Patent Description:
Prior art calibration slides comprising a plurality of etched features are commercially available as "Combined Resolution and Distortion Test Target R1L3S5P" from Thorlabs.

A selection of optional features of the invention is set out in the dependent claims.

Insofar as the term invention or embodiment is used in the following, or features are presented as being optional this should be internreted in such a wav that the only protection sought is that of the invention claimed References to "embodiment(s)" throughout the description which are not under the scope of the appended claims merely represent possible exemplary executions and are not part of the present invention.

The details of the present invention, both as to its structure and operation, may be gleaned in part by study of the accompanying drawings, in which like reference numerals refer to like parts, and in which:.

Embodiments of a physical calibration slide for calibrating a scanning system are described herein. After reading this description, it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, although various embodiments of the present invention will be described herein, it is understood that these embodiments are presented by way of example and illustration only, and not limitation. As such, this detailed description of various embodiments should not be construed to limit the scope or breadth of the present invention as set forth in the appended claims.

<FIG> is a block diagram illustrating an example processor-enabled slide-scanning system <NUM> that may be used in connection with various embodiments described herein. Alternative forms of scanning system <NUM> may also be used as will be understood by the skilled artisan. In the illustrated embodiment, scanning system <NUM> is presented as a digital imaging device that comprises one or more processors <NUM>, one or more memories <NUM>, one or more motion controllers <NUM>, one or more interface systems <NUM>, one or more movable stages <NUM> that each support one or more glass slides <NUM> with one or more samples <NUM>, one or more illumination systems <NUM> that illuminate sample <NUM>, one or more objective lenses <NUM> that each define an optical path <NUM> that travels along an optical axis, one or more objective lens positioners <NUM>, one or more optional epi-illumination systems <NUM> (e.g., included in a fluorescence-scanning embodiment), one or more focusing optics <NUM>, one or more line-scan cameras <NUM>, and/or one or more area-scan cameras <NUM>, each of which define a separate field of view <NUM> on sample <NUM> and/or glass slide <NUM>. The various elements of scanning system <NUM> are communicatively coupled via one or more communication busses <NUM>. Although there may be a plurality of each of the various elements of scanning system <NUM>, for simplicity in the description that follows, these elements will be described in the singular, except when needed to be described in the plural to convey the appropriate information.

Processor <NUM> may include, for example, a central processing unit (CPU) and a separate graphics processing unit (GPU) capable of processing instructions in parallel, or a multicore processor capable of processing instructions in parallel. Additional separate processors may also be provided to control particular components or perform particular functions, such as image processing. For example, additional processors may include an auxiliary processor to manage data input, an auxiliary processor to perform floating-point mathematical operations, a special-purpose processor having an architecture suitable for fast execution of signal-processing algorithms (e.g., digital-signal processor), a slave processor subordinate to the main processor (e.g., back-end processor), an additional processor for controlling line-scan camera <NUM>, stage <NUM><NUM>, objective lens <NUM>, and/or a display (e.g., a console comprising a touch panel display integral to scanning system <NUM>). Such additional processors may be separate discrete processors or may be integrated into a single processor.

Memory <NUM> provides storage of data and instructions for programs that can be executed by processor <NUM>. Memory <NUM> may include one or more volatile and/or non-volatile computer-readable storage mediums that store the data and instructions. These mediums may include, for example, random-access memory (RAM), read-only memory (ROM), a hard disk drive, a removable storage drive (e.g., comprising flash memory), and/or the like. Processor <NUM> is configured to execute instructions that are stored in memory <NUM>, and communicate via communication bus <NUM> with the various elements of scanning system <NUM> to carry out the overall function of scanning system <NUM>.

Communication bus <NUM> may be configured to convey analog electrical signals and/or digital data. Accordingly, communications from processor <NUM>, motion controller <NUM>, and/or interface system <NUM>, via communication bus <NUM>, may include both electrical signals and digital data. Processor <NUM>, motion controller <NUM>, and/or interface system <NUM> may also be configured to communicate with one or more of the various elements of scanning system <NUM> via a wireless communication link.

Motion control system <NUM> is configured to precisely control and coordinate X, Y, and/or Z movement of stage <NUM> (e.g., within an X-Y plane), X, Y, and/or Z movement of objective lens <NUM> (e.g., along a Z axis orthogonal to the X-Y plane, via objective lens positioner <NUM>), rotational movement of a carousel described elsewhere herein, lateral movement of a push/pull assembly described elsewhere herein, and/or any other moving component of scanning system <NUM>. For example, in a fluorescence-scanning embodiment comprising epi-illumination system <NUM>, motion control system <NUM> may be configured to coordinate movement of optical filters and/or the like in epi-illumination system <NUM>.

Interface system <NUM> allows scanning system <NUM> to interface with other systems and human operators. For example, interface system <NUM> may include a console (e.g., a touch panel display) to provide information directly to an operator via a graphical user interface and/or allow direct input from an operator via a touch sensor. Interface system <NUM> may also be configured to facilitate communication and data transfer between scanning system <NUM> and one or more external devices that are directly connected to scanning system <NUM> (e.g., a printer, removable storage medium, etc.), and/or one or more external devices that are indirectly connected to scanning system <NUM>, for example, via one or more networks (e.g., an image storage system, a Scanner Administration Manager (SAM) server and/or other administrative server, an operator station, a user station, etc.).

Illumination system <NUM> is configured to illuminate at least a portion of sample <NUM>. Illumination system <NUM> may include, for example, one or more light sources and illumination optics. The light source(s) could comprise a variable intensity halogen light source with a concave reflective mirror to maximize light output and a KG-<NUM> filter to suppress heat. The light source(s) could comprise any type of arc-lamp, laser, or other source of light. In an embodiment, illumination system <NUM> illuminates sample <NUM> in transmission mode, such that line-scan camera <NUM> and/or area-scan camera <NUM> sense optical energy that is transmitted through sample <NUM>. Alternatively or additionally, illumination system <NUM> may be configured to illuminate sample <NUM> in reflection mode, such that line-scan camera <NUM> and/or area-scan camera <NUM> sense optical energy that is reflected from sample <NUM>. Illumination system <NUM> may be configured to be suitable for interrogation of sample <NUM> in any known mode of optical microscopy.

In an embodiment, scanning system <NUM> includes an epi-illumination system <NUM> to optimize scanning system <NUM> for fluorescence scanning. It should be understood that, if fluorescence scanning is not supported by scanning system <NUM>, epi-illumination system <NUM> may be omitted. Fluorescence scanning is the scanning of samples <NUM> that include fluorescence molecules, which are photon-sensitive molecules that can absorb light at a specific wavelength (i.e., excitation). These photon-sensitive molecules also emit light at a higher wavelength (i.e., emission). Because the efficiency of this photoluminescence phenomenon is very low, the amount of emitted light is often very low. This low amount of emitted light typically frustrates conventional techniques for scanning and digitizing sample <NUM> (e.g., transmission -mode microscopy).

Advantageously, in an embodiment of scanning system <NUM> that utilizes fluorescence scanning, use of a line-scan camera <NUM> that includes multiple linear sensor arrays (e.g., a time-delay-integration (TDI) line-scan camera) increases the sensitivity to light of line-scan camera <NUM> by exposing the same area of sample <NUM> to each of the plurality of linear sensor arrays of line-scan camera <NUM>. This is particularly useful when scanning faint fluorescence samples with low levels of emitted light. Accordingly, in a fluorescence-scanning embodiment, line-scan camera <NUM> is preferably a monochrome TDI line-scan camera. Monochrome images are ideal in fluorescence microscopy because they provide a more accurate representation of the actual signals from the various channels present on sample <NUM>. As will be understood by those skilled in the art, a fluorescence sample can be labeled with multiple florescence dyes that emit light at different wavelengths, which are also referred to as "channels.

Furthermore, because the low-end and high-end signal levels of various fluorescence samples present a wide spectrum of wavelengths for line-scan camera <NUM> to sense, it is desirable for the low-end and high-end signal levels that line-scan camera <NUM> can sense to be similarly wide. Accordingly, in a fluorescence-scanning embodiment, line-scan camera <NUM> may comprise a monochrome <NUM>-bit <NUM>-linear-array TDI line-scan camera. It should be noted that a variety of bit depths for line-scan camera <NUM> can be employed for use with such an embodiment.

Movable stage <NUM> is configured for precise X-Y movement under control of processor <NUM> or motion controller <NUM>. Movable stage <NUM> may also be configured for Z movement under control of processor <NUM> or motion controller <NUM>. Movable stage <NUM> is configured to position sample <NUM> in a desired location during image data capture by line-scan camera <NUM> and/or area-scan camera <NUM>. Movable stage <NUM> is also configured to accelerate sample <NUM> in a scanning direction to a substantially constant velocity, and then maintain the substantially constant velocity during image data capture by line-scan camera <NUM>. In an embodiment, scanning system <NUM> may employ a high-precision and tightly coordinated X-Y grid to aid in the location of sample <NUM> on movable stage <NUM>. In an embodiment, movable stage <NUM> is a linear-motor-based X-Y stage with high-precision encoders employed on both the X and the Y axes. For example, very precise nanometer encoders can be used on the axis in the scanning direction and on the axis that is in the direction perpendicular to the scanning direction and on the same plane as the scanning direction. Stage <NUM> is also configured to support glass slide <NUM> upon which sample <NUM> is disposed.

Sample <NUM> can be anything that may be interrogated by optical microscopy. For example, glass microscope slide <NUM> is frequently used as a viewing substrate for specimens that include tissues and cells, chromosomes, deoxyribonucleic acid (DNA), protein, blood, bone marrow, urine, bacteria, beads, biopsy materials, or any other type of biological material or substance that is either dead or alive, stained or unstained, labeled or unlabeled. Sample <NUM> may also be an array of any type of DNA or DNA-related material, such as complementary DNA (cDNA) or ribonucleic acid (RNA), or protein that is deposited on any type of slide or other substrate, including any and all samples commonly known as microarrays. Sample <NUM> may be a microtiter plate (e.g., a <NUM>-well plate). Other examples of sample <NUM> include integrated circuit boards, electrophoresis records, petri dishes, film, semiconductor materials, forensic materials, and machined parts.

Objective lens <NUM> is mounted on objective positioner <NUM>, which, in an embodiment, employs a very precise linear motor to move objective lens <NUM> along the optical axis defined by objective lens <NUM>. For example, the linear motor of objective lens positioner <NUM> may include a fifty-nanometer encoder. The relative positions of stage <NUM> and objective lens <NUM> in X, Y, and/or Z axes are coordinated and controlled in a closed-loop manner using motion controller <NUM> under the control of processor <NUM> that employs memory <NUM> for storing information and instructions, including the computer-executable programmed steps for overall operation of scanning system <NUM>.

In an embodiment, objective lens <NUM> is a plan apochromatic ("APO") infinity-corrected objective lens which is suitable for transmission-mode illumination microscopy, reflection-mode illumination microscopy, and/or epi-illumination-mode fluorescence microscopy (e.g., an Olympus 40X, <NUM>. 75NA or 20X, <NUM> NA). Advantageously, objective lens <NUM> is capable of correcting for chromatic and spherical aberrations. Because objective lens <NUM> is infinity-corrected, focusing optics <NUM> can be placed in optical path <NUM> above objective lens <NUM> where the light beam passing through objective lens <NUM> becomes a collimated light beam. Focusing optics <NUM> focus the optical signal captured by objective lens <NUM> onto the light-responsive elements of line-scan camera <NUM> and/or area-scan camera <NUM>, and may include optical components such as filters, magnification changer lenses, and/or the like. Objective lens <NUM>, combined with focusing optics <NUM>, provides the total magnification for scanning system <NUM>. In an embodiment, focusing optics <NUM> may contain a tube lens and an optional 2X magnification changer. Advantageously, the 2X magnification changer allows a native 20X objective lens <NUM> to scan sample <NUM> at 40X magnification.

Line-scan camera <NUM> comprises at least one linear array of picture elements <NUM> ("pixels"). Line-scan camera <NUM> may be monochrome or color. Color line-scan cameras typically have at least three linear arrays, while monochrome line-scan cameras may have a single linear array or plural linear arrays. Any type of singular or plural linear array, whether packaged as part of a camera or custom-integrated into an imaging electronic module, can also be used. For example, a three linear array ("red-green-blue" or "RGB") color line-scan camera or a ninety-six linear array monochrome TDI may also be used. TDI line-scan cameras typically provide a substantially better signal-to-noise ratio ("SNR") in the output signal by summing intensity data from previously imaged regions of a specimen, yielding an increase in the SNR that is in proportion to the square-root of the number of integration stages. TDI line-scan cameras comprise multiple linear arrays. For example, TDI line-scan cameras are available with <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or even more linear arrays. Scanning system <NUM> also supports linear arrays that are manufactured in a variety of formats including some with <NUM> pixels, some with <NUM>,<NUM> pixels, and others having as many as <NUM>,<NUM> pixels. Similarly, linear arrays with a variety of pixel sizes can also be used in scanning system <NUM>. The salient requirement for the selection of any type of line-scan camera <NUM> is that the motion of stage <NUM> can be synchronized with the line rate of line-scan camera <NUM>, so that stage <NUM> can be in motion with respect to line-scan camera <NUM> during the digital image capture of sample <NUM>.

In an embodiment, the image data generated by line-scan camera <NUM> is stored in a portion of memory <NUM> and processed by processor <NUM> to generate a contiguous digital image of at least a portion of sample <NUM>. The contiguous digital image can be further processed by processor <NUM>, and the processed contiguous digital image can also be stored in memory <NUM>.

In an embodiment with two or more line-scan cameras <NUM>, at least one of the line-scan cameras <NUM> can be configured to function as a focusing sensor that operates in combination with at least one of the other line-scan cameras <NUM> that is configured to function as an imaging sensor. The focusing sensor can be logically positioned on the same optical axis as the imaging sensor or the focusing sensor may be logically positioned before or after the imaging sensor with respect to the scanning direction of scanning system <NUM>. In such an embodiment with at least one line-scan camera <NUM> functioning as a focusing sensor, the image data generated by the focusing sensor may be stored in a portion of memory <NUM> and processed by processor <NUM> to generate focus information, to allow scanning system <NUM> to adjust the relative distance between sample <NUM> and objective lens <NUM> to maintain focus on sample <NUM> during scanning. Additionally, in an embodiment, the at least one line-scan camera <NUM> functioning as a focusing sensor may be oriented such that each of a plurality of individual pixels <NUM> of the focusing sensor is positioned at a different logical height along the optical path <NUM>.

In operation, the various components of scanning system <NUM> and the programmed modules stored in memory <NUM> enable automatic scanning and digitizing of sample <NUM>, which is disposed on glass slide <NUM>. Glass slide <NUM> is securely placed on movable stage <NUM> of scanning system <NUM> for scanning sample <NUM>. Under control of processor <NUM>, movable stage <NUM> accelerates sample <NUM> to a substantially constant velocity for sensing by line-scan camera <NUM>, where the speed of stage <NUM> is synchronized with the line rate of line-scan camera <NUM>. After scanning a stripe of image data, movable stage <NUM> decelerates and brings sample <NUM> to a substantially complete stop. Movable stage <NUM> then moves orthogonal to the scanning direction to position sample <NUM> for scanning of a subsequent stripe of image data (e.g., an adjacent stripe). Additional stripes are subsequently scanned until an entire portion of sample <NUM> or the entire sample <NUM> is scanned.

For example, during digital scanning of sample <NUM>, a contiguous digital image of sample <NUM> is acquired as a plurality of contiguous fields of view that are combined together to form an image stripe. A plurality of adjacent image stripes are similarly combined together to form a contiguous digital image of a portion or the entire sample <NUM>. The scanning of sample <NUM> may include acquiring vertical image stripes or horizontal image stripes. The scanning of sample <NUM> may be either top-to-bottom, bottom-to-top, or both (i.e., bi-directional), and may start at any point on sample <NUM>. Alternatively, the scanning of sample <NUM> may be either left-to-right, right-to-left, or both (i.e., bi-directional), and may start at any point on sample <NUM>. It is not necessary that image stripes be acquired in an adjacent or contiguous manner. Furthermore, the resulting image of sample <NUM> may be an image of the entire sample <NUM> or only a portion of the sample <NUM>.

In an embodiment, computer-executable instructions (e.g., programmed modules and software) are stored in memory <NUM> and, when executed, enable scanning system <NUM> to perform the various functions (e.g., display the graphical user interface, execute the disclosed processes, control the components of scanning system <NUM>, etc.) described herein. In this description, the term "computer-readable storage medium" is used to refer to any media used to store and provide computer-executable instructions to scanning system <NUM> for execution by processor <NUM>. Examples of these media include memory <NUM> and any removable or external storage medium (not shown) communicatively coupled with scanning system <NUM> either directly (e.g., via a universal serial bus (USB), a wireless communication protocol, etc.) or indirectly (e.g., via a wired and/or wireless network).

<FIG> illustrates a line-scan camera <NUM> having a single linear array <NUM>, which may be implemented as a charge-coupled device ("CCD") array. Single linear array <NUM> comprises a plurality of individual pixels <NUM>. In the illustrated embodiment, the single linear array <NUM> has <NUM>,<NUM> pixels <NUM>. In alternative embodiments, linear array <NUM> may have more or fewer pixels. For example, common formats of linear arrays include <NUM>, <NUM>,<NUM>, and <NUM>,<NUM> pixels. Pixels <NUM> are arranged in a linear fashion to define a field of view <NUM> for linear array <NUM>. The size of field of view <NUM> varies in accordance with the magnification of scanning system <NUM>.

<FIG> illustrates a line-scan camera <NUM> having three linear arrays <NUM>, each of which may be implemented as a CCD array. The three linear arrays <NUM> combine to form a color array <NUM>. In an embodiment, each individual linear array in color array <NUM> detects a different color intensity, including, for example, red, green, or blue. The color image data from each individual linear array <NUM> in color array <NUM> is combined to form a single field of view <NUM> of color image data.

<FIG> illustrates a line-scan camera <NUM> having a plurality of linear arrays <NUM>, each of which may be implemented as a CCD array. The plurality of linear arrays <NUM> combine to form a TDI array <NUM>. Advantageously, a TDI line-scan camera may provide a substantially better SNR in its output signal by summing intensity data from previously imaged regions of a specimen, yielding an increase in the SNR that is in proportion to the square-root of the number of linear arrays <NUM> (also referred to as integration stages). A TDI line-scan camera may comprise a larger variety of numbers of linear arrays <NUM>. For example, common formats of TDI line-scan cameras include <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and even more linear arrays <NUM>.

The disclosed physical calibration slide may be used for setting up, aligning, and calibrating scanning systems <NUM>, such as the bright-field slide scanners produced by Leica Biosystems® of Nussloch, Germany. In an embodiment, the physical calibration slide may be a glass slide (e.g., like glass slide <NUM>) with physical dimensions of <NUM> millimeters (mm) x <NUM> x <NUM>. However, it should be understood that other sizes are possible.

The physical calibration slide may comprise a plurality of features, with each feature being targeted towards one or more calibration operations. A scanning system <NUM> may automatically or with manual intervention scan one or more of the plurality of features on the physical calibration slide. Scanning system <NUM> may then use the image of each scanned feature to perform calibration operations (e.g., by processor <NUM>), such as calculating calibration parameters, aligning cameras <NUM> and/or <NUM>, and/or the like. Any calculated calibration parameters may be stored in a configuration file (e.g., in memory <NUM>) on scanning system <NUM>, to be loaded and reused during each initialization of scanning system <NUM>.

<FIG> illustrates a physical calibration slide <NUM>, according to an embodiment. In the illustrated embodiment, physical calibration slide <NUM> has a width (i.e., along the short axis) of <NUM> and a length (i.e., along the long axis) of <NUM>. It should be understood that, in the illustrated embodiments, the short and long axes of physical calibration slide <NUM> correspond to the X and Y axes of scanning system <NUM>, respectively. However, in an alternative embodiment, the short and long axes of physical calibration slide <NUM> could correspond to the Y and X axes, respectively. Physical calibration slide <NUM> may comprise a label area <NUM> and a feature box <NUM>.

Label area <NUM> may comprise text and/or images that identify the purpose of physical calibration slide <NUM> (e.g., "setup-calibration slide"), a model of physical calibration slide <NUM> (e.g., model number, revision number, etc.), a manufacturer of physical calibration slide <NUM> or scanning systems <NUM> with which physical calibration slide <NUM> is intended to be used, and/or the like. Label area <NUM> may correspond in size and position to the label area (e.g., comprising a barcode) of a typical glass slide <NUM> used for digital pathology.

Feature box <NUM> may be distinguished by a line that defines the boundary or scaling factor of the coordinate system to be used for calibration. In an embodiment, feature box <NUM> may be <NUM> x <NUM> and be delineated by a <NUM> micrometer (µm) thick, clear boundary line (e.g., chrome-etched). Feature box <NUM> comprises a plurality of features <NUM>. Each feature <NUM> is targeted towards one or more calibration operations to be performed by a scanning system <NUM>. One or more - including potentially all of - features <NUM> may comprise a pattern created by chrome etching. For example, chromium may be deposited on the glass substrate of physical calibration slide <NUM>, and the patterns representing features <NUM> may be etched into the deposited chromium using any well-known method. In alternative embodiments, techniques, other than chrome-etching, may be used for creating the patterns representing features. Such techniques include, without limitation, laser writing, E-beam writing, and/or the like. However, while such techniques may provide more precision, they are generally more expensive.

<FIG> illustrates a specific example of physical calibration slide <NUM>, according to an embodiment. While a specific selection and arrangement of features <NUM> are illustrated in <FIG>, different selections and/or arrangements of features <NUM> may also be used. One or more of features <NUM> may be designed for scanning along both axes (i.e., short and long axes) of physical calibration slide <NUM>. One notable exception may be the Ronchi rulings features (slanted and/or straight) which may be oriented to favor the scanning axis (e.g., which may correspond to either the long or short axis of physical calibration slide <NUM>).

The various lines etched on physical calibration slide <NUM> (e.g., to form feature box <NUM>, the patterns of features <NUM>, etc.) may all generally be of uniform thickness, unless otherwise specified. In a preferred embodiment, the lines are all generally <NUM> thick. In an alternative embodiment, the lines may all generally be <NUM> thick. However, different line thicknesses may also be used, including different thicknesses for different lines, depending on the particular design and cost goals.

In an embodiment, no coverslip is used for physical calibration slide <NUM>. A coverslip could introduce imaging variation across physical calibration slide <NUM> due to nonuniform gluing and air bubbles and/or glue cracks from aging. Such variation would impact some of the tests of scanning system <NUM>. However, a coverslip may be added for modulation transfer function (MTF) tests if the scanner is optimized for scanning cover-slipped samples.

In an embodiment, physical calibration slide <NUM> comprises slanted Ronchi rulings features 222A and 222B. Slanted Ronchi rulings features 222A and 222B may be used by scanning system <NUM> to perform MTF tests and/or analyze the analytical model for scanning system <NUM>. <FIG> illustrates a close up of a portion of slanted Ronchi rulings features 222A and 222B, according to an embodiment. As illustrated, slanted Ronchi rulings feature 222A comprises parallel Ronchi rulings slanted at <NUM>° with respect to the short axis of physical calibration slide <NUM>, whereas slanted Ronchi rulings feature 222B comprises parallel Ronchi rulings slanted at <NUM>° with respect to the short axis. In an embodiment, the resolution of slanted Ronchi rulings features 222A and/or 222B may be <NUM> line pairs per millimeter (LP/mm) or <NUM> LP/inch. Each of slanted Ronchi rulings features 222A and 222B may be <NUM> x <NUM> with the longer dimension parallel to the long axis of physical calibration slide <NUM>.

In an embodiment, physical calibration slide <NUM> comprises straight Ronchi rulings features 222C and 222D. Straight Ronchi rulings features 222C and 222D may be used by scanning system <NUM> alignment, motion tests, chromatic aberration tests, focus checks, vibration tests, left and right focus tests, and/or a macro focus (MF) limits setup. One straight Ronchi rulings feature 222C may comprise parallel lines that extend parallel to the long axis of physical calibration slide <NUM>, whereas the other straight Ronchi rulings feature 222D may comprise parallel lines that extend parallel to the short axis of physical calibration slide <NUM>. In other words, the separate straight Ronchi rulings features 222C and 222D may be orthogonal to each other. The resolution of straight Ronchi rulings features 222C and/or 222D may be <NUM> LP/mm. Each of straight Ronchi rulings features 222C and 222D may be <NUM> x <NUM> with the longer dimension parallel to the long axis of physical calibration slide <NUM>.

As mentioned above, straight Ronchi rulings features 222C and 222D may be used for checking focus. For instance, <FIG> illustrates the same straight Ronchi rulings feature 222C both in focus and out of focus, from left to right.

As mentioned above, straight Ronchi rulings features 222C and 222D may be used for vibration testing (e.g., measurement). For instance, <FIG> illustrates the same straight Ronchi rulings feature 222C both with no vibration and vibration, from left to right. As illustrated, the variable distance between adjacent parallel Ronchi lines can be used to measure the effect of vibrations in scanning system <NUM>. For example, the effect may be represented as an error that is calculated based on the difference in distance between adjacent parallel Ronchi lines in a scanned image of physical calibration slide and the actual, known distance between those adjacent parallel Ronchi lines on physical calibration slide <NUM>.

As mentioned above, straight Ronchi rulings features 222C and 222D may be used for chromatic aberration tests. For instance, <FIG> illustrates a lateral chromatic test using straight Ronchi rulings feature 222C.

As mentioned above, straight Ronchi rulings features 222C and 222D may be used for motion tests. For instance, <FIG> illustrates a scan velocity test and trilinear sensor spacing test using straight Ronchi rulings feature 222D. The left side illustrates an image of straight Ronchi rulings feature 222D acquired using the correct scan speed, whereas the right side illustrates an image of the same straight Ronchi rulings feature 222D acquired using the wrong scan speed. To achieve the correct image aspect ratio, the scan velocity of scanning system <NUM> (e.g., the velocity of stage <NUM>) should be synchronized with the line rate of line scan camera <NUM>. The color shift can be corrected by adjusting the line spatial correction parameter in line scan camera <NUM>, but the image aspect ratio will be incorrect.

As mentioned above, straight Ronchi rulings features 222C and 222D may be used for alignment. For instance, <FIG> illustrates the detection of camera rotation on the short axis of line scan camera <NUM> using straight Ronchi rulings feature 222D. The left side shows rotation in one direction, the right side shows rotation in the opposite direction, and the middle shows alignment (i.e., no rotation).

In an embodiment, physical calibration slide <NUM> comprises star target feature 222E. Star target feature 222E may be used as an alignment mark and as a target for focusing tests by scanning system <NUM>. For instance, <FIG> illustrates a close up of star target feature 222E, according to an embodiment. As illustrated, star target feature 222E comprises a circle <NUM> with thirty-six (<NUM>) wedge pairs, i.e., seventy-two (<NUM>) total wedges. Each wedge pair comprises both a fully etched wedge 520A and a chrome wedge 520B, with each wedge representing <NUM>° of the circle <NUM>. In addition, circle <NUM> may comprise a core <NUM>. In an embodiment, the outer diameter of the circle <NUM> is <NUM>, and the core <NUM> of the circle <NUM> is fully etched with a diameter of <NUM>. It should be understood that different numbers and sizes of wedges <NUM> and/or different sizes of core <NUM> (e.g., <NUM> diameter) may be used, depending on the manufacturable resolution.

As illustrated in <FIG>, star target feature 222E may comprise one or more etched inner circles <NUM> between the core <NUM> and the circumference of the circle <NUM>. For example, at least one inner circle <NUM> may have a line thickness of <NUM> with an inner diameter of <NUM>, for an equivalent <NUM> LP/mm macro image resolution. During macro focus alignment, star target feature 222E should be resolvable outside this inner circle <NUM>. Thus, this inner circle <NUM> can be used for pass/fail testing on macro imaging.

<FIG> is a close up of the core <NUM> of star target feature 222E, according to an embodiment. In an embodiment, the core <NUM> of star target feature 222E may comprise a dot <NUM> (e.g., chrome) in the center of star target feature 222E. For example, dot <NUM> may have a <NUM> diameter. The core <NUM> and/or dot <NUM> may be used for fine alignment, positioning, and/or registration (e.g., macro corner index alignment).

In an embodiment, physical calibration slide <NUM> comprises crosshair feature 222F. For instance, <FIG> illustrates a close up of crosshair feature 222F, according to an embodiment. As illustrated, crosshair feature 222F may comprise an outer rectangle <NUM> surrounding a centered crosshair <NUM>() that almost fills the outer rectangle <NUM>. In addition, a smaller, offset crosshair <NUM> may be positioned in one corner (e.g., right-lower corner) within the outer rectangle <NUM>. Each crosshair <NUM> and <NUM> may comprise two orthogonal, bisecting lines. The lines of the outer rectangle <NUM> and/or centered crosshair <NUM> may be <NUM> thick, whereas the lines of the offset crosshair <NUM> may be <NUM> thick (e.g., corresponding to approximately <NUM> pixels with <NUM>/pixel resolution). The centered crosshair <NUM> may be <NUM> x <NUM>, whereas the offset crosshair <NUM> may be <NUM> x <NUM>. In an embodiment, the outer rectangle <NUM> is <NUM> wide (i.e., along the short axis).

The centered crosshair <NUM> in crosshair feature 222F may define the center of the coordinate system in macro imaging, and, for example, be located at the precise center of feature box <NUM>. The offset crosshair <NUM> may be used for fine alignment between high-resolution cameras (e.g., imaging sensor of line-scan camera <NUM> and/or focusing camera <NUM>).

In an embodiment, physical calibration slide <NUM> comprises clear area feature <NUM>. Clear area feature <NUM> comprises a blank or clear area (e.g., fully etched) that may be used for illumination correction. As illustrated, a region between slanted Ronchi rulings feature 222B and straight Ronchi rulings feature 222C may be used as the clear area of feature <NUM>. As an example, clear area feature <NUM> may be <NUM> x <NUM> with the longer dimension parallel to the long axis of physical calibration slide <NUM>.

In an embodiment, physical calibration slide <NUM> comprises letter-O feature <NUM>. Letter-O feature <NUM> comprises a letter "<NUM>" for macro imaging. <FIG> illustrates this letter "<NUM>", according to an embodiment. Letter-O feature <NUM> may be rotationally symmetric and independent of the scan axis. The line thickness of the letter "<NUM>" may be <NUM>¡. u11 or greater (e.g., <NUM>), approximating <NUM> pixels for easy identification, and the diameter of the letter "<NUM>" may be 3mni. The letter "<NUM>" may be formed by chrome etching, so that the line of the letter "O" remains in chromium. Advantageously, the edges of the letter "O" can be used to judge macro focusing.

In an embodiment, physical calibration slide <NUM> comprises a resolution target feature <NUM>. Resolution target feature <NUM> may comprise one or more resolution targets for macro and/or micro imaging. In the embodiment illustrated in <FIG>, resolution target feature <NUM> comprises seven (<NUM>) resolution targets that conform to the 1010A standard of the National Institute of Standards and Technology (NIST) and National Bureau of Standards (NBS). These resolution targets may include an <NUM> cycles/mm target (e.g., with line width of <NUM>". 5uni) as a location mark, <NUM>, <NUM>, and <NUM> cycles/mm targets for macro imaging (e.g., with each line width representing approximately <NUM> pixels), a <NUM> cycles/mm target for 4x imaging, a <NUM> cycles/mm target for 10x imaging (e.g., for best effort), and a <NUM> cycles/mm (or <NUM> or <NUM> cycles/mm) target for 20x imaging (e.g., with line width of <NUM>).

In an embodiment, one or more of the resolution targets in resolution target feature <NUM> may be encircled for easier identification on a magnified image. For example, a circle may be etched around both the <NUM> cycle/mm and <NUM> cycles/mm resolution targets, as illustrated in <FIG>.

In an embodiment, physical calibration slide <NUM> comprises a bullseye feature 222J. Bulleye feature 222J may be used for distortion tests, stitching error tests, camera walk-off tests, area camera rotation tests, and/or stripe alignment tests. Bullseye feature 222J may comprise a sea of bullseyes (i.e., a two-dimensional array of bullseyes). The sea may be a <NUM>,<NUM> x <NUM>,<NUM> square of tightly packed bullseyes (i.e., one million bullseyes). However, other dimensions are possible, depending on the design and cost constraints. <FIG> illustrates a <NUM> x <NUM> portion of the sea of bulls eyes, according to an embodiment. Each bullseye may comprise a <NUM> dot in the center of a <NUM> circle, and the entire sea of bullseyes in bullseye feature 222J may be a <NUM> x <NUM> square that is symmetric along both axes (i.e., along both the short and long axes of physical calibration slide <NUM>).

As mentioned above, bullseye feature 222J may be used for distortion testing. <FIG> illustrates examples of one-dimensional and two-dimensional distortion using a sea of bullseyes.

As mentioned above, bullseye feature 222J may be used for stitching error tests. <FIG> illustrates examples of up-and-down stitching error and left-and-right stitching error using a portion of the sea of bulls eyes.

As mentioned above, bullseye feature 222J may be used for a camera walk-off test (e.g., to test unsynchronized cameras in the case that scanning system <NUM> comprises a plurality of cameras <NUM> and/or <NUM>). <FIG> illustrates an example of a camera walk-off test using a sea of bullseYes.

As mentioned above, bullseye feature 222J may be used to detect rotation of area scan camera <NUM>. <FIG> illustrates an example of an area camera rotation test. The left side shows rotation in one direction, the right side shows rotation in the opposite direction, and the middle shows alignment (i.e., no rotation).

In an embodiment, physical calibration slide <NUM> comprises at least one triangle feature <NUM>. Triangle feature <NUM> may be used to match fields of view (FOV) between two cameras <NUM> (e.g., a main imaging and focusing sensor) and/or <NUM>, and/or macro image focusing. As illustrated in <FIG>, according to an embodiment, triangle feature <NUM> may comprise a <NUM>° isosceles triangle. As an example, the triangle may have a base that is <NUM> wide, a height of <NUM>, and be formed by lines that are <NUM> thick (e.g., resolved with <NUM> pixels at <NUM>/pixel macro image resolution). In an embodiment, there may be two triangle features <NUM>, that are oriented <NUM>° with respect to each other (e.g., on opposite sides of feature 222F along the short axis of physical calibration slide <NUM>).

In an embodiment, physical calibration slide <NUM> comprises one or more sets of symmetric corner features <NUM>, such as arrays of dots and/or squares arranged in an "L" pattern, that are symmetric along both the short and long axes of physical calibration slide <NUM>. Each corner feature <NUM> may be used for increased sensitivity in rigid-body (e.g., lateral and/or rotational) alignment, i.e., fine alignment.

<FIG> illustrates two examples of corner feature <NUM>. Physical calibration slide <NUM> may comprise one, both, or none of these examples. In both examples, corner feature <NUM> comprises two sets of fully etched geometric shapes in two opposite corners of a box, with the sets of geometric shapes being symmetrical to each other about both diagonals of the box. In each corner, the geometric shapes are arranged with the largest shape in the corner, and additional shapes extending from the corner, with decreasing size, in an "L" pattern. Center-to-center the shapes may be spaced at <NUM> from each other and cover a total of <NUM> FOV.

In the left-side example, the geometric shapes are squares, with a corner square and two arrays of squares that are spaced apart (e.g., <NUM> center-to-center) and extend from the corner square in orthogonal directions. The corner square may have a width of <NUM>, and the squares in both arrays may have widths of <NUM>, <NUM>, <NUM>, and <NUM>, respectively, from farthest from the corner square to closest to the corner square.

In the right-side example, the geometric shapes are dots, with a corner dot and two arrays of dots that are spaced apart (e.g., <NUM> center-to-center) and extend from the corner dot in orthogonal directions. The corner dot may have a diameter of <NUM>, and the dots in both arrays may have diameters of <NUM>, <NUM>, <NUM>, and <NUM>, respectively, from farthest from the corner dot to closest to the corner dot. It should be understood that different geometric shapes may be used, other than dots or squares, including different shapes within the same corner feature <NUM>.

In an embodiment, corner feature <NUM> may be used to check a sensor's perpendicularity for both a color sensor (e.g., main imaging sensor of line scan camera <NUM>) and a second sensor (e.g., focusing sensor of line scan camera <NUM> and/or area scan camera <NUM>). Specifically, a scanned image of corner feature <NUM> can demonstrate whether or not the respective sensor is skewed (i.e., not perpendicular).

In an embodiment, corner feature <NUM> can be used to align two cameras, such as the main imaging sensor and focusing sensor of line scan camera <NUM> and/or area camera <NUM>. Specifically, images of corner feature <NUM> from both cameras can be overlaid on each other. If there is blurriness, then the two cameras are not aligned. Lateral shifting and/or rotation of the cameras can then be performed until the two overlaid images align (i.e., are no longer blurry).

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles described herein can be applied to other embodiments without departing from the scope of the invention. Thus, it is to be understood that the description and drawings presented herein represent a presently preferred embodiment of the invention and are therefore representative of the subject matter which is broadly contemplated by the present invention. It is further understood that the scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art and that the scope of the present invention is solely defined by the appended claims.

Claim 1:
A physical calibration slide, having a long axis along a long side and short axis along a short side, the calibration slide comprising a plurality of etched features, the plurality of etched features comprising:
a first Ronchi ruling feature (222A) comprising at least one set of parallel lines that are slanted at a first angle with respect to the short axis;
a second Ronchi ruling feature (222B) positioned adjacent to the first Ronchi ruling feature (222A), the second Ronchi ruling feature (222B) comprising at least one set of parallel lines that are slanted at a second angle with respect to the short axis that is different than the first angle;
a clear area (<NUM>) positioned adjacent to the second Ronchi ruling feature (222B);
a third Ronchi ruling feature (222C) positioned adjacent to the clear area (<NUM>), the third Ronchi ruling feature (222C) comprising at least one set of parallel lines that are parallel to the long axis;
a fourth Ronchi ruling feature (222D) positioned adjacent to the third Ronchi ruling feature (222C), the fourth Ronchi ruling feature (222D) comprising at least one set of parallel lines that are parallel to the short axis;
a star target feature (222E) comprising a circle with a plurality of wedge pairs, wherein each of the plurality of wedge pairs comprises an etched wedge and a non-etched wedge;
a crosshair feature (222F) comprising at least one crosshair;
a letter-O feature (<NUM>) comprising a circle;
a two-dimensional array of bullseyes, wherein each of the bullseyes comprises a circle with a dot in the centre of the circle, the array preferably comprising 1000x1000 bullseyes; and
a triangle feature (<NUM>) comprising at least one isosceles triangle.