Patent Description:
The present disclosure generally relates to digital pathology, and more particularly relates to identifying a tissue area on a glass slide using a digital slide scanning apparatus.

Digital pathology is an image-based information environment which is enabled by computer technology that allows for the management of information generated from a physical slide. Digital pathology is enabled in part by virtual microscopy, which is the practice of scanning a specimen on a physical glass slide and creating a digital slide image that can be stored, viewed, managed, and analyzed on a computer monitor. With the capability of imaging an entire glass slide, the field of digital pathology has exploded and is currently regarded as one of the most promising avenues of diagnostic medicine in order to achieve even better, faster, and cheaper diagnosis, prognosis, and prediction of important diseases, such as cancer.

In the field, an image acquiring apparatus (<CIT>) is known for acquiring images of a sample. The apparatus includes a macro imaging unit for acquiring a macro image, a dark field light source for acquiring a dark field macro image, a macro image processing unit for generating a reference macro image. It further includes an image pickup condition setting unit which sets an image acquiring range as an image pickup condition of a micro image of the sample by referring to the reference macro image.

<CIT> relates to a microscope that includes dark field and bright field illumination to illuminate a preparate of a sample covered with a cover glass and an image capturing unit to acquire a dark field image and a bright field image of the preparate. It further includes a magnified portion image acquisition area determination unit to detect an edge of the cover glass in the preparate and to determine an internal area of the detected edge of the cover glass.

<CIT> discloses an information processing apparatus which can compose a microscopically observed image having a wide field of view and a high resolution by highly accurately stitching a plurality of digital images together. An image acquisition unit in the information processing apparatus acquires a first and second partial image, a stitching position adjustment unit adjusts a stitching position of the second partial image with respect to the first partial image.

A conventional digital slide scanning apparatus typically includes a high-resolution camera sensor that is used for scanning a high-resolution image of the specimen on the slide. A conventional digital slide scanning apparatus also typically includes a low-resolution camera sensor that is used for scanning a low-resolution macro image of the specimen on the slide. Typically, the macro image is used to identify the area of the glass slide that is occupied by the specimen, and may also be used to generate a thumbnail image of the whole slide. A drawback of the conventional digital slide scanning apparatus is that the inclusion of the low-resolution camera sensor adds cost to the apparatus. One solution that has been proposed is to use the high-resolution camera sensor to capture a high-resolution macro image.

However, a disadvantage of having a high-resolution macro image obtained by the high-resolution camera is that the high-resolution macro image often includes unwanted image artifacts from physical items on the slide or slide cover slip, such as dust, fingerprints, and/or the like. These artifacts can be introduced during slide preparation or handling.

These unwanted image artifacts in the macro image can significantly impact the image processing that is performed on the macro image of the specimen to determine, for example, the location of the specimen, the area of the glass slide to be scanned, and an initial focus point on the specimen. Furthermore, if an initial focus point (e.g., for constructing a focal surface) happens to be set to a location of an unwanted image artifact, the quality of the resulting digital slide image can be negatively impacted. Therefore, what is needed is a system and method that overcomes these significant problems found in the conventional systems as described above.

To solve the problems associated with conventional macro image capture in a digital slide scanning apparatus, solutions are described herein that utilize first and second illumination systems to capture two images of the slide. According to one aspect, a digital slide scanning apparatus according to claim <NUM> is provided. According to another aspect, a method for generating a macro image in a digital slide scanning apparatus according to claim <NUM> is provided.

Other features and advantages of the present invention will become more readily apparent to those of ordinary skill in the art after reviewing the following detailed description and accompanying drawings.

The structure and operation of the present invention will be understood from a review of the following detailed description and the accompanying drawings in which like reference numerals refer to like parts and in which:.

Certain embodiments disclosed herein provide systems and methods for capturing a high-resolution macro image of a glass slide that is free from unwanted image artifacts. For example, one method disclosed herein allows for a first high-resolution macro image to be captured using a bottom-illumination system and a second high-resolution macro image to be captured using a top-illumination system. The two high-resolution macro images are processed to identify unwanted image artifacts in the second high-resolution macro image and remove the identified unwanted image artifacts from the first high-resolution macro image to produce a high-resolution macro image with no unwanted image artifacts. 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 only, and not limitation. As such, this detailed description of various alternative 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 digital slide scanning apparatus <NUM> for identifying a tissue area of a sample <NUM> on a glass slide <NUM>, according to an embodiment. In the illustrated embodiment, the scanning apparatus <NUM> includes a high-resolution camera <NUM> that defines a first optical path <NUM> in combination with a first lens <NUM>. The first optical path <NUM> is configured for capturing high-resolution macro images of the sample <NUM> on the slide <NUM>. The first optical path <NUM> fully encompasses a field of view <NUM> of the high-resolution camera <NUM> on the sample <NUM>. The combination of the high-resolution camera <NUM> and the first lens <NUM> with the first optical path <NUM> is such that the field of view <NUM> covers substantially the entire width of the slide <NUM>, such that a single pass of the slide <NUM> under the first optical path <NUM> allows for imaging of the entire slide <NUM>. A macro image is typically captured at low magnification and includes the entire slide <NUM> in the macro image. The high-resolution camera <NUM> also defines a second optical path <NUM> in combination with a second lens <NUM>. The second optical path <NUM> is for capturing high-resolution images at high magnification.

In an embodiment, the high-resolution camera can be a line scan camera or an area scan camera or any of a variety of high-resolution cameras such as a time delay integration (TDI) camera, a color camera, or the like. For ease of discussion, the high-resolution camera will be referred to herein as a line scan camera <NUM>.

The digital slide scanning apparatus <NUM> also includes a first illumination system <NUM> that may optionally include one or more diffusers <NUM>. The first illumination system <NUM> is positioned below the slide <NUM> and is configured for transmission-mode illumination, such that light from the first illumination system <NUM> passes through the sample <NUM> and into the first optical path <NUM>. The light from the first illumination system <NUM> may also pass through the one or more diffusers <NUM> prior to passing through the sample <NUM>. Advantageously, the one or more diffusers <NUM> function to provide uniform illumination in the first optical path <NUM>.

The digital slide scanning apparatus <NUM> also includes a second illumination system <NUM>. The second illumination system <NUM> is positioned above the slide <NUM> and is configured for reflection-mode illumination, such that substantially all light from the second illumination system <NUM> reflects off of the slide <NUM> and the sample <NUM> and away from the first optical path <NUM>. Advantageously, the desired reflected light from debris will pass into the first optical path <NUM>. However, some undesired reflected light may also pass into the first optical path <NUM>. Accordingly, the digital slide scanning apparatus <NUM> is configured such that undesired reflected light from the second illumination system <NUM> does not land on any individual pixel sensor of the high-resolution camera <NUM>.

In an embodiment, during the macro image acquisition process, the stage (not shown) travels a linear out-and-back path (e.g., along an X axis that is parallel to the longitudinal axis of slide <NUM>). The out-and-back path moves the glass slide <NUM> under a macro imaging position, defined by the first optical path <NUM>, traveling in a first direction from a starting position (e.g., at which a first field of view <NUM>, representing one end of slide <NUM> or sample <NUM>, is under the macro imaging position) to an ending position (e.g., at which a second field of view <NUM>, representing the opposite end of slide <NUM> or sample <NUM>, is under the macro imaging position). Subsequently, the out-and-back path moves the glass slide <NUM> under the macro imaging position traveling in a second direction that is opposite the first direction from the ending position back to the starting position. On the "out" portion of the trip, a first macro image is captured using the first transmission-mode illumination system <NUM> and the first lens <NUM> along the first optical path <NUM>. On the "back" portion of the trip, a second macro image is captured using the second reflection-mode illumination system <NUM> and the first lens <NUM>, along the first optical path <NUM>. Alternatively, the second macro image could be captured first on the "out" portion of the trip, and the first macro image could be captured second on the "back" portion of the trip. In another alternative embodiment, the first and second macro images may be captured while the stage remains stationary (i.e., without movement of the stage relative to the first lens <NUM>), for example, by an area scan camera. In any case, while, in the interest of simplicity, the first macro image will be generally described herein as being captured first and the second macro image will be generally described herein as being captured second, it should be understood that the order of image capture may be reversed, such that first macro image is captured second and the second macro image is captured first.

Advantageously, the second illumination system <NUM> provides oblique illumination from the top of the glass slide <NUM> and uses an angled scatter light to highlight only the unwanted debris on top of the glass slide <NUM> and/or coverslip. The positioning of the second illumination system <NUM> and the direction of its scatter light are carefully configured to minimize undesired reflected light from traveling into the first optical path <NUM>. Additionally, the positioning of the high-resolution camera <NUM> within the first optical path <NUM> is carefully configured to minimize or eliminate undesired reflected light in the optical path <NUM> from reaching a sensor of the high-resolution camera <NUM>.

Advantageously, the digital slide scanning apparatus <NUM> is configured to provide very high image quality that can be used to identify the area of the glass slide <NUM> that is occupied by the specimen <NUM>. Because light from the second illumination system <NUM> may reflect off of the elements of the first illumination system <NUM> and/or the diffusers <NUM>, the first illumination system <NUM>, diffusers <NUM>, second illumination system <NUM>, and the high-resolution camera <NUM> are carefully aligned to avoid the individual sensors of the high-resolution camera <NUM> from receiving any light from the second illumination system <NUM> that is reflected from the diffusers <NUM>, the first illumination system <NUM>, the glass slide <NUM>, or the sample <NUM>. This results in the individual sensors of the high-resolution camera <NUM> capturing an image that emphasizes only the debris on the top of the glass slide and/or coverslip when the second illumination system <NUM> is used.

A processor <NUM> in the digital slide scanning apparatus <NUM> processes the two high-resolution macro images captured during the "out" and "back" portions of the trip under the first optical path <NUM>. Unwanted image artifacts corresponding to debris are identified in the macro image captured using the second illumination system <NUM>, and the identified unwanted image artifacts are corrected in the macro image captured using the first illumination system <NUM>. The result is a clean high-resolution macro image that is free from unwanted image artifacts and that can be subsequently used for tissue finding and initial focus point selection.

<FIG> is a block diagram illustrating an example first optical path <NUM> and high-resolution camera sensor <NUM>, according to an embodiment. As previously discussed, the elements of the digital slide scanning apparatus <NUM> are carefully aligned such that the individual sensors of the high-resolution camera <NUM> are positioned in the first optical path <NUM>, such that the individual sensors do not receive undesired reflected light <NUM> from the second illumination system <NUM>.

<FIG> is a block diagram illustrating an example digital slide scanning system <NUM> for identifying a tissue area <NUM> of a glass slide <NUM> with a first illumination system <NUM> turned on, according to an embodiment. In the illustrated embodiment, the first illumination system <NUM> is turned on, and light produced by the first illumination system <NUM> passes through one or more diffusers <NUM> that are configured to uniformly illuminate the slide <NUM> and the first optical path <NUM>. The line scan camera <NUM> is logically aligned such that its field of view <NUM> is positioned on a portion of the slide <NUM> that is uniformly lit by the first illumination system <NUM>.

<FIG> is a block diagram illustrating an example digital slide scanning system <NUM> for identifying a tissue area <NUM> of a glass slide <NUM> with a second illumination system <NUM> turned on, according to an embodiment. In the illustrated embodiment, the second illumination system <NUM> is turned on, and light produced by the second illumination system <NUM> illuminates the slide <NUM>, the sample <NUM>, and the coverslip <NUM>. The second illumination system <NUM> is positioned such that light from the second illumination system <NUM> reflects off of the slide <NUM>, the sample <NUM>, the coverslip <NUM>, the diffusers <NUM>, the first illumination system <NUM>, and any other objects within its illumination field, and the reflected light does not pass into the first optical path <NUM>. However, some undesired reflected light <NUM> may pass into the first optical path <NUM>. The line scan camera <NUM> is aligned such that the undesired reflected light <NUM> that does pass into the first optical path <NUM> is not received by any of the individual sensors of the line scan camera <NUM>.

<FIG> is a flow diagram illustrating an example process for scanning a macro image of a glass slide in a digital slide scanning apparatus, according to an embodiment. In the illustrated embodiment, the process may be carried out by a system such as those described with respect to <FIG> and <FIG>. Initially, in step <NUM>, the system turns on the first illumination system <NUM> for illuminating a slide from below. This is transmission-mode illumination.

Next, in step <NUM>, the stage that supports the glass slide <NUM> with the specimen is moved through the macro imaging position. The macro imaging position is defined by the field of view <NUM> of the high-resolution camera <NUM> that is positioned in the first optical path <NUM> that is created by the combination of the macro imaging lens <NUM> and the high-resolution camera <NUM>. As the stage moves the slide <NUM> through the macro imaging position, a first macro image of the entire slide <NUM> is captured during a single pass, as shown in step <NUM>. Advantageously, the field of view <NUM> of the high-resolution camera <NUM> is wide enough to capture substantially the entire width of the slide <NUM>.

In an alternative embodiment, the first macro image of the entire slide <NUM> may be captured while the stage remains stationary with respect to the objective lens, for example, by an area scan camera. For example, the first macro image may be captured as a series of mosaic tiles, with each tile being captured while the stage remains stationary. Alternatively, the first macro image may be captured as a single image that is captured while the stage remains stationary. An area scan camera may also capture the first macro image (as a single image or a series of mosaic tiles) while the stage is in motion, for example, using strobing illumination.

Next, in step <NUM>, the first illumination system <NUM> is turned off, and then, in step <NUM>, the second illumination system <NUM> is turned on. The second illumination system <NUM> illuminates the slide <NUM> at an angle from above. This is reflection-mode illumination. Next, in step <NUM>, the stage moves the glass slide <NUM> through the macro imaging position a second time, and, in step <NUM>, a second macro image of the entire slide <NUM> is captured during a single pass. Again, in an alternative embodiment, the second macro image of the entire slide <NUM> may be captured while the stage remains stationary, for example, by an area scan camera.

The second macro image of the entire slide <NUM>, captured using reflection mode illumination, is then analyzed in step <NUM>, and, in step <NUM>, unwanted image artifacts corresponding to debris are identified in the second macro image. Next, in step <NUM>, the same unwanted image artifacts that were identified in the second macro image are identified in the first macro image. The first macro image is then corrected, in step <NUM>, to remove or otherwise account for the unwanted image artifacts that are present in the first macro image. Finally, in step <NUM>, the corrected first macro image is stored as the clean macro image for the glass slide <NUM>. Advantageously, the corrected first macro image can be subsequently used to identify the area of the glass slide <NUM> that is occupied by the sample <NUM>.

While <FIG> illustrates the acquisition of the first macro image before the second macro image, it should be understood that the second macro image can be acquired before the first macro image. For example, steps <NUM>-<NUM> may be switched with steps <NUM>-<NUM>, and step <NUM> may comprise turning off the second illumination system <NUM>, instead of the first illumination system <NUM>. In addition, it should be understood that steps <NUM> and <NUM> could be performed at any time after the acquisition of the second macro image in step <NUM>, and therefore, do not necessarily need to occur after acquisition of the first macro image in step <NUM>.

<FIG> is a flow diagram illustrating an example process for determining a scan area for a glass slide having a sample thereon, according to an embodiment. <FIG> is a block diagram illustrating an example set of images used to determine a scan area for a glass slide having a sample thereon, according to an embodiment. <FIG> will be described together below. In the illustrated embodiments, the process of <FIG> may be carried out by a system such as those described with respect to <FIG> and <FIG>.

Initially, the first macro image <NUM> (the "bottom-lit image" in <FIG>, corresponding to example image <NUM> in <FIG>) is obtained (e.g., acquired via steps <NUM>-<NUM> in <FIG>), and the second macro image <NUM> (the "top-lit image" in <FIG>) is obtained (e.g., acquired via steps <NUM>-<NUM> in <FIG>). The first macro image <NUM> may be pre-processed for illumination correction, background offset, and/or background noise removal to generate a pre-processed first macro image <NUM> (corresponding to example image <NUM> in <FIG>). Advantageously, illumination-correction processing corrects for non-uniformity in illumination, and the background-offset processing reduces the background level to zero. The background noise is estimated by calculating the standard deviation of the negative pixels after the background-offset processing is completed. When the first macro image <NUM> is pre-processed, a predetermined image of an empty stage with no slide may be used as an illumination profile for macro images captured using the first illumination system <NUM>.

Similarly, the second macro image <NUM> is pre-processed for background offset to generate a pre-processed second macro image <NUM>. The pre-processed second macro image <NUM> is then further processed to identify noise in the image. The noise in the pre-processed second macro image <NUM> corresponds to unwanted image artifacts resulting from debris. In an embodiment, the pre-processed second macro image <NUM> is de-noised to highlight unwanted image artifacts and canny edge detection is used to identify object edges of the unwanted image artifacts, suppressing small peaks usually from noise, and connecting broken edges and/or lines. Accordingly, an artifacts mask <NUM> of unwanted image artifacts is generated from the pre-processed second macro image <NUM>. The artifacts mask <NUM> may be created by assigning artifacts a value of <NUM> and others a value of <NUM>. An image of an example artifacts mask <NUM> is illustrated in image <NUM> in <FIG>. In an embodiment, the image <NUM> is reconstructed by the edge image and the pre-processed macro image <NUM>. In an embodiment, the artifact mask <NUM> is delated, to account for pixel shifts between the first and second macro images.

Once the pre-processed first macro image <NUM> and the artifact mask <NUM> have been generated, these two images are processed to generate a corrected first macro image <NUM> that is free from unwanted image artifacts. In an embodiment, this image processing may be accomplished by multiplying the pre-processed first macro image <NUM> by (<NUM> - the artifact mask <NUM>), to remove the unwanted image artifacts from the pre-processed first macro image <NUM> and thereby generate the corrected first macro image <NUM> (corresponding to example image <NUM> in <FIG>).

In an embodiment, the process may also separate the pre-processed first macro image <NUM> into pieces depending on the amount of unwanted image artifacts present. Thus, a tissue reconstruction procedure may also be performed by evaluating the intensity level surrounding the pixels that are identified with unwanted image artifacts. Those pixels are then added back with the surrounding intensity levels to generate a reconstructed corrected first macro image <NUM> (corresponding to example image <NUM> in <FIG>).

Next, in an embodiment, a line detector is used for the coverslip detection, and a morphological operation is used for small object detection. The identified coverslip and small objects are then removed to generate a final first macro image mask <NUM>, where tissue is identified by a value of <NUM> and non-tissue is identified by a value of <NUM>. Advantageously, the final first macro image mask <NUM> can be used to identify the area of scanning <NUM>.

In an example embodiment, the intermediate image processing can result in faint macro slide images with unwanted image artifacts. In <FIG>, image <NUM> shows the first macro image <NUM>, and image <NUM> shows the binary image mask after pre-processing the first macro image <NUM> for background offset and background noise removal. Similarly, in <FIG>, image <NUM> shows an example of a processed unwanted-image-artifacts mask <NUM>, after the second macro image <NUM> has been pre-processed for background offset and to denoise the image data. Advantageously, artifact mask image <NUM> highlights the unwanted image artifacts and the unwanted slide label. The two masks <NUM> and <NUM> are then used for unwanted image artifact detection and removal from the first macro image <NUM> (also shown as image <NUM> in <FIG>). The two masks <NUM> and <NUM> can also be used for tissue reconstruction as shown in image <NUM>, as well as coverslip and small object detection and removal to generate image <NUM>, which is the final tissue mask. In the illustrated example, the identified scanning area <NUM> is shown as the area within the rectangle in image <NUM>.

<FIG> is a block diagram illustrating an example processor-enabled device <NUM> that may be used in connection with various embodiments described herein. Alternative forms of the device <NUM> may also be used as will be understood by the skilled artisan. In the illustrated embodiment, the device <NUM> is presented as a digital imaging device (also referred to herein as a scanner system, scanning system, digital scanning apparatus, digital slide scanning apparatus, etc.) 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 the sample, 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 scanner system), one or more focusing optics <NUM>, one or more line scan cameras <NUM> (e.g., comprising line scan camera <NUM>) and/or one or more area scan cameras <NUM>, each of which define a separate field of view <NUM> on the sample <NUM> and/or glass slide <NUM>. The various elements of the scanner system <NUM> are communicatively coupled via one or more communication busses <NUM>. Although there may be one or more of each of the various elements of the scanner 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.

The one or more processors <NUM> may include, for example, a central processing unit (CPU) and a separate graphics processing unit (GPU) capable of processing instructions in parallel, or the one or more processors <NUM> may include 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 the line scan camera <NUM>, the stage <NUM>, the objective lens <NUM>, and/or a display (not shown). Such additional processors may be separate discrete processors or may be integrated with the processor <NUM>.

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

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

The motion control system <NUM> is configured to precisely control and coordinate X, Y, and/or Z movement of the stage <NUM> (e.g., within an X-Y plane) and/or the objective lens <NUM> (e.g., along a Z axis orthogonal to the X-Y plane, via the objective lens positioner <NUM>). The motion control system <NUM> is also configured to control movement of any other moving part in the scanner system <NUM>. For example, in a fluorescence scanner embodiment, the motion control system <NUM> is configured to coordinate movement of optical filters and the like in the epi-illumination system <NUM>.

The interface system <NUM> allows the scanner system <NUM> to interface with other systems and human operators. For example, the interface system <NUM> may include a user interface to provide information directly to an operator and/or to allow direct input from an operator. The interface system <NUM> is also configured to facilitate communication and data transfer between the scanning system <NUM> and one or more external devices that are directly connected (e.g., a printer, removable storage medium) or external devices such as an image server system, an operator station, a user station, and an administrative server system that are connected to the scanner system <NUM> via a network (not shown).

The illumination system <NUM> is configured to illuminate a portion of the sample <NUM>. The illumination system <NUM> may include, for example, one or more light sources, including the first illumination system <NUM> and the second illumination system <NUM>, 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, the illumination system <NUM> illuminates the sample <NUM> in transmission mode such that the line scan camera <NUM> and/or area scan camera <NUM> sense optical energy that is transmitted through the sample <NUM>. Alternatively, or in combination, the illumination system <NUM> may also be configured to illuminate the sample <NUM> in reflection mode such that the line scan camera <NUM> and/or area scan camera <NUM> sense optical energy that is reflected from the sample <NUM>. The illumination system <NUM> may be configured to be suitable for interrogation of the microscopic sample <NUM> in any known mode of optical microscopy.

In an embodiment, the scanner system <NUM> optionally includes an epi-illumination system <NUM> to optimize the scanner system <NUM> for fluorescence scanning. 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 (excitation). These photon-sensitive molecules also emit light at a higher wavelength (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 the sample <NUM> (e.g., transmission-mode microscopy). Advantageously, in an optional fluorescence scanner system embodiment of the scanner system <NUM>, use of a line scan camera <NUM> that includes multiple linear sensor arrays (e.g., a TDI line scan camera) increases the sensitivity to light of the line scan camera by exposing the same area of the sample <NUM> to each of the multiple linear sensor arrays of the line scan camera <NUM>. This is particularly useful when scanning faint fluorescence samples with low emitted light.

Accordingly, in a fluorescence scanner system embodiment, the line scan camera <NUM> is preferably a monochrome TDI line scan camera. Advantageously, monochrome images are ideal in fluorescence microscopy because they provide a more accurate representation of the actual signals from the various channels present on the sample. As will be understood by those skilled in the art, a fluorescence sample <NUM> can be labeled with multiple florescence dyes that emit light at different wavelengths, which are also referred to as "channels.

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

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

The sample <NUM> (e.g., corresponding to sample <NUM>) can be anything that may be interrogated by optical microscopy. For example, a glass microscope slide <NUM> (e.g., corresponding to slide <NUM>) is frequently used as a viewing substrate for specimens that include tissues and cells, chromosomes, 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. The sample <NUM> may also be an array of any type of DNA or DNA-related material such as cDNA or RNA or protein that is deposited on any type of slide or other substrate, including any and all samples commonly known as a microarrays. The sample <NUM> may be a microtiter plate (e.g., a <NUM>-well plate). Other examples of the sample <NUM> include integrated circuit boards, electrophoresis records, petri dishes, film, semiconductor materials, forensic materials, or machined parts.

Objective lens <NUM> is mounted on the objective positioner <NUM>, which, in an embodiment, employs a very precise linear motor to move the objective lens <NUM> along the optical axis defined by the objective lens <NUM>. For example, the linear motor of the objective lens positioner <NUM> may include a <NUM> nanometer encoder. The relative positions of the stage <NUM> and the 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 the processor <NUM> that employs memory <NUM> for storing information and instructions, including the computer-executable programmed steps for overall scanning system <NUM> operation.

In an embodiment, the objective lens <NUM> is a plan apochromatic ("APO") infinity corrected objective with a numerical aperture corresponding to the highest spatial resolution desirable, where the objective lens <NUM> 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 the optical path <NUM> above the objective lens <NUM> where the light beam passing through the objective lens <NUM> becomes a collimated light beam. The focusing optics <NUM> focus the optical signal captured by the objective lens <NUM> onto the light-responsive elements of the line scan camera <NUM> and/or the area scan camera <NUM> and may include optical components such as filters, magnification changer lenses, and/or the like. The objective lens <NUM>, combined with the focusing optics <NUM>, provides the total magnification for the scanning system <NUM>. In an embodiment, the 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 the sample <NUM> at 40X magnification.

The line scan camera <NUM> comprises at least one linear array of picture elements ("pixels"). The line scan camera 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, <NUM> linear array ("red-green-blue" or "RGB") color line scan camera or a <NUM> 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. The scanner system <NUM> also supports linear arrays that are manufactured in a variety of formats including some with <NUM> pixels, some with <NUM> pixels, and others having as many as <NUM> pixels. Similarly, linear arrays with a variety of pixel sizes can also be used in the scanner system <NUM>. The salient requirement for the selection of any type of line scan camera <NUM> is that the motion of the stage <NUM> can be synchronized with the line rate of the line scan camera <NUM>, so that the stage <NUM> can be in motion with respect to the line scan camera <NUM> during the digital image capture of the sample <NUM>.

The image data generated by the line scan camera <NUM> is stored in a portion of the memory <NUM> and processed by the processor <NUM> to generate a contiguous digital image of at least a portion of the sample <NUM>. The contiguous digital image can be further processed by the processor <NUM> and the revised contiguous digital image can also be stored in the 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 the scanner 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 is stored in a portion of the memory <NUM> and processed by the one or more processors <NUM> to generate focus information, to allow the scanner system <NUM> to adjust the relative distance between the sample <NUM> and the objective lens <NUM> to maintain focus on the sample 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 of the focusing sensor is positioned at a different logical height along the optical path <NUM>.

In operation, the various components of the scanner system <NUM> and the programmed modules stored in memory <NUM> enable automatic scanning and digitizing of the sample <NUM>, which is disposed on a glass slide <NUM>. The glass slide <NUM> is securely placed on the movable stage <NUM> of the scanner system <NUM> for scanning the sample <NUM>. Under control of the processor <NUM>, the movable stage <NUM> accelerates the sample <NUM> to a substantially constant velocity for sensing by the line scan camera <NUM>, where the speed of the stage is synchronized with the line rate of the line scan camera <NUM>. After scanning a stripe of image data, the movable stage <NUM> decelerates and brings the sample <NUM> to a substantially complete stop. The movable stage <NUM> then moves orthogonal to the scanning direction to position the 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 the sample <NUM> or the entire sample <NUM> is scanned.

For example, during digital scanning of the sample <NUM>, a contiguous digital image of the 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 the sample <NUM> may include acquiring vertical image stripes or horizontal image stripes. The scanning of the sample <NUM> may be either top-to-bottom, bottom-to-top, or both (bi-directional), and may start at any point on the sample. Alternatively, the scanning of the sample <NUM> may be either left-to-right, right-to-left, or both (bi-directional), and may start at any point on the sample. Additionally, it is not necessary that image stripes be acquired in an adjacent or contiguous manner. Furthermore, the resulting image of the 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 the memory <NUM> and, when executed, enable the scanning system <NUM> to perform the various functions 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 the scanning system <NUM> for execution by the processor <NUM>. Examples of these media include memory <NUM> and any removable or external storage medium (not shown) communicatively coupled with the scanning system <NUM> either directly or indirectly, for example via a network (not shown).

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

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

Claim 1:
A digital slide scanning apparatus (<NUM>) comprising:
a stage (<NUM>) configured to support a glass slide (<NUM>) comprising a sample (<NUM>);
a high-resolution camera (<NUM>);
a first lens (<NUM>) defining a macro image optical path (<NUM>) to the high-resolution camera, wherein the first lens is configured to provide a field of view (<NUM>) to the high-resolution camera, and wherein the field of view (<NUM>) of the high-resolution camera is wide enough to capture substantially the entire width of the glass slide (<NUM>);
a first illumination system (<NUM>) optically coupled with the first lens and configured to provide transmission-mode illumination of the field of view from below the glass slide on the stage;
a second illumination system (<NUM>) optically coupled with the first lens and configured to provide reflection-mode illumination of the field of view at an angle from above the glass slide on the stage, wherein the first illumination system, the second illumination system and the high-resolution camera are aligned such that substantially all of the illumination light from the second illumination system that is reflected from the slide, the sample, and elements of the first illumination system is directed away from the macro image optical path (<NUM>) to the high-resolution camera; and
at least one processor (<NUM>) configured to, while the stage is supporting the glass slide:
capture a first macro image (<NUM>) of the sample on the glass slide using the high-resolution camera while the glass slide is under the first lens while the field of view is illuminated by the first illumination system,
capture a second macro image of the sample on the glass slide using the high-resolution camera while the glass slide is under the first lens while the field of view is illuminated by the second illumination system,
identify image artifacts in the second macro image, and
based on the identified image artifacts in the second macro image, correct the first macro image to generate a modified first macro image.