Apparatus, system, and method for illuminating and imaging an object

Abstract

The present invention provides an apparatus, system and method for illuminating and imaging an object. The system comprises: a surface configured to have an imaging target placed thereon; an illumination module configured to illuminate the imaging target, the illumination module comprising: a light source configured to emit a visible light beam, wherein the light source comprises one or more light emitting diodes (LEDs); a beam splitter coupled to and positioned in front of a lens and configured to split the visible light beam from the light source; at least one illumination module mirror; and an excitation filter coupled to and positioned in front of the light source; and an imaging system comprising: an image capture device configured to capture an image of the imaging target, the image capture device comprising one or more filters, a lens, one or more sensors and a camera.

Description

[0001] This application is a divisional application of the invention patent application with application number 201710960925.4, application date 2017.10.13, and invention title "Apparatus, System and Method for Illuminating and Imaging an Object". Technical Field

[0002] This disclosure relates to apparatus, systems, and methods for illuminating an object and obtaining a high-resolution image of the object. This disclosure also relates to methods for correcting image non-uniformity. Background Technology

[0003] Imaging devices, systems, and methods are needed to provide high-resolution images of objects without relying on methods such as digital magnification or the use of zoom lenses. Digital magnification typically leads to image pixelation as the image is enlarged. Zoom lenses are difficult to use in many cases because it is often difficult to consistently meet various requirements such as large aperture, focal length, working distance, distortion, field curvature, and signal attenuation.

[0004] There is also a need for illumination devices, systems, and methods capable of providing two or more beams of light to illuminate an imaging target without simultaneously using two or more light sources, particularly in uniform illumination methods. Using multiple light sources typically results in beams of varying optical power being applied to the imaging target, as separate maintenance is required when using two light sources, and they may possess different optical properties after being manufactured or configured within the device or system. Furthermore, using multiple light sources often leads to higher overall illuminance nonuniformity of the imaging target, and the illumination system also exhibits higher mechanical complexity, which in turn increases maintenance requirements and the likelihood of nonuniform illumination. Another common problem during imaging (regardless of the imaging mode) is image nonuniformity. For example, when the same sample is placed at different locations on the imaging surface or in the field of view, even if the same sample emits the same signal, the corresponding images will appear nonuniform based on location. Image nonuniformity needs to be addressed in this art. Summary of the Invention

[0005] This invention discloses an illumination system. The illumination system includes a surface, a light source, a beam splitter, a first reflector, and a second reflector. The surface is configured to have an imaging target disposed thereon. The light source is configured to emit a beam of light. The beam splitter is configured to separate the beam from the light source into a first beam and a second beam. The first reflector is configured to reflect the first beam to provide a reflected first beam illuminating the surface. The second reflector is configured to reflect the second beam to provide a reflected second beam illuminating the surface.

[0006] In another embodiment, the illumination system includes a surface, a light source, a beam splitter, and a first reflector. The surface is configured to have an imaging target disposed thereon. The light source is configured to emit a beam of light. The beam splitter is configured to split the beam from the light source into a first beam and a second beam. The second beam illuminates the surface. The first reflector is configured to reflect the first beam from the beam splitter to provide a reflected first beam that illuminates the surface.

[0007] A method for illumination is also disclosed. The method includes providing a surface having an imaging target disposed thereon. The method further includes providing a beam of light via a light source. The method further includes splitting the beam of light into a first beam and a second beam. The method further includes illuminating the surface. The illumination includes: (i) reflecting the first beam using a first mirror to produce a reflected first beam illuminating the surface, and (ii) reflecting the second beam using a second mirror to produce a reflected second beam illuminating the surface.

[0008] In another embodiment, the illumination method includes providing a beam of light via a light source. The method further includes splitting the beam of light into a first beam and a second beam. The method also includes illuminating a surface of an imaging target placed thereon. The illumination includes reflecting the first beam using a first mirror to produce a reflected first beam that illuminates the surface. A second beam is split from the beam such that the second beam illuminates the surface.

[0009] An imaging system is also disclosed. The imaging system includes an imaging surface, a mirror, and a capturing device. The imaging surface is configured to mount an imaging target thereon. The capturing device is configured to capture an image of the imaging target via a path of emitted light extending from the imaging target, reflecting off the mirror, and reaching the capturing device. The mirror, the capturing device, or both are configured to move diagonally relative to the imaging surface to shorten the length of the emitted light path.

[0010] In another embodiment, the imaging system includes an imaging surface, a mirror, a mirror axis, a capture device, a capture device axis, and a drive block. The imaging surface is configured to mount an imaging target thereon. The mirror is configured to move along the mirror axis in a first diagonal direction. The capture device is configured to capture an image of the imaging target via a path of emitted light extending from the imaging target, reflecting off the mirror, and reaching the capture device. The capture device is configured to move along the capture device axis in a second diagonal direction. The drive block transmits the motion between the mirror and the capture device, causing the mirror and the capture device to move simultaneously.

[0011] An imaging method is also disclosed. The method includes placing an imaging target on an imaging surface. The method further includes moving a capture device, a mirror, or both, diagonally relative to the imaging surface. The method also includes capturing an image of the imaging target using the capture device along a path of emitted light extending from the imaging target, reflecting off the mirror, and reaching the capture device.

[0012] An illumination and imaging system is also disclosed. The system includes a surface configured to have an imaging target disposed thereon. A light source is configured to emit a beam of light. A beam splitter is configured to separate the beam from the light source into a first beam and a second beam. A first illumination mirror is configured to reflect the first beam to provide a reflected first beam illuminating the surface. A second illumination mirror is configured to reflect the second beam to provide a reflected second beam illuminating the surface. A capturing device is configured to capture an image of the imaging target via a path extending from the imaging target, reflecting off an emitting mirror, and reaching the capturing device. The emitting mirror, the capturing device, or both are configured to move diagonally relative to the surface to shorten the path length.

[0013] A method for illumination and imaging is also disclosed. The method includes placing an imaging target on a surface. The method also includes emitting a beam of light from a light source. The method further includes splitting the beam of light into a first beam and a second beam. The method also includes illuminating the imaging target. Illumination includes: (i) reflecting the first beam using a first illumination mirror to produce a reflected first beam illuminating the surface, and (ii) reflecting the second beam using a second illumination mirror to produce a reflected second beam illuminating the surface. The method also includes capturing an image of the imaging target using a capturing device along a path extending from the imaging target, reflecting off an emitting mirror, and reaching the capturing device.

[0014] This disclosure describes, in some embodiments, a method for generating an image corrected for non-uniformity. In some embodiments, non-uniformity is shown as an image having varying intensity signals for the same signal measured at different locations in the field of view.

[0015] The non-uniformity correction method of this disclosure can be applied to images obtained from a variety of samples, including biological samples containing biomolecules such as proteins, peptides, glycoproteins, modified proteins, nucleic acids, DNA, RNA, carbohydrates, lipids, lipopolysaccharides, biopolymers, other metabolites produced by cells and tissues, and combinations thereof. Biomolecules or biological samples containing biomolecules can be imaged individually, or can be dispersed, positioned, or embedded in membranes, gels, filter paper, slides, microplates, or matrices such as polyacrylamide gels or nitrocellulose or PDVF membrane blots, agarose gels, agar plates, cell culture plates, or tissue sections for imaging. The non-uniformity correction method of this disclosure can be applied to images obtained from any of the aforementioned samples.

[0016] The non-uniformity correction method disclosed herein can be applied to images generated by changes in the chemiluminescence of biological samples or by changes in the fluorescence of samples. The non-uniformity correction method disclosed herein can also be applied to images generated by bioluminescence imaging, transillumination, or reflected light imaging.

[0017] In one embodiment, a method for generating an image corrected for non-uniformity includes: calculating the relative illumination of an imaging lens of a plurality of pixels on an imaging sensor; generating a flat field correction matrix based on the relative illumination; capturing or acquiring an image of one or more biological samples, wherein the image is non-uniform; and adjusting the captured image using the flat field correction matrix to generate an image corrected for non-uniformity.

[0018] In one embodiment, generating the flat field correction matrix includes taking the reciprocal of the relative illuminance to generate the flat field correction matrix. In some embodiments, the relative illuminance is calculated using a formula obtained by fitting a linear or nonlinear curve regression. This curve may be a first-order polynomial, a second-order polynomial, a third-order polynomial, etc. The flat field correction matrix is ​​then calculated to generate flat field correction matrix values.

[0019] Adjusting a captured image using a flat-field correction matrix involves multiplying an image of one or more captured or acquired biological samples by the value of the flat-field correction matrix. In some embodiments, adjusting a captured or acquired image using a flat-field correction matrix further includes multiplying the image of one or more captured or acquired biological samples by the value of the flat-field correction matrix on a pixel-to-pixel basis to generate a flat-field corrected image. In some embodiments, the flat-field corrected image displays the accuracy of the signal level of each image of one or more captured or acquired biological samples, regardless of their position in the field of view.

[0020] In one embodiment, this disclosure provides a method for generating a non-uniformity-corrected image, the method comprising: calculating the relative illumination of an imaging lens of a plurality of pixels on an imaging sensor; taking the reciprocal of the relative illumination to generate a flat field correction matrix; capturing or acquiring an image of a biological sample; and multiplying the image of the captured or acquired biological sample by the value of the flat field correction matrix pixel-to-pixel to generate a flat field-corrected image.

[0021] In one embodiment, this disclosure describes a method for generating a non-uniformity-corrected image, the method comprising: capturing or acquiring images of one or more biological samples, wherein the images are non-uniform; and adjusting the captured or acquired images using a flat-field correction matrix to generate a non-uniformity-corrected image. Adjusting the captured or acquired images may include multiplying the images of the captured one or more biological samples by the value of the flat-field correction matrix on a pixel-to-pixel basis to generate a flat-field-corrected image.

[0022] In some embodiments, the flat field correction matrix is ​​present in the imaging device or imaging system. The flat field correction matrix is ​​available to the user using the imaging device. The flat field correction matrix values ​​may be stored in the imaging device. In some embodiments, the flat field correction matrix values ​​may be stored in software or computer components of the imaging device. The flat field correction matrix values ​​are available to the user using the imaging system to perform non-uniformity correction. In some embodiments, the flat field correction matrix is ​​a master flat field correction matrix.

[0023] In one embodiment, a method for generating a flat-field correction matrix for correcting image non-uniformity includes: calculating the relative illumination of an imaging lens for a plurality of pixels on an imaging sensor; generating a flat-field correction matrix based on the relative illumination; and normalizing the flat-field correction matrix based on the maximum pixel intensity value in the matrix. In one embodiment, generating the flat-field correction matrix includes taking the reciprocal of the relative illumination and normalizing the values ​​in the matrix. For example, a manufacturer or user may generate a flat-field correction matrix for future use.

[0024] Further objects and advantages of the invention will be set forth in part in the following description, and these objects and advantages will be apparent in part from the description or may be learned by practice of the invention. The objects and advantages of the invention will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0025] It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only and do not limit the claimed invention. Attached Figure Description

[0026] The accompanying drawings are incorporated in and form part of this specification, used to illustrate the invention and together with this description to explain the principles of the invention.

[0027] Figures 1 and 2 show perspective views (from different angles) of an imaging system according to one embodiment.

[0028] Figures 3 and 4 show perspective views of an imaging system according to one embodiment, wherein some components in Figures 1 and 2 are omitted to better show the axes of the coupling components.

[0029] Figures 5, 6, 7 and 8 show cross-sectional side views of an imaging system with continuously increasing scaling according to one embodiment.

[0030] Figure 9 shows a beam of light emitted from the illumination module of an imaging system according to one embodiment.

[0031] Figure 10 illustrates an incident illumination beam, at least partially blocked by a reflector, when the center of the path of the emitted light remains centered on the reflector as the emitting mirror moves during the scaling process, according to one embodiment.

[0032] Figure 11 shows a simplified schematic side view of a light beam emitted from the illumination module of the imaging system shown in Figure 9 according to one embodiment.

[0033] Figure 12 shows a simplified schematic top view of a light beam emitted from an illumination module with an additional reflector according to one embodiment.

[0034] Figure 13 shows a simplified schematic top view of a light beam emitted from an illumination module having two beam splitters according to one embodiment.

[0035] Figure 14 shows a cross-sectional side view of a beam before it reaches the beam splitter through the aperture, according to one embodiment.

[0036] Figure 15 shows a perspective view of the photometer reference plate.

[0037] Figure 16A shows a stacked image of a spot on a photometer reference plate at different locations in the field of view, and Figure 16B shows a graph representing the signal of the same spot at different locations.

[0038] Figure 17 shows the relative illumination of the imaging lens within an imaging system according to one embodiment, and the best fit obtained by nonlinear regression.

[0039] Figure 18A shows a modified image of Figure 16A obtained by applying a flat-field correction principal matrix according to one embodiment, and Figure 18B shows a graph representing the ratio of the intensity of the spot to the highest intensity of the spot.

[0040] Figure 19A shows an image of a chemiluminescent sample in the center of the field of view at 1x zoom; Figure 19B shows an image of a chemiluminescent sample in the center of the field of view and the upper right diagonal position at 1x zoom; and Figure 19C shows an image of a chemiluminescent sample in the upper right diagonal position of the field of view at 1x zoom.

[0041] Figure 20A shows an image of quantitative analysis of two rows of bands according to one embodiment, and Figure 20B shows... Figure 20A Figure 20C shows the intensity of the first row of the band in Figure 20A before flat correction, Figure 20D shows the intensity of the second row of the band in Figure 20A before flat correction, and Figure 20E shows the intensity of the second row of the band in Figure 20A after flat correction.

[0042] Figure 21A shows a table of relative illumination of the imaging lens versus sensor image height at 1x magnification according to one embodiment, and Figure 21B shows a table of relative illumination of the imaging lens versus sensor image height at 2x magnification.

[0043] Figure 22 shows a graph of relative illumination symmetrical with respect to the CCD center according to one embodiment.

[0044] Figure 23A shows a graph of the best-fit nonlinear regression curve at a magnification of 1x according to one embodiment, and Figure 23B shows a graph of the best-fit nonlinear regression curve at a magnification of 2x.

[0045] Figure 24 shows a simulated image at 1x magnification according to one embodiment.

[0046] Figure 25 shows a flat-field corrected master image according to one embodiment.

[0047] Figures 26A, 26B, and 26C are images after applying flat field correction. Figure 26A shows an image at the center of the field of view at 1x magnification according to one embodiment; Figure 26B shows an image at 2x magnification at a position between the center of the field of view and the upper right diagonal position; and Figure 26C shows an image at 1x magnification at the upper right diagonal position of the field of view.

[0048] Figure 27 shows an image of two rows of eight stripes according to one embodiment.

[0049] Figure 28A shows the first row of Figure 27 before flattening correction at 1x magnification according to one embodiment; Figure 28B shows the first row of Figure 27 after flattening correction at 1x magnification; Figure 28C shows the second row of Figure 27 before flattening correction at 1x magnification; and Figure 28D shows the second row of Figure 27 after flattening correction at 1x magnification.

[0050] Figure 29A shows the first row of Figure 27 before flattening correction at 2x magnification according to one embodiment; Figure 29B shows the first row of Figure 27 after flattening correction at 2x magnification; Figure 29C shows the second row of Figure 27 before flattening correction at 2x magnification; and Figure 29D shows the second row of Figure 27 after flattening correction at 2x magnification.

[0051] Figure 30 shows a graph of the membrane position (e.g., center, top right corner) before and after flat field correction according to one embodiment. Detailed Implementation

[0052] The exemplary embodiments of the invention will now be described in detail with reference to the accompanying drawings. Where possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. In the following description, reference is made to the accompanying drawings, which form a part therein, and in which specific exemplary embodiments in which the invention can be practiced are shown by means of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it should be understood that other embodiments may be utilized and changes may be made without departing from the scope of the invention. Therefore, the following description is merely exemplary.

[0053] Figures 1 and 2 show perspective views of portions of an imaging system 100 obtained from different angles according to one embodiment. The imaging system 100 may include an imaging surface 110. In one example, the imaging surface 110 may be or include a tray or screen. The imaging surface 110 may be planar and substantially horizontal (i.e., parallel to the ground). An imaging target 112 may be placed on the imaging surface 110. The imaging target 112 may be or include biological materials such as nucleic acids and / or proteins associated with polyacrylamide gels, agarose gels, nitrocellulose membranes, and PVDF membranes. The imaging target 112 may also be or include non-biological materials such as manufactured articles and documents.

[0054] The imaging system 100 may also include a mirror 120. The mirror 120 may be positioned (e.g., directly) above the imaging surface 110 and the imaging target 112. The mirror 120 may include a reflective surface. As shown, the reflective surface may be planar; however, in other embodiments, the reflective surface may be curved. When the reflective surface of the mirror 120 is planar, the reflective surface of the mirror 120 may be oriented at an angle relative to the imaging surface 110 (i.e., relative to the horizontal direction). This angle may be from about 10° to about 80°, from about 20° to about 70°, or from about 30° to about 60°. For example, the angle may be about 45°.

[0055] The imaging system 100 may also include an acquisition device 130. The acquisition device 130 may include a detector housing 140, one or more filters (one of which is shown as 150), and a camera 160. The detector housing 140 may be positioned above the imaging surface 110 and laterally (e.g., horizontally) offset from the reflector 120. The detector housing 140 may include a lens 142. The detector housing 140 may also include a filter wheel, a motor, and / or sensors for controlling the focus and aperture of the lens 142. The lens 142 may be planar, and its central longitudinal axis may intersect with the reflective surface of the reflector 120. Accordingly, the path of emitted light may extend vertically between the imaging target 112 and the reflector 120, and laterally between the reflector 120 and the lens 142 of the detector housing 140. As used in this article, "path of emitted light" refers to the path in the field of view from the imaging target 112 through the lens 142 to the camera 160.

[0056] Filter 150 can be coupled to and positioned behind detector housing 140, and the path of emitted light can extend through detector housing 140 and into filter 150. Filter 150 can be an electromagnetic (“EM”) filter that transmits only light of a selected wavelength to camera 160. Positioning filter 150 behind lens 142 allows filter 150 to be smaller than when filter 150 is positioned in front of lens 142. Both excitation and emitted light can enter lens 142. Excitation light blocked by filter 150 can strike lens 142 and surrounding surfaces, and a certain amount of excitation light can return to filter 150 and pass through filter 150 at this time. In another embodiment, the filter can be positioned in front of lens 142. Because the excitation is blocked by the filter in front of lens 142, very little excitation light may be present after the filter; for example, almost no excitation light propagates into lens 142 and reaches camera 160. This makes it easy to control stray light and reduce background signal. The filter in front of lens 142 may be larger than the filter 150 behind the lens. Therefore, the filter wheel may be larger and occupy more space. In some embodiments, a second filter may also be placed in front of lens 142. In such embodiments, the second filter of a notch filter may be placed in front of lens 142, while filter 150 is placed behind lens 142. These embodiments can provide the advantage of two filters operating simultaneously to minimize the impact of stray light, including stray excitation light, on the emission captured by camera 160.

[0057] Camera 160 may be coupled to and positioned behind filter 150, and the path of emitted light may extend through filter 150 and into camera 160, wherein camera 160 may capture one or more (e.g., filtered) images of imaging target 112.

[0058] The imaging system 100 may further include a first sensor 190 located at a first position and a second sensor 192 located at a second position (shown in FIG. 1). The first sensor 190 may be a limiting sensor configured to limit the travel distance of the detector housing 140, filter 150, and camera 160. The second sensor 192 may be a homing sensor configured to set the detector housing 140, filter 150, and camera 160 to an initial default position.

[0059] The imaging system 100 may further include an illumination module 200 (as shown in FIG. 1). The illumination module 200 may be or include a direct illumination module and / or a transmitted illumination module. The illumination module 200 may include a light source 210. The light source 210 may be or include one or more light-emitting diodes (“LEDs”). The illumination module 200 may further include an excitation filter 220 coupled to and positioned in front of the light source 210. The excitation filter 220 may be configured to limit the wavelength range of light from the light source 210. The illumination module 200 may further include a lens 230 coupled to and positioned in front of the excitation filter 220. In at least one embodiment, the lens 230 may be or include an annular lens. The illumination module 200 may further include a beam splitter 240 coupled to and positioned in front of the lens 230. Beam splitter 240 can be configured to separate or divide a beam from light source 210 into two or more beam portions. Illumination module 200 may also include a near-infrared (“NIR”) illumination module and a reflector 250, excitation filter 220, lens 230, beam splitter 240, or combinations thereof, which may be positioned close to (e.g., below) light source 210. The NIR illumination module and reflector 250 may include LEDs that provide light in the NIR range. The NIR illumination module and reflector 250 may also reflect NIR light into beam splitter 240 at substantially the same angle as visible light. Illumination module 200 may also include a rear reflector 260, excitation filter 220, lens 230, beam splitter 240, or combinations thereof, positioned below capture device 130 and / or above light source 210. Illumination module 200 may also include a front reflector 262. Imaging surface 110 may be laterally (e.g., horizontally) positioned between one side of light source 210, excitation filter 220, lens 230, beam splitter 240, and the other side of front reflector 262. Front reflector 262 may also be positioned above imaging surface 110. Although not shown, illumination module 200 may also include a transmissive illumination module and a light source (e.g., LED). One or more light sources for transmissive illumination may be positioned below imaging surface 110 to provide illumination through imaging surface 110 and imaging target 112.

[0060] Figures 3 and 4 show perspective views of an imaging system 100 according to one embodiment, with some components (e.g., mirror 120 and capture device 130) omitted to better show the axes 124, 134 of the coupling components.

[0061] A reflector 120 (not shown in Figures 3 and 4) may be coupled to a reflector support structure 122, and a capture device 130 (also not shown in Figures 3 and 4) may be coupled to a capture device support structure 132. The reflector support structure 122 may be coupled to and configured to slide back and forth along the reflector axis 124 in an axis aligned (e.g., parallel) to the reflector axis 124. The capture device support structure 132 may be coupled to and configured to slide back and forth along the capture device axis 134 in an axis aligned (e.g., parallel) to the capture device axis 134. A drive block 180 may be coupled to and configured to slide back and forth along the drive block axis 184 in an axis aligned (e.g., parallel) to the drive block axis 184. In at least one embodiment, the reflector axis 124, the capture device axis 134, the drive block axis 184, or a combination thereof may be in a single plane.

[0062] The mirror axis 124 may be diagonally oriented relative to the upper surface of the imaging surface 110. As used herein, "diagonal" means a direction that is neither parallel nor perpendicular to the imaging surface 110. More specifically, the mirror axis 124 may be oriented at an angle relative to the imaging surface 110, which is approximately 10° to approximately 170°, approximately 40° to approximately 140°, or approximately 70° to approximately 110° (when viewed from the directions shown in Figures 3 and 4). For example, angle 126 may be approximately 91° (when viewed from the angles shown in Figures 3 and 4).

[0063] The acquisition device axis 134 may also be diagonally oriented relative to the imaging surface 110 (i.e., relative to the horizontal direction). More specifically, the acquisition device axis 134 may be oriented at an angle relative to the imaging surface 110, which is approximately 10° to approximately 80°, approximately 20° to approximately 70°, or approximately 30° to approximately 60° (when viewed from the directions shown in Figures 3 and 4). For example, angle 136 may be approximately 35° (when viewed from the angles shown in Figures 3 and 4). The angle 127 between the mirror axis 124 and the acquisition device axis 134 may be approximately 80° to approximately 140°, approximately 90° to approximately 130°, or approximately 100° to approximately 120°. For example, angle 127 may be approximately 123°.

[0064] The drive block shaft 184 can be positioned between the mirror shaft 124 and the acquisition device shaft 134 (i.e., within angle 127). The drive block shaft 184 can also be diagonally or vertically (i.e., vertically) oriented relative to the upper surface of the imaging surface 110.

[0065] Referring to Figure 4, a first drive shaft 138 may be coupled to and extends between the capture device support structure 132 and the drive block 180. The capture device support structure 132 (and the capture device 130), the drive block 180, or a combination thereof may be configured to slide axially along the first drive shaft 138. A second drive shaft 128 may be coupled to and extends between the mirror support structure 122 and the drive block 180. The mirror support structure 122 (and the mirror 120), the drive block 180, or a combination thereof may be configured to slide axially along the first drive shaft 128.

[0066] Imaging system 100 may include one or more motors (one of which is shown as 170 in Figure 3). Motor 170 causes mirror 120 and / or acquisition device 130 (e.g., detector housing 140, filter 150, and camera 160) to move relative to imaging surface 110 and imaging target 112. In the illustrated embodiment, a single motor 170 can cause mirror 120 and acquisition device 130 to move simultaneously. This simultaneous movement can be achieved using a drive shaft and drive block connecting mirror 120 and acquisition device 130, such as the first drive shaft 138, second drive shaft 128, and drive block 180 described above in conjunction with Figure 4. This approach offers the advantage of controlling the movement of mirror 120 and acquisition device 130 using a single motor and controlling the movement in a synchronized manner without relying on separate control mechanisms such as control software, thereby providing advantages such as reduced complexity and cost, reduced maintenance requirements, and improved ability to maintain a consistent image center at different scaling levels. In another embodiment, a first motor moves the reflector 120, and a second motor moves the capture device 130, with the ratio of the movement of the reflector 120 relative to the capture device 130 being fixed. This fixed ratio of movement can be achieved by software control of the first and second motors, enabling synchronized movement while maintaining image centering during scaling. A drive block 180 can be coupled to the reflector 120 and the capture device 130. When using a single motor 170, the drive block 180 can coordinate the movement of the reflector 120 and the capture device 130, as detailed in Figures 3 and 4. In other embodiments, one or more belt drives or other devices can be used to move the reflector 120 and the capture device 130.

[0067] Referring again to Figures 3 and 4, the motor 170 can be coupled to the lead screw 172 via a coupler 174. The coupler 174 transmits the rotational motion of the motor 170 to the lead screw 172, thereby causing the lead screw 172 to rotate. The lead screw 172 can be parallel to the capture device shaft 134. When the lead screw 172 rotates in a first direction, it can push the capture device support structure 132 (and the capture device 130) along the capture device shaft 134 in a first axial direction. Conversely, when the lead screw 172 rotates in a second (i.e., opposite) direction, it can pull the capture device support structure 132 (and the capture device 130) along the capture device shaft 134 in a second (i.e., opposite) direction.

[0068] When the capture device support structure 132 (and the capture device 130) moves along the capture device shaft 134 in a first axial direction, the first drive shaft 138 can cause the drive block 180 to move along the drive block shaft 184 in a first axial direction. Conversely, when the capture device support structure 132 (and the capture device 130) moves along the capture device shaft 134 in a second axial direction, the first drive shaft 184 can cause the drive block 180 to move along the drive block shaft 184 in a second (i.e., opposite) axial direction.

[0069] When the transmission block 180 moves along the transmission block shaft 184 in the first axial direction, the second transmission shaft 128 can move the mirror support structure 122 (and the mirror 120) along the mirror shaft 124 in the first axial direction. Conversely, when the transmission block 180 moves along the transmission block shaft 184 in the second axial direction, the second transmission shaft 128 can move the mirror support structure 122 (and the mirror 120) along the mirror shaft 124 in the second (i.e., opposite) axial direction.

[0070] Therefore, it should be understood that the reflector 120 and the acquisition device 130 can move simultaneously. When the reflector 120 and the acquisition device 130 move along their respective first axes, the total path length of the emitted light from the imaging target 112 (reflected by the reflector 120) to the lens 142 of the detector housing 140 can be reduced, and when the reflector 120 and the acquisition device 130 move along their respective second axes, the total path length of the emitted light from the imaging target 112 (reflected by the reflector 120) to the lens 142 of the detector housing 140 can be increased.

[0071] Figures 5, 6, 7, and 8 show cross-sectional side views of an imaging system 100 with progressively increasing scaling according to one embodiment. More specifically, Figure 5 shows the unscaled imaging system 100. In one embodiment, when unscaled, the total length of the center of the path 115 of the emitted light from the imaging target 112 (reflected by mirror 120) to the lens 142 of the detector housing 140 can be, for example, about 455 mm, but the total length of the center of the path 115 will depend on the overall configuration of the system and its components, including the characteristics of the acquisition device 130. When unscaled, the path 114 of the emitted light may contact a first portion (e.g., a surface area) of mirror 120. The first portion (e.g., the surface area) can be about 50% to about 100%, about 75% to about 99%, or about 85% to about 95% of the total surface area of ​​mirror 120.

[0072] Referring now to FIG. 6, the capturing device 130 and the reflector 120 can move along their respective first axes to reduce the total length of the center of the emitted light path 115 (i.e., magnify the imaging target 112 on the imaging surface 110). The center of the emitted light path 115 between the imaging target 112 and the reflector 120 can remain fixed as the reflector 120 moves diagonally (i.e., the vertical arrow is the same as in FIG. 5 and FIG. 6). Therefore, the point on the reflector that the center of the emitted light path 115 contacts can change / move as the reflector 120 and the capturing device 130 move along their respective first axes. For example, the center of the emitted light path 115 can contact the reflector 120 at point 116A in FIG. 5 and at point 116B in FIG. 6. Additionally, the portion (e.g., surface area) of the reflector 120 that the emitted light path 114 contacts can decrease as the reflector 120 and the capturing device 130 move along their first axes.

[0073] Referring now to Figure 7, the reflector 120 and the capturing device 130 can also move along their respective first axes to further reduce the total length of the center of the emitted light path 115 (i.e., magnify the imaging target 112 on the imaging surface 110). The center of the emitted light path 115 between the imaging target 112 and the reflector 120 can remain fixed as the reflector 120 moves diagonally (i.e., the vertical arrow is the same as in Figures 5-7). Therefore, the point on the reflector that the center of the emitted light path 114 contacts can change / move as the reflector 120 and the capturing device 130 move along their respective first axes. For example, the center of the emitted light path 114 can contact the reflector 120 at point 116C in Figure 7. In addition, the portion (e.g., surface area) of the reflector 120 that the emitted light path 114 contacts can continue to decrease as the reflector 120 and the capturing device 130 move further along their first axes.

[0074] Referring now to Figure 8, the reflector 120 and the capturing device 130 minimize the total length of the center of the emitted light path 115 (i.e., maximize the magnification of the imaging target 112 on the imaging surface 110). The center of the emitted light path 115 between the imaging target 112 and the reflector 120 can remain fixed as the reflector 120 moves diagonally (i.e., the vertical arrow is the same as in Figures 5-8). Therefore, the point on the reflector that the center of the emitted light path 115 contacts can vary / move as the reflector 120 and the capturing device 130 move along their respective first axes. For example, the center of the emitted light path 115 can contact the reflector 120 at point 116D in Figure 8. In one example, when the magnification is at its maximum, the total length of the center of the emitted light path 115 from the imaging target 112 (reflected by the reflector 120) to the lens 142 of the detector housing 140 can be, for example, about 215 mm. Therefore, the imaging system 100 can be configured to magnify from about 1x to about 2x; however, in other embodiments, the imaging system 100 can be configured to further magnify (i.e., greater than 2x). Additionally, the portion (e.g., surface area) of the reflector 120 contacted by the path 114 of the emitted light can decrease with increasing magnification. For example, at maximum magnification, this portion (e.g., surface area) can be about 5% to about 80%, about 10% to about 70%, or about 20% to about 60% of the total surface area of ​​the reflector 120.

[0075] Figure 9 illustrates a beam of light (e.g., incident illumination beam) 212 emitted from illumination module 200 according to one embodiment. Beam 212 may be emitted from a light source 210 of illumination module 200 (see Figure 1). Beam 212 may be split into a first beam 213 and a second beam 214 by beam splitter 240. The first beam 213 may be reflected by rear reflector 260 and illuminate imaging target 112, and the second beam 214 may be reflected by front reflector 262 and illuminate imaging target 112. This is described in more detail below in conjunction with Figure 11. In another embodiment, beam 212 may be emitted from and reflected by a reflector in NIR illumination module 250 to reach imaging target 112. In at least one embodiment, the light beam 212 may extend through the path 114 of the emitted light to illuminate the imaging target 112, which may reflect the illumination light or may include a fluorescent component that emits light when excited by the incident illumination.

[0076] When the reflector 120 and the capturing device 130 are at their maximum magnification, as shown in FIG9, the lower end 139 of the capturing device 130 can be positioned below the lower end 129 of the reflector 120. Therefore, the reflector 120 does not obstruct the beam 214 at any point along the reflector axis 124.

[0077] Figure 10 illustrates an incident illumination beam 212 that is at least partially blocked by a reflector 120 according to one embodiment. If the center of the path 114 of the emitted light remains fixed at the same point on the reflector 120 as the reflector moves (e.g., point 116A in Figure 5), then when the capturing device 130 and the reflector 120 are at their maximum magnification, the lower end 129 of the reflector 120 can be positioned below the lower end 139 of the capturing device 130. Therefore, the reflector 120 can at least partially block the beam 212. Thus, as shown in Figures 5-9, the center of the path 114 of the emitted light can move / change on the reflector 120 as the reflector 120 moves to avoid blocking the beam 212.

[0078] Epi-illumination and / or excitation are used to determine the fluorescence pattern for protein analysis. Many fluorescent dyes are used for protein staining and / or blotting, and different dyes have different excitation spectral distributions, thus requiring excitation light of different colors. Certain excitation powers can provide a fluorescence imaging signal within an acceptable imaging exposure time. If the illumination and / or excitation power varies too much in the field of view, one or more dark areas may exist where the banding / stripes of the sample are difficult to see, or the banding / stripes may be visible in the dark areas, but the signal in the brighter areas becomes saturated. Therefore, substantially uniform illumination improves image quality.

[0079] There are two types of incident illumination: axial and off-axis (i.e., tilted). Axial illumination can generate bright spots on an image because some light is reflected from the sample. Off-axis illumination is one way to address this problem. In some embodiments, the off-axis angle can be greater than or equal to a predetermined amount to avoid generating bright spots.

[0080] Figure 11 shows a simplified schematic side view of a light beam 212 emitted from the illumination module 200 shown in Figure 9 according to one embodiment. The light beam 212 may be emitted from a light source 210 (see Figure 1) of the illumination module 200. The light source 210 may include a first LED for fluorescence excitation and a second LED for near-infrared excitation. In another embodiment, a halogen tungsten lamp may be used to cover both spectra. For any given channel, only one beam of light may be present. The light source 210 may have a single color. In at least one embodiment, the light source 210 may be a white light source, and filters may be used to generate different colors.

[0081] Beam 212 can be split into a first beam 213 and a second beam 214 by beam splitter 240. Although not shown, in other embodiments, beam splitter 240 may be configured to split beam 212 into three or more beams. As used herein, the term "beam splitter" includes one or more optical components capable of separating or otherwise separating a beam, and includes, but is not limited to, prisms, plates, dielectric mirrors, metal-coated mirrors, beam splitter cubes, fiber optic beam splitters, and optical fibers configured to collimate and bundle light before generating two or more output beams.

[0082] Beam splitter 240 can evenly separate or divide the intensity of the resulting beams, or can separate them into different proportions of intensity. In the illustrated embodiment, beam splitter 240 is a plate, and a first beam 213 is reflected from beam splitter 240, while a second beam 214 passes through beam splitter 240. Beam splitter 240 may include a coating and / or a filter (e.g., a linearly variable filter) such that one end / side of beam splitter 240 may have different characteristics than the opposite end / side. The first beam 213 may include about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, or 60%) of the optical power of beam 212, and the second beam 214 may include about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, or 60%) of the optical power of beam 212. Therefore, in some embodiments, the first beam 213 and the second beam 214 may uniformly separate the optical power of the beam 212 (50% for the first beam 213 and 50% for the second beam 214). In other embodiments, the first beam 213 may have a larger or smaller percentage of the optical power of the beam 212 compared to the second beam 214. The angle between the center of the first beam 213 and the center of the second beam 214 may be from about 62° to about 68°, from about 70° to about 90°, or from about 90° to about 110°. The first beam 213 may be reflected by the rear mirror 260 to produce a reflected first beam 215 that illuminates the imaging target 112 on the imaging surface 110. The second beam 214 may be reflected by the front mirror 262 to produce a reflected second beam 216 that illuminates the imaging target 112 on the imaging surface 110. The angle between the center of the reflected first beam 215 and the center of the reflected second beam 216 can be from about 80° to about 100°, from about 106° to about 114°, or from about 120° to about 140°. Although not shown, in at least one embodiment, the second beam 214 can directly illuminate the imaging target 112 on the imaging surface 110 without being reflected off the front reflector 262 and generating the reflected second beam 216.

[0083] The reflected first beam 215 and the reflected second beam 216 can provide off-axis illumination of the imaging target 112 on the imaging surface 110. More specifically, the reflected first beam 215 and the reflected second beam 216 can provide substantially symmetrical illumination of the imaging target 112 on the imaging surface 110. For example, the angle between the reflected first beam 215 and the imaging surface 110 can be within + / - 10° of the angle between the reflected second beam 216 and the imaging surface 110. The distance from the beam splitter 240 to the rear mirror 260 to the imaging surface 110 can be substantially equal to (e.g., within 10%) the distance from the beam splitter 240 to the front mirror 262 to the imaging surface 110. In at least one embodiment, the rear mirror 260 and / or the front mirror 262 can be moved in conjunction with the rotation of the beam splitter 240 to change the illumination of the imaging target 112 on the imaging surface 110.

[0084] Figure 12 shows a simplified schematic top view of a light beam 1212 emitted from an illumination module 1200 having additional reflectors 1261-1265 according to one embodiment. In the illustrated embodiment, the light beam 1212 can be emitted from a light source of the illumination module 1200 and can be split into a first beam 1213 and a second beam 1214 by a beam splitter 1240. The first beam 1213 can be reflected out of the beam splitter 1240, and the second beam 1214 can pass through the beam splitter 1240. The first beam 1213 is reflected out of the first reflector 1261 and the second reflector 1262 before illuminating the imaging target 112 on the imaging surface 110. The second beam 1214 is reflected out of the third reflector 1263, the fourth reflector 1264, and the fifth reflector 1265 before illuminating the imaging target 112 on the imaging surface 110. Similar to the embodiment in FIG11, bundles 1213 and 1214 can provide off-axis illumination of the imaging target 112 on the imaging surface 110. In addition, bundles 1213 and 1214 can provide substantially symmetrical illumination of the imaging target 112 on the imaging surface 110.

[0085] Figure 13 shows a simplified schematic top view of a light beam 1312 emitted from an illumination module 1300 having two beam splitters 1340, 1342, according to one embodiment. In the illustrated embodiment, the light beam 1312 may be emitted from a light source of the illumination module 1300 and may be split by the first beam splitter 1340 into a first beam 1313 and a second beam 1314. The first beam 1313 may be reflected out of the first beam splitter 1340, while the second beam 1314 may pass through the first beam splitter 1340. In at least one embodiment, the first beam 1313 may include about 15% to about 35% (e.g., 15%, 20%, 25%, 30%, or 35%) of the optical power of the beam 1312, and the second beam 1314 may include about 65% to about 85% (e.g., 65%, 70%, 75%, 80%, or 85%) of the optical power of the beam 1312.

[0086] The first beam 1313 is then reflected by the first mirror 1360 to produce a reflected first beam 1315 illuminating the imaging target 112 on the imaging surface 110. The second beam 1314 can be split into a third beam 1316 and a fourth beam 1317 by the second beam splitter 1342. The third beam 1316 is reflected by the second beam splitter 1342, while the fourth beam 1317 passes through the second beam splitter 1342. In at least one embodiment, the third beam 1316 may include about 20% to about 40% (e.g., 33%) of the optical power of the beam 1314, and the fourth beam 1317 may include about 60% to about 80% (e.g., 66%) of the optical power of the beam 1314. The third beam 1316 is then reflected by the second mirror 1362 to produce a reflected third beam 1318 illuminating the imaging target 112 on the imaging surface 110. Similar to the embodiment in FIG11, bundles 1315 and 1318 can provide off-axis illumination of the imaging target 112 on the imaging surface 110. In addition, bundles 1315 and 1318 can provide substantially symmetrical illumination of the imaging target 112 on the imaging surface 110.

[0087] The fourth beam 1317 can also illuminate the imaging target 112 on the imaging surface 110. As shown, the fourth beam 1317 may not reflect off the mirror before illuminating the imaging target 112 on the imaging surface 110. In one embodiment, the angle between the first beam 1313 and the fourth beam 1317 may be within about 10° to about 40° of the angle between the third beam 1316 and the fourth beam 1317. Similarly, in one embodiment, the angle between the reflected first beam 1315 and the fourth beam 1317 may be within about 10° to about 40° of the angle between the reflected third beam 1318 and the fourth beam 1317.

[0088] Although Figure 13 shows beams 1315, 1317, and 1318 illuminating the imaging target 112 on the imaging surface 110, in another embodiment, four or more beams may illuminate the imaging target 112 on the imaging surface 110. For example, four beams may illuminate the imaging target 112 on the imaging surface 110 from the front, rear, left, and right sides.

[0089] Figure 14 shows a cross-sectional side view of the light beam 212 in Figure 9 according to one embodiment before it reaches the beam splitter 240 after passing through the aperture 1418 of the beam shaper 1410. The beam shaper 1410 may include one or more lenses (three lenses as shown in 1412, 1414, and 1416). As shown, the aperture 1418 may be positioned between the second lens 1414 and the third lens 1416; however, in other embodiments, the aperture 1418 may be positioned anywhere within the beam shaper 1410 or outside the beam shaper 1410 before the light reaches the beam splitter. The dimensions (e.g., cross-sectional area or diameter) of the aperture 1418 may be fixed. In another embodiment, the dimensions of the aperture 1418 may be varied to change the illumination dimensions (e.g., cross-sectional area or diameter) of the imaging target 112 on the imaging surface 110. The intensity of the beam 212 from the light source can also be changed to change the illumination intensity of the imaging target 112 on the imaging surface 110.

[0090] Flat field calibration The imaging devices or systems disclosed herein can be used to image a variety of biomolecules and biological samples, such as proteins, peptides, glycoproteins, modified proteins, nucleic acids, DNA, RNA, carbohydrates, lipids, lipopolysaccharides, biopolymers, and other metabolites produced by cells and tissues. Biological samples can be imaged individually or in the presence of membranes, gels, filter paper, slides, microplates, or matrices such as polyacrylamide gels, nitrocellulose or PDVF membrane blots, agarose gels, agar plates, cell culture plates, or tissue sections.

[0091] The imaging system disclosed herein can image biomolecules and biological samples in several imaging modes, including fluorescence imaging, chemiluminescence imaging, bioluminescence imaging, and transmission or reflected light imaging. In some imaging modes, the sample emits light or displays changes in the emitted light (wavelength, frequency, or intensity), allowing imaging without external illumination or excitation. In other imaging modes, the sample emits light or changes the emitted light (wavelength, frequency, or intensity) and is then exposed to external illumination or an excitation source for imaging. In still other imaging modes, the sample reflects light or changes the reflected light (wavelength, frequency, or intensity) and is then exposed to external illumination for imaging.

[0092] A common problem encountered during imaging (regardless of the imaging mode) is that different locations in the image appear non-uniform when the sample is placed at different locations on the imaging surface or in the field of view. The imaging surface is exemplified in one embodiment by component 110 in Figure 1, and is also referred to herein as the imaging area, field of view, sample screen, or sample tray. In some embodiments, image non-uniformity is shown as images with varying intensity signals for the same signal measured at different locations on the imaging surface or in the field of view. In some embodiments, image non-uniformity is shown as images with different signal levels for the same signal measured at different locations on the imaging surface or in the field of view. This position-dependent image non-uniformity is partly caused by a characteristic of the imaging lens, namely relative illumination.

[0093] Non-uniformity in images based on the position of the sample on the imaging surface hinders accurate quantitative measurements of biomolecules. This disclosure describes systems, algorithms, and methods for calibrating lens assemblies in imaging systems, which eliminate lens-exhibited non-uniformity to obtain accurate data from biomolecule sample images. Calibrating lens assemblies using the methods and systems of this disclosure eliminates lens-exhibited non-uniformity to obtain accurate data from sample images.

[0094] As described in the embodiments above, the imaging and illumination devices and systems of this disclosure provide imaging correction functions to improve data accuracy through image analysis. These functions, alone or in further combination with methods and systems, calibrate image non-uniformity to provide excellent and accurate quantitative measurements of biological samples in electrophoretic gels or membranes, and subsequently provide reliable data analysis and image information derived from the samples.

[0095] In one embodiment, a method for generating an image corrected for non-uniformity includes: calculating the relative illumination of an imaging lens of a plurality of pixels on an imaging sensor; generating a flat field correction matrix based on the relative illumination; capturing an image of one or more biological samples, wherein the image is non-uniform; and adjusting the captured image using the flat field correction matrix to generate an image corrected for non-uniformity.

[0096] In one embodiment, adjusting the captured image using a flat field correction matrix includes multiplying the image of the captured biological sample by the value of the flat field correction matrix on a pixel-to-pixel basis to generate a flat field corrected image.

[0097] In some embodiments, a method for generating a non-uniformity-corrected image may include: calculating the relative illumination of an imaging lens of a plurality of pixels on an imaging sensor; taking the reciprocal of the relative illumination to generate a flat-field correction matrix; and providing the flat-field correction matrix to a user; wherein the user may multiply an image of a captured biological sample by the value of the flat-field correction matrix pixel-to-pixel to generate a flat-field corrected image. The user may obtain a captured image using an imaging device before performing flat-field correction processing on the image using the flat-field correction matrix. In some embodiments, the user may choose to generate a non-uniformity-corrected image. In some embodiments, the user may be instructed to generate a non-uniformity-corrected image by providing the user with a pre-calculated value of the flat-field correction matrix and instructing the user to multiply the image of the captured biological sample pixel-to-pixel by the value of the flat-field correction matrix to generate a flat-field corrected image. In some embodiments, the imaging device or imaging software manufacturer may provide the flat-field correction matrix to the user. In some embodiments, the user may calculate the value of the flat-field correction matrix using the methods and algorithms described herein.

[0098] In one embodiment, a method for generating an image corrected for non-uniformity includes: calculating the relative illumination of an imaging lens covering a plurality of pixels on an imaging sensor; and taking the reciprocal of the relative illumination to generate a flat-field correction matrix. For example, an imaging device manufacturer or user may generate a flat-field correction matrix for future use.

[0099] The flat-field calibrated images obtained by the methods of this disclosure show the accuracy of the signal level for each image of the captured biological sample, regardless of its position on the imaging surface, imaging region, or field of view. Flat-field calibrated images are images that have undergone non-uniformity correction.

[0100] Examples of flat field correction calibration An exemplary application of the imaging device or imaging system disclosed herein is as an imaging device for imaging biomolecules (e.g., but not limited to proteins and nucleic acids in gels or blotting) in chemiluminescence mode, where the chemiluminescent sample emits light without external illumination and excitation. As described above, one problem encountered when performing chemiluminescence imaging is the non-uniformity of the image, where the chemiluminescent sample is placed on an imaging surface (such as...). Figure 1 When the components (110, imaging area, field of view, sample screen or sample tray) are in different positions, the image signal will be different even for the same sample (such as the same protein or nucleic acid band in a gel or blot).

[0101] This problem is illustrated using a photometer reference microplate. In one example, Figure 15 shows a perspective view of a photometer reference plate 1500. However, any photometer reference plate known in the art can be used to illustrate this problem. The photometer reference plate 1500 has one or more radiation spots. The eight radiation spots shown on the photometer 1500 are numbered 1501-1508. The radiation is blocked by seven of the radiation spots (e.g., 1501-1507). Only one (e.g., the brightest) spot 1508 is imaged at different locations on the imaging surface (diagonally across the imaging screen). Spot 1508 of the photometer reference plate 1500 is positioned at different locations within the field of view (or sample screen). Images of spot 1508 of the photometer 1500 are acquired at various portions of the imaging surface using constant exposure times, and the images are stacked.

[0102] Figure 16A shows an image 1600 of the light spot 1508 of the photometer 1500. Figure 16B shows a diagram 1610 of the signals of the light spot 1508 acquired from various locations on the imaging surface of the imaging device of the present disclosure according to one embodiment. Figure 1610 of Figure 16B shows that signals from the same light spot 1508 appear to have different intensities at different locations on the imaging screen, even though all signals are the same, because these signals are emitted by the same emitting light spot 1508 on the photometer 1500. As shown in Figures 16A and 16B, the same signal from a chemiluminescent sample appears different due to imaging at different locations on the imaging screen / sample screen. This signal difference caused by location is due to a characteristic of the imaging lens, namely relative illumination. Given the non-uniformity of the image, the user cannot determine whether the imaging difference is caused by differences in the concentration of biomolecules (proteins, nucleic acids, DNA, etc.) in various luminescent samples, or whether the signal difference is caused by the imaging of sample bands at different locations on the sample screen. Therefore, current imaging methods cannot obtain reliable and accurate quantitative information.

[0103] The aforementioned signal differences may be caused by the relative illumination of the imaging lens in the imaging system of this disclosure. Relative illumination is a way of representing the combined effects of vignetting and roll-off in the imaging lens, and is typically expressed as a percentage of illumination at any point on the sensor, normalized to the location in the field of view with maximum illumination. Vignetting and roll-off are two separate components of relative illumination. Since the aforementioned signal differences are caused by relative illumination (i.e., one of the characteristics of the imaging lens), these differences can be corrected if the relative illumination is known. The correction process involves creating a flat-field correction master matrix file based on the relative illumination data of the imaging lens, normalizing the flat-field correction master matrix based on the maximum value in the matrix, and applying this flat-field correction (FF) master matrix to an image captured from the system.

[0104] Figure 17 illustrates a relative illumination of an imaging lens according to one embodiment of the present disclosure, Figure 1700. A formula for the curve in Figure 1700 can be obtained through linear or nonlinear curve fitting regression. For discrete data points, a regression method can be applied to fit a curve to a series of points to find the best-fit formula. The identified formula can then be used to calculate the relative illumination at any location on the imaging sensor. In one embodiment of the present disclosure, the flat-field correction algorithm includes the following steps: • Step 1 - Calculate the relative illumination of all pixels on the imaging sensor based on the formula identified from the curve fitting regression; • Step 2 - Take the reciprocal of the numbers from Step 1, and normalize the reciprocal based on the maximum value to generate the flat-field correction principal matrix; and • Step 3 - During image acquisition, the image of the captured biological sample is multiplied pixel-to-pixel by the matrix created in Step 2 to generate the final image with flat-field correction applied.

[0105] The image after flat-field correction displays the accuracy of signal levels for all strips in the sample.

[0106] Figure 18A shows an image 1800 of Figure 16A after flat-field correction according to one embodiment, and Figure 18B Figure 18A shows the ratio of spot intensity to the central spot. In other words, Figure 18B shows the relative percentages of those spots before and after flat-field correction. As shown, before applying flat-field correction, the ratio of each spot intensity to the spot with the maximum intensity value (Figure 18A) is approximately 0.7, while after flat-field correction, this ratio increases to greater than 0.95. Thus, significant illuminance compensation is achieved after applying flat-field correction.

[0107] Figure 19A shows an image 1900 of a chemiluminescent sample at the center of the field of view at a 1x scaling ratio, Figure 19C shows an image 1910 of a chemiluminescent sample at the upper right corner of the field of view at a 1x scaling ratio, and Figure 19B shows images 1920 of a chemiluminescent sample at both the center and upper right corner of the field of view at a 1x scaling ratio.

[0108] Figure 20A shows image 2000 of quantitative analysis of two rows of bands according to one embodiment; Figure 20B shows the intensity of the first row of bands before flat-field correction (Figure 2010); Figure 20C shows the intensity of the first row of bands after flat-field correction (Figure 2020); Figure 20D shows the intensity of the second row of bands before flat-field correction (Figure 2030); and Figure 20E shows the intensity of the second row of bands after flat-field correction (Figure 2040). As shown, the intensity of a single band varies with the position of the sample on the imaging surface (with the same sample), while after flat-field correction, the intensity does not vary with the position on the imaging surface.

[0109] Generate the flat field correction (FF) master matrix Figure 21A shows a table 2100 of the relative illumination of the imaging lens and the sensor image height at 1x magnification according to one embodiment, and Figure 21B shows a table 2110 of the relative illumination of the CCD sensor imaging lens and the image height at 2x magnification.

[0110] Figure 22 shows a graph 2200 of relative illumination symmetrical with respect to the image sensor center according to one embodiment. The maximum image height is approximately 8 mm. Simulations were performed from 0 mm to 8 mm.

[0111] Figure 23A shows Figure 2300 of the best-fit curve at 1x magnification according to one embodiment, and Figure 23B Figure 2310 shows the best-fit curve at a magnification of 2x. The curve can be a first-order polynomial, a second-order polynomial, a third-order polynomial, etc. The image height is calculated using this formula: Where h represents the height of the center pixel of the distance detection sensor (in mm), x c Let x represent the x-coordinate of the center pixel, and y represent the x-coordinate of the center pixel. c This represents the y-coordinate of the center pixel. In this example, the pixel height is 3.69 µm / pixel. Taking this into account, at 1x magnification, the relative illumination can be calculated using the formula for the best-fit curve: Where RI represents relative illumination (%), 0 ≤ RI ≤ 100.

[0112] For the Bin 1x1 image: Width = 3360 pixels → x c = 1690 Height = 2704 pixels → y c = 1352 For pixel (1,1) = 7.98 mm = 51.9% Figure 24 shows a simulated image 2400 at 1x magnification according to one embodiment.

[0113] Figure 25 illustrates a flat-field corrected main image 2500 according to one embodiment. The value of each pixel in the main image can be equal to RI. -1 .

[0114] Application of flat field correction main image Figure 26A shows image 2600 of a sample at 1x magnification with the sample in the center of the field of view according to one embodiment; Figure 26C shows image 2610 of a sample at 1x magnification with the sample in the upper right corner of the field of view; and Figure 26B shows image 2620 of samples in both the center and upper right corners of the field of view. In this example, the sample is a protein blot membrane with an equivalent amount of protein to be visualized as a chemiluminescent substrate, a bin number of 5, a scaling factor of 1x or 2x, a high gain (e.g., 55), and a shelf life of 60 seconds.

[0115] The flat-field principal matrix is ​​applied to the images in Figures 26A-26C. A rectangular mask can be drawn on the selected bands, and the average pixel intensity is measured. Macros are used to ensure that the measurement area for each band is in the same location for both the images before and after applying the flat-field principal matrix. Although the protein blot membrane is prepared with equal protein content, signal values ​​still vary between different bands. For each individual band, the signal intensity within the same row should be similar, regardless of membrane location.

[0116] Figure 27 shows an image 2700 of two rows of eight sample strips according to one embodiment.

[0117] Figure 28A shows Figure 2800 in the first row before flat field correction according to one embodiment; Figure 28B shows Figure 2810 in the first row after applying the flat field correction master matrix; Figure 28C shows Figure 2820 in the second row before applying the flat field correction master matrix; and Figure 28D shows Figure 2830 in the second row after applying the flat field correction master matrix. The magnification in Figures 28A-28D is 1x. Before applying the flat field correction master matrix, the difference in ADU (analog-to-digital conversion unit) values ​​is caused by the position of the film on the imaging device surface / screen. After applying the flat field correction master matrix, the ADU values ​​are similar and independent of the film position.

[0118] Figure 29A shows the first row of Figure 2900 before applying the flat-field correction master matrix according to one embodiment. Figure 29B Figure 2910 shows the first row after applying the flat-field correction master matrix, Figure 29C shows the second row before applying the flat-field correction master matrix, and Figure 29D shows the second row after applying the flat-field correction master matrix, Figure 2930. The magnification in Figures 29A-29D is 2x.

[0119] Figure 30 illustrates the film position (e.g., the middle position and upper right corner position of the film and its sample strips on the imaging device surface / screen) before and after applying the flat-field correction master matrix according to one embodiment. As shown, the upper right corner position is blurry before applying the flat-field correction master matrix, and the strips have similar brightness after applying the flat-field correction master matrix.

[0120] While the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​described in specific examples are reported as precisely as possible. However, any numerical value inherently contains a certain degree of error, which is necessarily caused by the standard deviation present in their respective experimental determinations. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the range “less than 10” can encompass any and all subranges between a minimum value of zero and a maximum value of 10; that is, any and all subranges having a minimum value equal to or greater than zero and a maximum value equal to or less than 10 (e.g., 1 to 5). In some cases, numerical values ​​stated for parameters may be negative. In such cases, example values ​​of the range stated as “less than 10” may be negative, such as -1, -2, -3, -10, -20, -30, etc.

[0121] While the teachings of exemplary embodiments have been referenced, those skilled in the art will be able to make various modifications to the described embodiments without departing from the true spirit and scope. The terminology and descriptions used herein are for illustrative purposes only and are not intended to be limiting. Specifically, although methods have been described by way of example, method steps may be performed in a different order or simultaneously. Furthermore, the terms “including,” “having,” “has,” or variations thereof, used in the embodiments and claims, are intended to be inclusive in a manner similar to the term “including.” As used herein, the term “one or more of” in a list of all articles (A and B) means A alone, B alone, or A and B. Those skilled in the art will recognize that these and other variations may be within the spirit and scope as defined in the following claims and their equivalents.

[0122] Other embodiments of the invention will become apparent to those skilled in the art from the description and practical considerations of the invention disclosed herein. It is intended that the description and examples be considered exemplary only, wherein the true scope and spirit of the invention are indicated by the following claims.

[0123] As used herein, the terms “inside” and “outside,” “up” and “down,” “upward” and “downward,” “above” and “below,” “inward” and “outward,” and other similar terms used herein refer to their relative positions to each other and are not intended to indicate a particular direction or spatial orientation. The terms “coupled,” “coupled,” “connect,” “connected,” “connected to,” and “to connect” mean “directly connected to” or “connected through one or more intermediate elements or components.”

Claims

1. A system comprising: A surface configured to allow the imaging target to be placed on it; An illumination module, configured to illuminate the imaging target, the illumination module comprising: A light source configured to emit a visible light beam, wherein the light source includes one or more light-emitting diodes (LEDs). A beam splitter is coupled to and positioned in front of the lens and configured to split the visible light beam from the light source. At least one lighting module reflector; and An excitation filter is coupled to and positioned in front of the light source; and Imaging system, including: An image capturing device configured to capture an image of the imaging target, the image capturing device including one or more filters, lenses, one or more sensors, and a camera.

2. The system according to claim 1, wherein, The illumination module also includes a near-infrared (NIR) illumination module configured to emit a near-infrared (NIR) beam, and a second illumination module reflector; Optionally, the near-infrared (NIR) illumination module is positioned below the light source; and / or The beam splitter is further configured to split the near-infrared (NIR) beam from the near-infrared (NIR) illumination module.

3. The system according to any one of the preceding claims, wherein, The one or more filters are electromagnetic filters; and / or The one or more filters are placed behind the lens.

4. The system according to any one of the preceding claims, wherein the lighting module further comprises a front reflector; Optionally, the front reflector is positioned above the imaging surface; and / or A rear reflector, which is positioned below the image capturing device and / or above the light source.

5. The system according to any one of the preceding claims, wherein the one or more sensors include a homing sensor or a limiting sensor.

6. The system according to any one of the preceding claims, wherein the beam splitter comprises a prism, a plate, a dielectric mirror, a metal-coated mirror, a beam splitter cube, an optical fiber beam splitter, or an optical fiber configured to collimate and bundle light before generating two or more output beams.

7. The system according to any one of the preceding claims, wherein, The one or more light-emitting diodes include at least a first LED for visible fluorescence excitation and / or a second LED for near-infrared (NIR) excitation.

8. The system according to any one of the preceding claims, wherein, The beam splitter is configured to split a visible light beam from the light source into a first beam and a second beam, wherein the second beam illuminates the surface; and The illumination module reflector is configured to reflect the first beam to provide a reflected first beam for illuminating the imaging target; Optionally, the first beam is reflected from the beam splitter, and the second beam passes through the beam splitter. and / or The reflected first beam and the second beam provide substantially symmetrical illumination of the imaging target.

9. The system according to any one of the preceding claims, wherein, The image capturing device further includes an imaging mirror, and wherein the image capturing device is configured to capture an image of the imaging target via a path of emitted light extending from the imaging target, reflecting off the imaging mirror, and reaching the image capturing device, wherein the imaging mirror, the image capturing device, or both are configured to move diagonally relative to the imaging surface to shorten the length of the path of the emitted light.

10. The system according to claim 9, wherein, The imaging mirror moves in the diagonal direction, and wherein, as the imaging mirror moves, the reflecting surface of the imaging mirror remains diagonally oriented relative to the imaging surface.

11. The system according to any one of claims 9 or 10, wherein, The image capturing device and the imaging mirror move simultaneously in different diagonal directions.

12. The system according to any one of claims 9 to 11, wherein, The path of the emitted light reflects off an area on the imaging mirror, and the area decreases as the image capturing device, the imaging mirror, or both move diagonally relative to the surface on which the imaging target is placed, thereby shortening the length of the path of the emitted light.

13. The system according to any one of claims 9 to 12, wherein the center of the path of the emitted light reflects a point on the imaging mirror, and wherein the point moves to shorten the length of the path of the emitted light as the image capturing device, the imaging mirror, or both move diagonally relative to the surface on which the imaging target is placed.

14. The system according to any one of claims 9 to 13, further comprising: An imaging mirror axis, wherein the imaging mirror is configured to move along the imaging mirror axis in a first diagonal direction; as well as An image capture device axis, wherein the image capture device is configured to move along the image capture device axis in a second diagonal direction.

15. The system according to any one of claims 9 to 14, further comprising: A transmission block that transmits the motion between the imaging mirror and the image capturing device, thereby causing the imaging mirror and the image capturing device to move simultaneously; and Optionally, it may also include: a first drive shaft extending between the image capture device and the drive block, wherein the image capture device, the drive block, or both are configured to move along the first drive shaft; as well as A second drive shaft extends between the imaging mirror and the drive block, wherein the imaging mirror, the drive block, or both are configured to move along the second drive shaft.

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