providing an absorptive filter between the imaging lens and the sample surface. The absorptive filter is configured to separate the fluorescence generated by the dye from light generated by the light source and reflected by the sample surface.

[0030] In some circumstances, each reaction chamber contains or is configured to contain an equal amount of fluorescent dye, and if the amount of biological sample provided to each reaction chamber is also equal, the fluorescence generated by the reaction chambers may be symmetrical.

[0031] In other situations, the amount of dye in each reaction chamber is different. Some of these situations include: (a) a real-time polymerase chain reaction (PCR) system including an optical system according to various embodiments. Groups of reaction chambers may receive different concentrations of target deoxyribonucleic acid (DNA) (tagged with dye); and (b) a digital PCR system including an optical system according to various embodiments. Poisson statistics determine the average number of target DNA molecules that enter each chamber. As a result, there is a variation in the target DNA molecules (and therefore a variation in the dye tagged to the DNA molecules) across all reaction chambers.

[0032] However, it is desirable to provide symmetric illumination across all reaction chambers, regardless of differences in the amount of dye and associated target DNA molecules. This is because the reaction chambers need to be uniformly illuminated for comparison purposes so that it can be determined whether there is in fact a different fluorescence (due to different amounts of dye) from each reaction chamber. Uniform illumination is required to determine differences in dye concentration across the reaction chambers.

[0033] In various embodiments, the light source is configured to generate visible light, infrared light, or ultraviolet light, hi various embodiments, the light source is configured to generate any combination of visible light, infrared light, and ultraviolet light.

[0034] In various embodiments, the light source is a light emitting diode.

[0035] In one embodiment, the distance between the light source and the at least one lens is about 60 mm, the distance between the light source and the sample surface is about 220 mm, the angle of incidence is about 17°, and the illumination subsystem is rotated about 2.7° about the pivot point (in the second rotation or tilt).

[0036] In another embodiment, the distance between the light source and the at least one lens is about 30 mm, the distance between the light source and the sample surface is about 110 mm, the angle of incidence is about 22°, and the illumination subsystem is rotated about 1.3° about the pivot point.

[0037] 4 is a general diagram of an optical system 400 according to various embodiments. The optical system 400 includes an illumination subsystem 402 having a light source 404 and at least one lens 406. The illumination subsystem 402 has an optical axis with an angle of incidence greater than 0° and less than 90° with respect to a normal to the sample surface. The illumination subsystem 402 is pivoted or rotatable about a pivot point such that an adjusted illumination distribution generated by the illumination subsystem 402 on the sample surface has a higher symmetry compared to a reference illumination distribution on the sample surface generated by the illumination subsystem 402 without pivoting about a pivot point on the optical axis between the light source 404 and the sample surface. The optical system 400 is configured to illuminate the sample surface.

[0038] In other words, an optical system 400 can be provided that includes an illumination subsystem 402. The illumination subsystem 402 includes a light source 404 and at least one lens 406. The illumination subsystem 402 has an optical axis with an angle of incidence selected from a range greater than 0° and less than 90° with respect to a normal to the sample surface. The illumination subsystem 402 is pivoted or tilted about a pivot point on the optical axis such that an adjusted illumination distribution produced by an illumination subsystem 402 that is tilted or rotated about a pivot point is more symmetrical than a reference illumination distribution (i.e., an oblique illumination distribution) produced by an illumination subsystem 402 without tilt or rotation (i.e., a subsystem that is positioned such that its optical axis forms an angle of incidence with respect to a normal to the sample surface without pivoting about a pivot point).

[0039] The angle through which the illumination subsystem 402 rotates (due to the second tilt about the pivot point) depends on the angle of incidence, the light source 404, the at least one lens 406, the position of the pivot point along the optical axis, the distance between the light source 404 and the at least one lens 406, and / or the distance between the light source 404 and the sample surface.

[0040] Tilting or pivoting the illumination subsystem 402 about the pivot point causes the illumination subsystem 402 to align along an additional optical axis different from the optical axis. The pivot point may be located at the intersection of the optical axis and the additional optical axis. The additional optical axis has an additional angle of incidence with respect to the normal to the sample surface that is greater than 0° and less than 90°.

[0041] In various embodiments, the pivot point can be implemented as a pivot axis. The subsystem 402 further includes a frame that holds the light source 404 and at least one lens 406. The frame is attached to the pivot axis, and the light source 404 and at least one lens 406 are pivoted or can be pivoted about the pivot point. In various embodiments, the pivot angle of the illumination subsystem 402 can be determined during fabrication or assembly of the optical system 400. The optical system 400 can include a frame that holds the light source 404 and at least one lens 406.

[0042] In various embodiments, the adjusted illumination distribution at the sample plane produced by the illumination subsystem 402 may be a symmetric distribution, with the central point of the adjusted illumination distribution being the brightest.

[0043] In various embodiments, the light source 404 and the at least one lens 406 may be aligned along an optical axis.

[0044] In various embodiments, the illumination subsystem 402 includes an excitation filter between the light source 404 and the sample surface.

[0045] In various embodiments, the optical system 400 includes an imaging lens above the sample plane. The optical system 400 may include an absorptive filter between the imaging lens and the sample plane.

[0046] In various embodiments, the optical system 400 includes an array of reaction chambers that form a sample surface, each reaction chamber of the array of reaction chambers containing a fluorescent dye and configured to fluoresce when illuminated by an illumination subsystem.

[0047] In various embodiments, light source 404 is configured to generate visible light, infrared light, or ultraviolet light. Light source 404 may also be configured to generate any combination of visible light, infrared light, and ultraviolet light.

[0048] Light source 404 may be configured to generate light at a single wavelength, or may be configured to generate light at multiple wavelengths.

[0049] The light source 404 may be a light emitting diode.

[0050] In one embodiment, the distance between the light source 404 and the at least one lens 406 is about 60 mm, the distance between the light source 404 and the sample surface is about 220 mm, the angle of incidence is about 17°, and the illumination subsystem 402 is rotated about 2.7° about the pivot point (for a second rotation or tilt).

[0051] In another embodiment, the distance between the light source 404 and the at least one lens 406 is about 30 mm, the distance between the light source 404 and the sample surface is about 110 mm, the angle of incidence is about 22°, and the illumination subsystem 402 is rotated about 1.3° about the pivot point.

[0052] Various embodiments relate to an optical system having an illumination subsystem configured to generate a symmetric illumination distribution at a sample plane. The illumination subsystem includes a light source and at least one lens. The light source and the at least one lens are aligned or positioned such that a line passing through a center of the light source and a center of the at least one lens (i.e., a further optical axis) is at an angle between 0° and 90° with respect to a normal to the sample plane.

[0053] Various embodiments relate to a method of forming an optical system having an illumination subsystem configured to generate a symmetric illumination distribution at a sample plane. The illumination subsystem includes a light source and at least one lens. The light source and the at least one lens are aligned or positioned such that a line passing through a center of the light source and a center of the at least one lens (i.e., a further optical axis) is at an angle between 0° and 90° with respect to a normal to the sample plane.

[0054] The optical systems described herein are biological entity detection systems. The biological entity detection systems are configured to detect a biological sample that includes a biological entity, such as DNA. In various embodiments, the system includes one or more microfluidic channels configured to deliver the biological entity to the array of reaction chambers. The system also includes one or more microfluidic channels configured to deliver a fluorescent dye to the array of reaction chambers.

[0055] 5 is a schematic diagram of an optical system 500 illustrating the rotation of the illumination subsystem 502 about a pivot point or axis proximate a lens 506 of the illumination subsystem 502 in various embodiments. The rotation of the illumination subsystem 502 shown in FIG. 5 is counterclockwise. In various other embodiments, the rotation may be clockwise. The rotation of the illumination subsystem 502 results in improved symmetry of the illumination distribution in a central region of illumination at the sample plane. The illumination subsystem 502 includes a light source 504 and a lens 506.

[0056] The rotation of the illumination subsystem 502 is referred to as a "second tilt" and may be a rotation of the light source 504 and lens 506 about a pivot point located closer to the lens.

[0057] "First tilt" refers to the orientation of the illumination subsystem about a point on the sample surface, which can be the oblique angle of illumination, i.e., the angle of incidence measured relative to the surface normal of the sample surface.

[0058] As shown in FIG. 5, pivoting the illumination subsystem 502 about the pivot point may result in a new optical axis pointing away from the center of the sample surface, resulting in symmetric illumination near the center of the sample surface.

[0059] FIG. 6A is an optical design model and simulation of an optical system 600 according to various embodiments implemented on a breadboard. FIG. 6A shows the optical design model and a simulation of a light beam illuminating a sample surface that is about 40 mm long and about 30 mm wide. The illumination subsystem 602 includes a blue light emitting diode (LED) 604 and three plano-convex lenses 606 in front of the LED 604. FIG. 6B shows the illumination subsystem 602 of the optical system 600 of FIG. 6A according to various embodiments. As shown in FIG. 6B, a circular aperture with a diameter of 20 mm is located 5 mm in front of the diode 604. The aperture is defined by a ring with an inner diameter of 20 mm. The outer circumference of the ring is attached to the mechanical housing of the illumination subsystem 602. The three plano-convex lenses 606 are positioned to collect the light beam and project the surface of the LED 604 toward the sample surface. The sample surface is located about 220 mm from the surface of the LED and 160 mm from the front of the imaging lens 606. Diode 604 is manufactured by Luminus (part number PT-121-B). The emission of diode 604 has a narrow wavelength band peaking at approximately 460 nm. However, the use of any other suitable LED or light source is contemplated.

[0060] 6A, the optical system 600 also includes an excitation filter 608 between the light source 604 and the sample plane. The optical system 600 also includes an imaging lens 610 positioned above the sample plane. The optical system 600 further includes an absorption filter 612 between the imaging lens 610 and the sample plane.

[0061] The oblique incidence angle is about 17°, but the second tilt can be set to about 0° initially to show the simulated results of an asymmetric non-uniform illumination distribution. Figure 7A is an image showing a two-dimensional (2D) profile of a surface with an asymmetric non-uniform illumination distribution. Figure 7B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm), showing the y-dimensional profile of an asymmetric non-uniform illumination distribution.

[0062] Improved symmetry of the illumination can be achieved by maintaining the oblique angle at approximately 17° and providing the illumination subsystem with a second tilt of approximately 2.7° about the pivot point.

[0063] FIG. 8A is a schematic diagram of the optical system 600 as shown in FIG. 6A according to various embodiments, with a second tilt of 2.7°. The second tilt of 2.7° moves the illumination rays in the y-direction, resulting in an illumination distribution with improved symmetry. FIG. 8B is a schematic diagram of the optical illumination subsystem of the optical system 600 of FIG. 8A according to various embodiments. Corresponding features are labeled with the same reference numbers.

[0064] 9A is an image showing a two-dimensional (2D) profile of a surface of illumination with improved symmetry in accordance with various embodiments, and FIG 9B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm) showing the y-dimensional profile of an illumination distribution with improved symmetry.

[0065] FIG. 10 is an image of a breadboard on which the optical system 600 shown in FIGS. 6 and 8 is configured in various embodiments. In this configuration, a blue LED is used as the light source integrated into the illumination subsystem. A forward current of 1 amp is applied to the LED, resulting in bright illumination at the sample surface. The sample surface was a piece of white paper cut from a standard A4-sized white sheet often used for printing and copying documents. White paper was used as the sample because it naturally fluoresces when excited by blue illumination and emits a broad-wavelength green light that is brightly visible through an absorption filter and imaging lens. The focal plane of the imaging lens is equipped with a FLIR 20 megapixel complementary metal-oxide semiconductor (CMOS) sensor with a pixel size of 2.4 microns.

[0066] FIG. 11A is an image showing a two-dimensional (2D) profile of the surface of the while paper when the second tilt angle is 0°. FIG. 11B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm), showing the y-dimensional profile of the white paper when the second tilt angle is 0°. FIGS. 11A-B show an asymmetric illumination distribution. As shown, there is a drop in brightness towards the right edge of the sample surface.

[0067] FIG. 12A is an image showing a two-dimensional (2D) profile of the surface of while paper when the second tilt angle is 2.7°. FIG. 12B is a plot of intensity (relative units or rel. units) as a function of y coordinate (millimeters or mm) showing the y-dimensional profile of white paper when the second tilt angle is 2.7°. FIG. 12A-B shows a more symmetrical illumination distribution compared to FIG. 11A-B.

[0068] The intensity distribution observed in the images shown in Figures 12A-B may not be as uniform as the simulation shown in Figures 9A-B. Both the left and right edges of the actual image are darker than the simulation shows. The reason is that the imaging lens and image sensor (i.e., CMOS sensor) may also contribute to the non-uniformity. The imaging lens (part number 86-574 from supplier Edmund Optics) may have optical non-uniformity where the intensity at the edges drops to 80%-90% of the peak intensity. Also, the CMOS sensor may have some non-uniformity. The combination of these results in the overall non-uniformity shown in the images. However, it can be seen that when using oblique incidence as the illumination, a second tilt angle can eliminate or reduce the asymmetry of the non-uniformity distribution.

[0069] FIG. 13A shows a schematic diagram of a biological entity detection system or device 1300 in various embodiments. The system 1300 includes one or more illumination subsystems as described herein. FIG. 13A shows that the system 1300 may include five illumination subsystems 1302a-e. The system 1300 is configured to detect a biological sample that includes a biological entity, such as DNA. The system may have an array of reaction chambers 1316 configured to hold the biological sample. In various embodiments, the system may have one or more microfluidic channels configured to deliver the biological entity to the array of reaction chambers 1316. The system may also have one or more microfluidic channels configured to deliver a fluorescent dye to the array of reaction chambers 1316. The illumination subsystems 1302a-e and the array of reaction chambers 1316 are mounted within a housing 1318. The array of reaction chambers 1316 forms a sample surface. The system 1300 also includes a camera 1310 configured to capture or detect fluorescence emitted by the biological sample when the array of reaction chambers 1316 is illuminated by one or more illumination subsystems 1302a-e. The camera lens 1310 and other components, such as filters, are also mounted within a housing 1318. Figure 13B is a schematic diagram of the optical system 1302a and camera 1310 shown in Figure 13A in various embodiments.

[0070] The second tilt can be set to about 1.3 degrees, and the oblique angle can be set to about 22 degrees for each of the illumination subsystems 1302a-e. The subsystems 1302a-e also include three different color LEDs (blue, green, red). Thus, one illumination subsystem can provide illumination having a wavelength different from that of the illumination by another illumination subsystem. The LEDs can be scanned sideways (i.e., moved parallel above the sample surface). The LEDs can be scanned relative to a fixed LED lens (i.e., a lens between the LEDs and the sample surface) that projects illumination at oblique incidence onto the sample surface. While the present invention has been particularly shown and described with reference to specific embodiments, it will be understood by those skilled in the art that various changes in form and details can be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the present invention is indicated by the appended claims, and it is therefore intended to include all changes that come within the meaning and range of equivalence of the claims.

Claims

1. A method for illuminating a sample surface with an illumination subsystem having a light source and at least one lens, comprising: generating an adjusted illumination distribution on the sample plane by adjusting the illumination subsystem from a first tilt to a second tilt about a pivot point located between a light source of the illumination subsystem and a sample plane and located on a first optical axis of the illumination subsystem; the first optical axis has an incident angle with respect to a normal to the sample surface that is greater than 0° and smaller than 90°; the illumination subsystem has a second optical axis at the second tilt; 11. A method of illuminating a sample plane, wherein an intensity distribution of the adjusted illumination distribution is more symmetric than an intensity distribution of a reference illumination distribution produced by the illumination subsystem of the first tilt.

2. 2. The method of claim 1, wherein adjusting the illumination subsystem about a pivot point located between the light source and the sample surface and on the first optical axis includes tilting or rotating the illumination subsystem to the second tilt.

3. 3. The method of claim 2, wherein tilting or rotating the illumination subsystem comprises tilting or rotating the illumination system about the pivot point by between 1.3° and 2.7°.

4. 2. The method of claim 1, further comprising the step of aligning the light source and at least one lens of the illumination subsystem along the second optical axis after adjusting the illumination subsystem about the pivot point.

5. 2. A method for illuminating a sample surface according to claim 1, wherein the intensity distribution of the adjusted illumination distribution is brightest at the center.

6. 2. The method of claim 1, wherein the angle at which the illumination subsystem is adjusted is an angle that depends on at least one of the following: an angle of incidence, a light source, at least one lens of the illumination subsystem, a position of a pivot point on the first optical axis, a distance between the light source and the at least one lens, and a distance between the light source and the sample surface.

7. 2. The method of claim 1, wherein the sample surface is formed by an array of reaction chambers, each reaction chamber being configured to hold a biological sample for detecting a target biological entity in the biological sample.

8. 8. The method of claim 7, wherein each reaction chamber of the array of reaction chambers is configured to contain a fluorescent dye that fluoresces when illuminated by the illumination subsystem.

9. 2. The method of claim 1, wherein the light source is configured to generate visible, infrared or ultraviolet light.

10. 1. A method of illuminating a sample surface, comprising: Utilizing an illumination subsystem including a light source and at least one lens and having a first optical axis at a predetermined angle of incidence with respect to a surface normal of a sample surface at a first tilt, generating a reference illumination distribution having a non-uniform intensity distribution on the sample surface by the illumination subsystem at the first tilt; generating an adjusted illumination distribution exhibiting a uniform intensity distribution by adjusting the illumination subsystem to a second tilt exhibiting a second optical axis about a pivot point located between the light source and the sample surface and located on the first optical axis; and measuring the adjusted illumination distribution located on the second optical axis.

11. 11. The method of claim 10, wherein measuring the adjusted illumination distribution located at the second optical axis includes imaging a drop in luminance.

12. 12. The method of claim 11, wherein a decrease in brightness occurs towards an edge of the sample surface.

13. 11. The method of claim 10, wherein adjusting the illumination subsystem about a pivot point includes moving the illumination subsystem along a direction parallel to the sample surface to improve symmetry of an intensity distribution of the illumination subsystem.

14. The method of claim 10, wherein the illumination subsystem includes a plurality of light sources including the light source, and a plurality of lenses including the at least one lens; The method of illuminating a sample plane, wherein generating the reference illumination distribution and the adjusted illumination distribution includes projecting light from the multiple light sources through the multiple lenses.

15. An optical system comprising: an illumination subsystem including at least one light source and at least one lens, the illumination subsystem being positioned at a first inclination and having a first optical axis at an angle of incidence with respect to a normal to the sample surface that is greater than 0° and less than 90°; the illumination subsystem is configured to generate a reference illumination distribution at the first tilt; the illumination subsystem generates an adjusted illumination distribution at the sample plane when the illumination subsystem has a second optical axis at a second tilt adjusted about a pivot point located between the at least one light source and the sample plane and located on the first optical axis; An optical system, wherein an adjusted illumination distribution produced by the illumination subsystem at the second tilt is more symmetric than a reference illumination distribution produced by the illumination subsystem at the first tilt.

16. 16. The optical system of claim 15, wherein the at least one lens comprises a plurality of lenses arranged to collect light from the at least one light source and project the light onto the sample surface.

17. 16. The optical system of claim 15, further comprising: an imaging lens above the sample surface; a semiconductor sensor disposed in a focal plane of the imaging lens.

18. 20. The optical system of claim 17, wherein at least one of the imaging lens and the solid state sensor introduces a non-uniformity into the reference illumination distribution.

19. 16. The optical system of claim 15, wherein the at least one light source includes a plurality of light sources having different wavelengths.

20. 20. The optical system of claim 19, wherein different wavelengths correspond to different colors.

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