Patent Description:
<CIT> discloses systems and methods for capturing a digital image of a slide using an imaging line sensor and a focusing line sensor. Most auto-focus methods in microscope imaging systems can be divided into two categories: laser-based interferometer for sensing slide position, and image content analysis. With laser-based interferometer methods, measuring the reflection of the laser beam off a slide surface can provide only global focus information of the slide position or the cover slip position. It lacks focusing accuracy for tissue samples with large height variations. In addition, the image content analysis methods require multiple image acquisitions at different focus depths, and use algorithms to compare the images to determine the best focus. However, acquiring multiple images at different focus depths may create time delays between focusing and imaging. Therefore, what is needed is a system and method that overcomes these significant problems found in the conventional methods described above.

The present invention discloses a system for scanning a sample to acquire a digital image of the sample according to claim <NUM>. Further advantageous embodiments of the present invention are disclosed in the dependent claims.

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

The structure and operation of embodiments will be understood from a review of the following detailed description and the accompanying drawings in which like reference numerals refer to like parts, wherein not all of the embodiments comprise each and every feature of claim <NUM>, however, still help to understand some aspects of the invention, and in which:.

Certain embodiments are based on image content analysis (e.g., tissue finding and macro focus), and take advantage of line imaging and line focusing for accurate real-time auto-focusing. In one embodiment, full stripe focusing is performed during a retrace process of line scanning. In an alternative embodiment, focusing is performed during image scanning. Both embodiments eliminate time delays in image scanning, thereby speeding up the entire digital image scanning process. In addition, certain embodiments provide for real-time (i.e., instantaneous or near-instantaneous) focusing in line scan imaging using multiple linear detectors or other components. After reading this description it will become apparent to one skilled in the art how to implement various alternative embodiments and use those embodiments in alternative applications. However, although various embodiments 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 of the present application as set forth in the appended claims.

In an embodiment, one or more focus points are determined for a sample (e.g., a tissue sample prepared on a glass microscope slide). For example, a macro focus point or a plurality of focus points may be determined for the sample. Specifically, one or more positions on the sample may be determined. For each of these positions, the sample may be moved along X and Y axes (e.g., by a motorized stage), such that, that position on the sample is located under an objective lens. In an alternative embodiment, the objective lens may be moved along X and Y axes, or both the objective lens and the sample may be moved along X and Y axes, such that the objective lens is located above each position on the sample. In any case, for each position, an image of the region of the sample at that position may be acquired at a plurality of focus heights, while the sample is stationary in the X and Y axes, via a focusing sensor optically coupled with the objective lens as the objective lens is moved along a Z axis (i.e., orthogonal to both the X and Y axes) through the plurality of focus heights. Software may be used to compute the best focus height for each position, based on the images acquired at the plurality of focus heights for the position. A real-time focus mechanism may then constrain the objective lens at the computed best focus heights at the corresponding positions, via a feedback loop, while scanning the entire sample. It should be understood that while the term "focus height" or "Z height" may be used throughout to describe a distance of the objective lens with respect to the sample, this term does not limit the disclosed embodiments to an objective lens positioned above the sample, but should instead be understood to encompass any distance that represents a distance between the objective lens and a plane of the sample, regardless of their orientations to each other.

<FIG> is a block diagram illustrating an example side view configuration of a scanning system <NUM>, according to an embodiment. In the illustrated embodiment, scanning system <NUM> comprises a sample <NUM> (e.g., a tissue sample prepared on a glass microscope slide) that is placed on a motorized stage (not shown), illuminated by an illumination system (not shown), and moved in a scanning direction <NUM>. An objective lens <NUM> has an optical field of view (FOV) <NUM> that is trained on sample <NUM> and provides an optical path for light from the illumination system that passes through the specimen on the slide, reflects off of the specimen on the slide, fluoresces from the specimen on the slide, or otherwise passes through objective lens <NUM>.

<FIG> illustrates the relative positions between an imaging sensor <NUM> and a focusing sensor <NUM> in space. The light travels on the optical path through objective lens <NUM> to a beam splitter <NUM> that allows some of the light to pass through lens <NUM> to imaging sensor <NUM>. As illustrated in <FIG>, the light may be bent by a mirror <NUM> (e.g., at <NUM>°) between lens <NUM> and imaging sensor <NUM>. Imaging sensor <NUM> may be, for example, a line charge-coupled device (CCD) or a line complementary metal-oxide semiconductor (CMOS) device.

In addition, some of the light travels from beam splitter <NUM> through lens <NUM> to focusing sensor <NUM>. As illustrated in <FIG>, this light may be bent by beam splitter <NUM> (e.g., at <NUM>°) between objective lens <NUM> and lens <NUM>. Focusing sensor <NUM> may also be, for example, a line charge-coupled device (CCD) or line CMOS device.

In an embodiment, the light that travels to imaging sensor <NUM> and the light that travels to focusing sensor <NUM> each represents the complete optical field of view <NUM> from object lens <NUM>. Based on the configuration of the system, the scanning direction <NUM> of sample <NUM> is logically oriented with respect to imaging sensor <NUM> and focusing sensor <NUM> such that the logical scanning direction <NUM> causes the optical field of view <NUM> of objective lens <NUM> to pass over the respective focusing sensor <NUM> and imaging sensor <NUM>.

<FIG> is a block diagram illustrating an example configuration of focusing sensor <NUM> and imaging sensor <NUM> with respect to an optical field of view <NUM> having a circular illumination radius <NUM>, according to an embodiment. In the illustrated embodiment, the positioning of focusing sensor <NUM> is shown with respect to imaging sensor <NUM> and the logical scan direction <NUM>. In this case, the scan direction <NUM> refers to the direction in which the stage or specimen (e.g., a tissue sample) is moving with respect to sensors <NUM> and <NUM> in space. As illustrated, imaging sensor <NUM> is centered within optical field of view <NUM> of objective lens <NUM>, while focusing sensor <NUM> is offset from the center of optical field of view <NUM> of objective lens <NUM>. The direction in which focusing sensor <NUM> is offset from the center of optical field of view <NUM> of the objective lens <NUM> is the opposite of the logical scanning direction <NUM>. This placement logically orients focusing sensor <NUM> in front of imaging sensor <NUM>, such that, as a specimen on a slide is scanned, focusing sensor <NUM> senses the image data before, at the same time that, or after the imaging sensor <NUM> senses that same image data. Thus, a given portion (e.g., line) of sample <NUM> will reach focusing sensor <NUM> first, and subsequently reach imaging sensor <NUM> second.

When imaging sensor <NUM> and focusing sensor <NUM> are projected onto a same plane using, for example a beam-splitter, focusing sensor <NUM> is within the illumination circle, which has a radius R, of the optical field of view <NUM> at a location ahead of the primary imaging sensor <NUM> in terms of the logical scanning direction <NUM>. Thus, when a view of a section of a tissue sample passes focusing sensor <NUM>, focus data can be captured and the focus height for objective lens <NUM> can be calculated based on one or more predetermined algorithms, prior to, at the same time as, or after the time of the view of the same section of tissue sample passing imaging sensor <NUM>. The focus data and the calculated focus height for objective lens <NUM> can be used to control (e.g., by a controller) the height of objective lens <NUM> from sample <NUM> before the view of the same section of the tissue sample is sensed by imaging sensor <NUM> via objective lens <NUM>. In this manner, imaging sensor <NUM> senses the view of the section of the tissue sample while objective lens <NUM> is at the calculated focus height.

Circular illumination radius <NUM> preferably illuminates an optical field of view <NUM> covering both focusing sensor <NUM> and imaging sensor <NUM>. Radius <NUM> is a function of the field of view on sample <NUM> and the optical magnification of the focusing optical path Mfocusing. The function can be expressed as:
<MAT>.

For example, for Mfocusing = <NUM> and FOV = <NUM> (e.g., Leica PlanApo 20x objective), R = <NUM>. Imaging sensor <NUM> is projected in the middle of the optical field of view <NUM> for best image quality, while focusing sensor <NUM> is offset with respect to the center of optical field of view <NUM> by a distance h from imaging sensor <NUM>. There is a relationship among the distance h, the radius R, and the length L of focusing sensor <NUM>, such that:
<MAT>.

For example, for a sensor length = <NUM> and R = <NUM>, h ≤ <NUM>. It should be understood that, when h > <NUM>, any given region of sample <NUM> is sensed by focusing sensor <NUM> first and imaging sensor <NUM> second, whereas, when h < <NUM>, the given region of sample <NUM> is sensed by imaging sensor <NUM> first and focusing sensor <NUM> second. If h = <NUM>, the given region of sample <NUM> is sensed by imaging sensor <NUM> and focusing sensor <NUM> simultaneously as the stage moves along slide scan direction <NUM>. In an embodiment, the average of multiple line images of the same region of sample <NUM> may be used as the line image for that region.

The available time t for focusing sensor <NUM> to capture multiple camera lines, for focus height calculation and for moving objective lens <NUM> to the right focus height, is a function of the distance h between focusing sensor <NUM> and imaging sensor <NUM>, magnification Mfocusing, and scan speed v:
<MAT>.

For example, for a scan speed of <NUM>/s, the maximum time available is about <NUM> for Mfocusing = <NUM> and h = <NUM>. The maximum number of camera lines captured by focusing sensor <NUM>, available for the focus calculation is:
N = t * κ, where κ is the line rate of the focusing sensor <NUM>.

For example, for a camera line rate of <NUM>, Nmax = <NUM>,<NUM> lines, where objective lens <NUM> stays at the same height. Otherwise, N < Nmax to allow objective lens <NUM> to move to the next focus height.

At a high level, a sample <NUM> (e.g., tissue sample) is passed under objective lens <NUM> in an X direction. A portion of sample <NUM> is illuminated to create an illuminated optical field of view <NUM> in the Z direction of a portion of sample <NUM> (i.e., perpendicular to an X-Y plane of sample <NUM>). The illuminated optical field of view <NUM> passes through objective lens <NUM> which is optically coupled to both focusing sensor <NUM> and imaging sensor <NUM>, for example, using a beam splitter <NUM>. Focusing sensor <NUM> and imaging sensor <NUM> are positioned, such that focusing sensor <NUM> receives a region or line of the optical field of view <NUM> before, at the same time as, or after imaging sensor <NUM> receives the same region or line. In other words, as focusing sensor <NUM> is receiving a first line of image data, imaging sensor <NUM> is simultaneously receiving a second line of image data which was previously received by focusing sensor <NUM> and which is a distance h/Mfocusing on sample <NUM> from the first line of image data. It will take a time period Δt for imaging sensor <NUM> to receive the first line of image data after focusing sensor <NUM> has received the first line of image data, where Δt represents the time that it takes sample <NUM> to move a distance h/Mfocusing in the logical scan direction <NUM>.

During the period Δt, a processor of scanning system <NUM> calculates an optimal focus height in the Z direction for the first line of image data, and adjusts objective lens <NUM> to the calculated optimal focus distance before, at the time that, or after imaging sensor <NUM> receives the first line of image data.

In an embodiment, focusing sensor <NUM> is separate from imaging sensor <NUM> and is tilted at an angle θ with respect to a direction that is perpendicular to the optical imaging path. Thus, for each line of image data, focusing sensor <NUM> simultaneously receives pixels of image data at a plurality of Z height values. The processor may then determine the pixel(s) having the best focus within the line of image data (e.g., having the highest contrast with respect to the other pixels within the line of image data). After the optimal Z height value is determined, the processor or other controller may move objective lens <NUM> in the Z direction to the determined optimal Z height value before, simultaneously as, or after imaging sensor <NUM> receives the same line of image data.

As discussed above, focusing sensor <NUM> may be tilted within the optical field of view such that light from objective lens <NUM> is sensed by focusing sensor <NUM> at a plurality of Z height values. <FIG> is a block diagram illustrating an example top view configuration of imaging sensor <NUM> with respect to an imaging optical path <NUM>, according to an embodiment. <FIG> is a block diagram illustrating an example top view configuration of a tilted focusing sensor <NUM>, with respect to a focusing optical path <NUM>, according to an embodiment. As can be seen in <FIG>, focusing sensor <NUM> is tilted at an angle θ with respect to a direction that is perpendicular to focusing optical path <NUM>. <FIG> is a block diagram illustrating an example of a sensor, in which half of the sensor is used to acquire a main image, and the other half of the sensor is used to acquire a focusing image.

Thus, an image projected on tilted focusing sensor <NUM> and acquired as a line of image data by tilted focusing sensor <NUM> will have variable sharpness or contrast. This line of image data will have its highest focus (e.g., greatest sharpness or contrast) in a particular region or pixel location of tilted focusing sensor <NUM>. Each region or pixel location of tilted focusing sensor <NUM> may be directly mapped or otherwise correlated to a Z height of objective lens <NUM>, such that the Z height of objective lens <NUM> may be determined from a particular pixel location of tilted focusing sensor <NUM>. Thus, once the pixel location of highest focus (e.g., highest contrast) is determined, the Z height of objective lens <NUM> providing the highest focus may be determined by identifying the Z height of objective lens <NUM> that is mapped to that pixel location of highest focus. Accordingly, a feedback loop may be constructed. By this feedback loop, for a given region on sample <NUM>, the position of objective lens <NUM> may be automatically controlled (e.g., by increasing or decreasing the height of objective lens <NUM>) to always correspond to the position on tilted focusing sensor <NUM> having the highest focus for that region, before, at the time that, or after imaging sensor <NUM> senses the same region of sample <NUM>, such that the region of sample <NUM> being imaged by imaging sensor <NUM> is always at the best available focus.

<FIG> is a block diagram illustrating an example tilted focusing sensor <NUM>, according to an embodiment. In the illustrated embodiment, tilted focusing sensor <NUM> comprises a plurality of sensor pixels <NUM> within a range of focusing (z) on a tissue sample (e.g., <NUM>). As illustrated in <FIG>, tilted focusing sensor <NUM> may be positioned at a location where the entire focusing range (z) in the Z direction is transferred by optics to the entire array of sensor pixels <NUM> in tilted focusing sensor <NUM> in the Y direction. The location of each sensor pixel <NUM> is directly correlated or mapped to a Z height of objective lens <NUM>. As illustrated in <FIG>, each dashed line, p<NUM>, p<NUM>,. pn, across projected focusing range (d) represents a different focus value and corresponds to a different focus height of objective lens <NUM>. The pi having the highest focus for a given region of a sample can be used by scanning system <NUM> to determine the optimal focus height of objective lens <NUM> for that region of sample <NUM>.

The relationship between the projected focusing range (d) on tilted focusing sensor <NUM> and the focusing range (z) on sample <NUM> may be expressed as: d = z * Mfocusing<NUM>, where Mfocusing is the optical magnification of the focusing path. For instance, if z = <NUM> and Mfocusing= <NUM>, then d = <NUM>.

In order to cover the entire projected focusing range (d) by a tilted focusing sensor <NUM> that is a linear array sensor, the tilting angle θ should follow the relationship: sinθ =d/L, where L is the length of focusing sensor <NUM>. Using d = <NUM> and L = <NUM>, θ = <NUM>°. θ and L can vary as long as tilted focusing sensor <NUM> covers the entire focusing range (z).

The focusing resolution, or the minimum step of focus height motion Δz along the Z axis is a function of the sensor pixel size, e=minimum(ΔL). Derived from the above formulas: Δz=e*z/L. For instance, if e=<NUM>, L=<NUM>, and z=<NUM>, then Δz=<NUM><<NUM>.

In an embodiment, a scan line (e.g., one-dimensional image data), acquired by tilted focusing sensor <NUM> from sample <NUM>, is analyzed. A figure of merit (FOM) (e.g., contrast of the data) may be defined. The location (corresponding to a focus height value of objective lens <NUM>) of a pixel <NUM> of the maximum FOM on the sensor array can be found. In this manner, the focus height of objective lens <NUM>, corresponding to the location of the pixel <NUM> of the maximum FOM, can be determined for that scan line.

The relationship between the location Li on tilted focusing sensor <NUM> of a pixel i and the focus height Zi of objective lens <NUM> may be represented as follows: Li = Zi * Mfocusing<NUM> / sinθ.

If the focus distance is determined by a mean from L<NUM> to L<NUM>, according to the analysis of the data from tilted focusing sensor <NUM> discussed above, the focus height of objective lens <NUM> needs to be moved from Z<NUM> to Z<NUM> based on: Z<NUM> = Z<NUM> + (L<NUM>-L<NUM>)* sinθ / Mfocusing<NUM>.

Although the field of view (FOV) in the Y axis of focusing sensor <NUM> and imaging sensor <NUM> can be different, the centers of both sensors are preferably aligned to each other along the Y axis.

<FIG> is a time chart diagram illustrating an example interplay between a focusing sensor <NUM> and an imaging sensor <NUM> during scanning, according to an embodiment. Specifically, the timing of a scan using an imaging sensor <NUM> and focusing sensor <NUM> is illustrated. At time t<NUM>, the focus height of objective lens <NUM> is at Z<NUM> on tissue section X<NUM>, which is in the field of view of focusing sensor <NUM>. Focusing sensor <NUM> receives focusing data corresponding to the tissue section X<NUM>. The focus height Z<NUM> is determined to be the optimal focus height for tissue section X<NUM> using the focusing data and, in some embodiments, associated focusing algorithms. The optimal focus height is then fed to the Z positioner to move objective lens <NUM> to the height Z<NUM>, for example, using a control loop. At t<NUM>, tissue section X<NUM> is moved into the field of view of imaging sensor <NUM>. With the correct focus height, imaging sensor <NUM> will sense an optimally-focused image of tissue section X<NUM>. At the same time t<NUM>, focusing sensor <NUM> captures focusing data from tissue section X<NUM>, and the focusing data will be used to determine the optimal focus height Z<NUM> which in turn will be fed into the Z positioner prior to, at the time that, or after tissue section X<NUM> passes into the field of view of imaging sensor <NUM> at time t<NUM>. Such a process can continue until the entire tissue sample is scanned.

In general at time tn, tissue section Xn+<NUM> is in the field of view of focusing sensor <NUM>, tissue section Xn is in the field of view of imaging sensor <NUM>, and objective lens <NUM> is at a focus height of Zn. Furthermore, prior to, at the time, or after tn+<NUM>, the optimal focus height for tissue section Xn+<NUM> is determined and the focus height of objective lens <NUM> is adjusted to Zn+<NUM>. At time t<NUM>, focusing sensor <NUM> senses tissue section X<NUM> and determines the focus height as Z<NUM> for tissue section X<NUM>; at time t<NUM>, tissue section X<NUM> moves under imaging sensor <NUM> and objective lens <NUM> moves to focus height Z<NUM> while focusing sensor <NUM> senses tissue section X<NUM> and determines the focus height as Z<NUM> for tissue section X<NUM>; at time tn, tissue section Xn moves under imaging sensor <NUM> and objective lens <NUM> moves to focus height Zn while focusing sensor <NUM> senses tissue section Xn+<NUM> and determines the focus height as Zn+<NUM> for tissue section Xn+<NUM>. Xn-<NUM> and Xn do not necessarily represent consecutive or adjacent lines of image data, as long as a scan line is acquired by focusing sensor <NUM> and an optimal focus height for the scan line is determined and set prior to, at the same time as, or after the same scan line being acquired by imaging sensor <NUM>. In other words, focusing sensor <NUM> and imaging sensor <NUM> may be arranged such that one or more scan lines exist between the field of view of focusing sensor <NUM> and the field of view of imaging sensor <NUM>, i.e., that distance h between focusing sensor <NUM> and imaging sensor <NUM> comprises one or more scan lines of data. For instance, in the case that the distance h comprises five scan lines, tissue section X<NUM> would be in the field of view of focusing sensor <NUM> at the same time that tissue section X<NUM> is in the field of view of imaging sensor <NUM>. In this case, the focus height of objective lens <NUM> would be adjusted to the calculated optimal focus height after the tissue section X<NUM> is sensed by imaging sensor <NUM> but prior to, at the same time as, or after the tissue section X<NUM> being sensed by imaging sensor <NUM>. Advantageously, the focus height of objective lens <NUM> may be smoothly controlled between tissue section X<NUM> and X<NUM> such that there are incremental changes in focus height between X<NUM> and X<NUM> that approximate a gradual slope of the tissue sample.

<FIG> illustrates a tilted focusing sensor <NUM> that utilizes one or more beam splitters and one or more prism mirrors, according to an embodiment. The beam splitter(s) and mirror(s) are used to create a plurality of images of the same field of view on tilted focusing sensor <NUM>, with each of the plurality of images at a different focal distance, thereby enabling focusing sensor <NUM> to simultaneously sense multiples images of the same region of sample <NUM> at different foci (corresponding to different focus heights for objective lens <NUM>). Focusing sensor <NUM> may be a single large line sensor, or may comprise a plurality of line sensors (e.g., positioned in a row along a longitudinal axis).

Specifically, <FIG> illustrates a tilted focusing sensor <NUM> that utilizes beam splitters 620A and 620B and prism mirror <NUM> to guide a light beam <NUM> (illustrated as separate red, blue, and green channels, although they do not need to be separated channels) through a plurality of optical paths 610A-610C, each having different focal distances, onto a single tilted line sensor <NUM>. It should be understood that light beam <NUM> conveys a field of view from objective lens <NUM>. As illustrated, the optical paths, in order from highest focal distance to lowest focal distance, are 610A, 610B, and 610C. However, it should be understood that each optical path will reach tilted line sensor <NUM> at a range of focal distances, rather than at a single focal distance, due to the tilt of tilted line sensor <NUM>. In other words, the image acquired by tilted line sensor <NUM> on each optical path will comprise pixels acquired at a focal distance that increases from a first side of the image to a second, opposite side of the image. In the illustrated example, light beam <NUM> enters beam splitter 620A and is split into an optical path 610A, which proceeds to a first region of tilted focusing sensor <NUM> at a first focal distance, and an optical path which proceeds to beam splitter 620B. The optical path that proceeds to beam splitter 620B is split into an optical path 610B, which proceeds to a second region of tilted focusing sensor <NUM> at a second focal distance, and an optical path 610C, which is reflected off of mirror <NUM> onto a third region of tilted focusing sensor <NUM> at a third focal distance. Each of the first, second, and third focal distances and first, second, and third regions are different from each other. In this manner, a single tilted focusing sensor <NUM> simultaneously senses light beam <NUM> at a plurality of different focal distances (e.g., three in the illustrated example). It should be understood that fewer or more beam splitters <NUM> and/or mirrors <NUM> may be used to create fewer or more optical paths <NUM> with different focal distances (e.g., two optical paths or four or more optical paths, each with a different focal distance with respect to tilted focusing sensor <NUM>).

With respect to the embodiment illustrated in <FIG>, the best focus may be determined and correlated to a height of objective lens <NUM>, in the same manner as described above. Redundant information from a plurality of images at different focal distances may provide higher confidence to a focus result.

<FIG> and <FIG> illustrate alternatives to a tilted focusing sensor. Specifically, <FIG> and <FIG> illustrate a non-tilted focusing sensor <NUM> that utilizes one or more beam splitters and one or more prism mirrors to achieve the same results as a tilted focusing sensor, according to a couple of embodiments. The beam splitter(s) and mirror(s) are used to create a plurality of images of the same field of view on focusing sensor <NUM>, with each of the plurality of images at a different focal distance, thereby enabling focusing sensor <NUM> to simultaneously sense multiple images of the same region of sample <NUM> at different foci (corresponding to different focus heights for objective lens <NUM>). Focusing sensor <NUM> may be a single large line sensor, or may comprise a plurality of line sensors positioned in a row along a longitudinal axis.

<FIG> illustrates a non-tilted focusing sensor <NUM> that utilizes beam splitters 620A and 620B and prism mirrors 630A and 630B to guide a light beam <NUM> (illustrated as separate red, blue, and green channels, although they do not need to be separated channels) through a plurality of optical paths 610A-610C, each having different focal distances, onto a single line sensor <NUM>. It should be understood that light beam <NUM> conveys a field of view from objective lens <NUM>. As illustrated, the optical paths, in order from highest focal distance to lowest focal distance, are 610A, 610B, and 610C. In the illustrated example, light beam <NUM> enters beam splitter 620A and is split into an optical path 610B, which is reflected off of mirror 630A and through glass block 640A onto a first region of focusing sensor <NUM> at a first focal distance, and an optical path which proceeds to beam splitter 620B. The optical path that proceeds to beam splitter 620B is split into an optical path 610A which passes onto a second region of focusing sensor <NUM> (e.g., adjacent to the first region of focusing sensor <NUM>) at a second focal distance, and an optical path 610C, which is reflected off of mirror 630B and through glass block 640B onto a third region of focusing sensor <NUM> (e.g., adjacent to the second region of focusing sensor <NUM>) at a third focal distance. Each of the first, second, and third focal distances and first, second, and third regions are different from each other. In this manner, focusing sensor <NUM> simultaneously senses light beam <NUM> at a plurality of different focal distances (e.g., three in the illustrated example). It should be understood that fewer or more beam splitters <NUM>, mirrors <NUM>, glass blocks <NUM>, and/or regions of focusing sensor <NUM> may be used to create fewer or more optical paths <NUM> with different focal distances (e.g., two optical paths or four or more optical paths, each with a different focal distance with respect to focusing sensor <NUM>).

<FIG> illustrates a non-tilted focusing sensor <NUM> that utilizes beam splitters 620A and 620B and prism mirrors 630A and 630B to guide a light beam <NUM> (illustrated as separate red, blue, and green channels, although they do not need to be separated channels) through a plurality of optical paths 610A-610C, each having different focal distances, onto respective ones of a plurality of line sensors 30A-30C. As illustrated, the optical paths, in order from highest focal distance to lowest focal distance, are 610A, 610B, and 610C. In the illustrated example, light beam <NUM> enters beam splitter 620A and is split into an optical path 610B, which is reflected off of mirror 630A and through glass block 640A onto a first region of focusing sensor <NUM> at a first focal distance, and an optical path which proceeds to beam splitter 620B. The optical path that proceeds to beam splitter 620B is split into an optical path 610A which passes onto a second region of focusing sensor <NUM> at a second focal distance, and an optical path 610C, which is reflected off of mirror 630B and through glass block 640B onto a third region of focusing sensor <NUM> at a third focal distance. Each of the first, second, and third focal distances and the first, second, and third regions of focusing sensor <NUM> are different from each other. In this manner, focusing sensor <NUM> simultaneously senses light beam <NUM> at a plurality of different respective focal distances (e.g., three in the illustrated example). It should be understood that fewer or more beam splitters <NUM>, mirrors <NUM>, glass blocks <NUM>, and/or regions of focusing sensor <NUM> may be used to create fewer or more optical paths <NUM> with different focal distances (e.g., two optical paths or four or more optical paths, each with a different focal distance with respect to a different focusing sensor <NUM>).

In the embodiments illustrated in <FIG>, <FIG>, and <FIG>, the beam splitters and mirrors are positioned after the imaging lens in the optical path. Alternatively, tube lenses may be positioned after the beam splitting optics. In this alternative embodiment, the locations of individual images with the same field of view are defined by the focal lengths and positions of the lenses.

<FIG> illustrates an alternative non-tilted focusing sensor <NUM> in which the beam splitting optics are positioned before tube lenses, according to an embodiment. Specifically, non-tilted focusing sensor <NUM> utilizes beam splitters 620A, 620B, and 620C and prism mirror <NUM> to guide a light beam <NUM> through a plurality of optical paths 610A-610D, each having different focal distances, onto a single line sensor <NUM>. As illustrated, the optical paths, in order from highest focal distance to lowest focal distance, are 610A, 610B, 610C, and 610D. In the illustrated example, light beam <NUM> enters beam splitter 620A and is split into an optical path 610A, which is focused by lens 650A onto a first region of focusing sensor <NUM> at a first focal distance, and an optical path which proceeds to beam splitter 620B. The optical path that proceeds to beam splitter 620B is split into an optical path 610B, which is focused by lens 650B onto a second region of focusing sensor <NUM> at a second focal distance, and an optical path which proceeds to beam splitter 620C. The optical path that proceeds to beam splitter 620B is split into an optical path 610C, which is focused by lens 650C onto a third region of focusing sensor <NUM> at a third focal distance, and an optical path 610C, which is reflected off of mirror <NUM> and focused by lens 650D onto a fourth region of tilted focusing sensor <NUM> at a fourth focal distance. Each of the first, second, third, and fourth focal distances and the first, second, third, and fourth regions are different from each other. In this manner, focusing sensor <NUM> (e.g., comprising a single line sensor or a plurality of line sensors) simultaneously senses light beam <NUM> at a plurality of different focal distances (e.g., four in the illustrated example). It should be understood that fewer or more beam splitters <NUM>, mirrors <NUM>, and/or regions of focusing sensor <NUM> may be used to create fewer or more optical paths <NUM> with different focal distances (e.g., two optical paths, three optical paths, or five or more optical paths, each with a different focal distance with respect to focusing sensor <NUM>).

In the embodiments described above, a given region of a sample <NUM> is simultaneously acquired by different regions of a focusing sensor <NUM> at a plurality of different focal distances, producing a plurality of images at different focal distances. An algorithm can then be applied to this plurality of images to determine a best focal distance, which can be correlated to a focus height of objective lens <NUM> along the Z axis.

By aligning the optics (e.g., as discussed above), the plurality of images acquired by different regions of focusing sensor <NUM> can correlate or map to various focus spots from a focus buffer. The focus buffer may contain contrast measures, for the focus points, calculated from image data that has been continuously acquired while objective lens <NUM> moves along the Z axis (i.e., as the focus height of objective lens <NUM> changes). For instance, a measure of contrast (e.g., averaged contrast) for each focus height represented by the plurality of images may be plotted, as illustrated in an example by the points in <FIG>. The best focus (i.e., the peak of contrast measures in the focus buffer) can be determined by using a peak-finding algorithm (e.g., fitting, hill-climbing, etc.) to identify the peak of a curve that best fits the points, as illustrated in an example by the curve in <FIG>. The peak of the curve represents the best contrast measure, and maps to a particular focus height that provides the best focus.

<FIG> illustrates the focal relationship between a tilted focusing sensor <NUM> and imaging sensor <NUM>, according to an embodiment. Specifically, in an embodiment, a point P of tilted focusing sensor <NUM> is parfocal with imaging sensor <NUM>. Thus, when sensing a region of a sample <NUM> using tilted focusing sensor <NUM>, the appropriate focus height of objective lens <NUM> from sample <NUM> may be determined as the focus height of objective lens <NUM> that positions the pixel(s) having the best focus at point P of focusing sensor <NUM>. This determined focus height is what may then be used for objective lens <NUM> when sensing the same region using imaging sensor <NUM>.

<FIG> illustrate the focus functions for tilted focusing sensor <NUM> and imaging sensor <NUM>. The focus function may be a function of contrast within the images sensed by tilted focusing sensor <NUM> and imaging sensor <NUM>. For example, CI represents the contrast function for imaging sensor <NUM>, and CT represents the contrast function for tilted focusing sensor <NUM>. Thus, CI(x) returns a contrast measure for an image pixel at position x along the array of imaging sensor <NUM>, and CT(x) returns a contrast measure for an image pixel at position x along the array of tilted focusing sensor <NUM>. In both instances, the contrast measure may be a root mean square of contrast values at x. CD represents the difference between CT and CI (e.g., CT-CI). Thus, CD(x) represents the difference between CT and CI at position x along the arrays of imaging sensor <NUM> and tilted focusing sensor <NUM> (e.g., CT(x)-CI(x)). CD(x) removes tissue-dependent spatial variations. A ratio between the contrast functions of the two images can be used as well to remove tissue-dependent spatial variations (e.g., CT(x)/CI(x)). In addition, a threshold can be defined to remove influences from background noise.

<FIG> illustrates the contrast function CI<NUM> which represents contrast measures as a function of a position on imaging sensor <NUM>. Similarly, <FIG> illustrates the contrast function CT<NUM> which represents contrast measures as a function of a position on tilted focusing sensor <NUM>. <FIG> illustrates a ratio of the contrast function for tilted focusing sensor <NUM> to the contrast function for imaging sensor <NUM> (i.e., CT<NUM>/CI<NUM>).

When tilted focusing sensor <NUM> and imaging sensor <NUM> both sense the same region of sample <NUM>, the best focus will be at a position x on both sensors <NUM> and <NUM> at which the ratio of CT to CI (e.g., CT/CI) is <NUM>. A predetermined point P on tilted focusing sensor <NUM> is parfocal with imaging sensor <NUM>. This point P may be determined during system calibration.

In an embodiment, a figure-of-merit (FOM) function may be used to determine the best focus for a region of sample <NUM> based on data acquired by tilted focusing sensor <NUM>. Specifically, a peak of the CT function may be determined. This CT peak will correspond to a position x on tilted focusing sensor <NUM> and is correlated to a focus height within the Z range of objective lens <NUM>. Thus, when the CT peak is away from the parfocal point P on tilted focusing sensor <NUM> (i.e., CT(P) does not represent the peak value), a command may be initiated to move objective lens <NUM> in real time along an axis that is orthogonal to sample <NUM> (i.e., Z axis) until the peak of CT is at P (i.e., until CT(P) is the peak value for CT). In other words, de-focus is characterized by the shift of the peak value of CT away from parfocal point P on tilted focusing sensor <NUM>, and auto-focusing can be achieved via a feedback loop that moves objective lens <NUM> along the Z axis until the peak value of CT is at parfocal point P on tilted focusing sensor <NUM>.

In an embodiment, an image of a field of view acquired by imaging sensor <NUM> is compared to an image of the same field of view acquired by focusing sensor <NUM> (e.g., tilted focusing sensor <NUM>) using their ratio, difference, or other calculation.

In an embodiment, focusing sensor <NUM> may comprise a single line sensor or dual line sensors designed to acquire two images of the same field of view that are reversed in terms of the focal distances represented by the pixels of the images. For example, a first one of the two images has pixels representing the lowest focal distance at a first side (e.g., left side) of the captured field of view and pixels representing the highest focal distance at a second side of the captured field of view that is opposite the first side (e.g., right side), whereas the second one of the two images has pixels representing the highest focal distance at the first side (e.g., left side) of the captured field of view and pixels representing the lowest focal distance at the second side (e.g., right side) of the captured field of view. If the highest and lowest focal distances are the same for both images, then a line of pixels in the center of each image will be parfocal between the two images, and the corresponding lines of pixels emanating from the center to the sides edges (e.g., left and right edges) of the captured field of view of each image will also be parfocal between the two images but in opposite directions. For example, using left and right to arbitrarily define the edges of the images, for all distances D, a vertical line of pixels in the first image that is a distance D from the center to the left edge of the field of view represented in the first image will be parfocal with a vertical line of pixels in the second image that is a distance D from the center to the right edge of the field of view in the second image. It should be understood that, if the field of view is inverted or mirrored between the first and second images, then both images will have their highest and lowest focal distances on the same sides of the image, but on opposite sides of the field of view represented by the image.

<FIG> illustrates optical components for forming two images with reversed focal distances using two focusing sensors 30A and 30B, according to an embodiment. The tilt of focusing sensor 30A is reversed with respect to the tilt of focusing sensor 30B around the logical Z axis (i.e., the focus axis). It should be understood that the logical Z axis in <FIG> is not necessarily the same as the physical Z axis of objective lens <NUM>, since the light may be bent (e.g., orthogonally by a beam splitter or prism mirror) after it passes through objective lens <NUM>. In this embodiment, optical paths 610A and 610B provide the same optical field of view to focusing sensors 30A and 30B, but since focusing sensors 30A and 30B are reversed in terms of their tilt, the focal distances of pixels in the two images are reversed. This is illustrated by the sets of three arrows labeled Z<NUM>, Z<NUM>, and Z<NUM>, which each represent a different sample focal height. Thus, Z<NUM>a and Z<NUM>b both represent a first focal height, Z<NUM>a and Z<NUM>b both represent a second focal height, and Z<NUM>a and Z<NUM>b both represent a third focal height, where each of the first, second, and third focal heights are different from each other.

<FIG> illustrate optical components for forming two mirror images on a tilted focusing sensor <NUM>, according to two different embodiments. In the illustrated embodiments, one or more optical components may be used to form the field of view on a first region of tilted focusing sensor <NUM> and a reversed field of view on a second region of tilted focusing sensor <NUM>. It should be understood that tilted focusing sensor <NUM> may be a single line sensor or a plurality of adjacent line sensors.

<FIG> illustrates optical components for forming two mirror images on a tilted focusing sensor <NUM>, according to a first embodiment. As illustrated, a light beam <NUM> enters beam splitter <NUM> and is split into an optical path 610A, which is reflected off of mirror <NUM> onto a first region 30A of focusing sensor <NUM> such that a first image is acquired from first region 30A of focusing sensor <NUM>, and an optical path 610B, which passes through dove prism <NUM>. Dove prism <NUM> reverses light beam <NUM> so that a mirror image is formed on a second region 30B of focusing sensor <NUM> such that a second image is acquired from second region 30B of focusing sensor <NUM>. In other words, optical path 610A provides the field of view to first region 30A of focusing sensor <NUM> and a mirrored field of view to second region 30B of focusing sensor <NUM>. Thus, the second image is a mirror image of the first image around the logical Z axis. Since the angle of tilt (θ) is the same in both first region 30A and second region 30B of tilted focusing sensor <NUM>, the fields of view depicted in the first and second images are reversed in terms of the direction of the focal distances (e.g., from highest to lowest) at which they were acquired.

<FIG> illustrates optical components for forming two mirror images on a tilted focusing sensor <NUM>, according to a second embodiment. As illustrated, a light beam <NUM> enters beam splitter <NUM> and is split into an optical path 610A, which is reflected off of mirror 630A (e.g., a flat plate) to a first region 30A of tilted focusing sensor <NUM>, and an optical path 610B. Optical path 610B reflects off of two surfaces of mirror 630B back into beam splitter <NUM>, where it is reflected onto a second region 30B of tilted focusing sensor <NUM>. Light beam <NUM> traveling on optical path 610B is reversed such that it produces an image on second region 30B of tilted focusing sensor <NUM> that is the mirror image of the image formed in optical path 610A on first region 30A of tilted focusing sensor <NUM>. Since the angle of tilt (θ) is the same in both first region 30A and the second region 30B of tilted focusing sensor <NUM>, the fields of view depicted in the first and second images are reversed in terms of the direction (e.g., from highest to lowest) of the focal distances at which they were acquired.

<FIG> illustrates the directionality of the focal distances for the two images acquired by regions 30A and 30B of focusing sensor <NUM> in the embodiments illustrated in <FIG>, <FIG>, according to an embodiment. A comparison of these two images using a difference, a ratio, or another calculation may provide the amount of movement and direction of movement required to place objective lens <NUM> at a focus height on the Z axis that achieves the best focus for focusing sensor <NUM>. In an embodiment, the center or a parfocal point of each of the region(s) of focusing sensor <NUM> (i.e., each region corresponding to its own separate optical path <NUM>) is parfocal with each other, as well as with imaging sensor <NUM>. Thus, determining a best focus for a given region of sample <NUM> comprises identifying a focus height for objective lens <NUM>, such that the best foci for the two images acquired by focusing sensor <NUM> are at the center or a parfocal point of the respective regions of focusing sensor <NUM> that acquired the images. When the best foci for the regions of focusing sensor <NUM> are centered or parfocal in this manner, the focus height of objective lens <NUM> that corresponds to the centers or parfocal points of both regions is also the focus height at which imaging sensor <NUM> (which is parfocal with the centers of both regions of focusing sensor <NUM> or with a parfocal point, e.g., determined during system calibration) is at the best focus for the given region of sample <NUM>.

<FIG> illustrates the focus functions for two reversed images acquired by regions 30A and 30B of focusing sensor <NUM>. In embodiments in which a mirrored image is acquired (e.g., by region 30B in <FIG>), the mirrored image is inverted by software or other means prior to operations performed with respect to the two reversed images. This inversion of the mirrored image results in the two images no longer being mirror images of each other in terms of content. In other words, the two images represent the same field of view in the same orientation. However, even though the orientation of the field of view represented by the images are the same, the directions of their focal distances are reversed. For example, after this inversion process, the content on one side of a first one of the images will have been acquired at focal distance Z<NUM>, while the same content on the same side of the second one of the images will have been acquired at focal distance Z<NUM>, and the content on the other side of the first image will have been acquired at focal distance Z<NUM>, while the same content on the same side of the second image will have been acquired at Z<NUM>. The centers of the images will both have been acquired at Z<NUM>.

The focus function may be a function of contrast within the reversed images. The functions may return a contrast measure for a given position x along each region of focusing sensor <NUM> (e.g., regions 30A and 30B) that acquires one of the reversed images (e.g., a root mean square of contrast values at a position x). For example, Cb represents the contrast measures for region 30B of focusing sensor <NUM> which acquired the reversed image, and Ca represents the contrast measures for region 30A of focusing sensor <NUM> which acquired the non-reversed image. C<NUM>a and C<NUM>b represent the contrast measures for the middle portions of the reversed images, C<NUM>a and C<NUM>b represent the contrast measures for the corresponding portions of one side of the reversed images, and C<NUM>a and C<NUM>b represent the contrast measures for the corresponding portions of the other side of the reversed images.

If a ratio algorithm is used, C<NUM>a/C<NUM>b will be close to <NUM> across the entire field of view of focusing sensor <NUM> when the best foci for both images are centered in their corresponding regions (e.g., regions 30A and 30B) of focusing sensor <NUM>. When the minimum of C<NUM>a/C<NUM>b (i.e., C<NUM>a/C<NUM>b < <NUM>) is on the left side of parfocal point P, a command may be sent in a feedback loop to move objective lens <NUM> along the Z axis in a direction such that the minimum of C<NUM>a/C<NUM>b moves towards parfocal point P. When the maximum of C<NUM>a/C<NUM>b (i.e., C<NUM>a/C<NUM>b > <NUM>) is on the left side of parfocal point P, a command may be sent in a feedback loop to move objective lens <NUM> along the Z axis in a direction such that the maximum of C<NUM>a/C<NUM>b moves towards parfocal point P. The same algorithm may be applied to the other half of the ratio data centered to parfocal point P (i.e., the right-hand side of the curve). The second set of data can be used in cases where half of the field of view contains no tissue or non-useful data, or simply for redundancy to increase a success rate.

In any of the embodiments described herein as using multiple regions of a focusing sensor <NUM> (e.g., the embodiments illustrated <FIG>, <FIG>), focusing sensor <NUM> may be a single focusing sensor comprising the multiple regions, or a plurality of focusing sensors each consisting of one of the multiple regions. Furthermore, in embodiments in which a plurality of focusing sensors are used as the regions of focusing sensor <NUM>, each of the plurality of focusing sensors may be arranged in the same plane as each other, or in different planes from each other, depending on the particular design.

<FIG> illustrates a method for real-time focusing, according to an embodiment. Initially, a calibration step <NUM> may be performed. Calibration step <NUM> may comprise locating a parfocal point P (e.g., parfocal with imaging sensor <NUM>) on a tilted focusing sensor <NUM> (in embodiments which utilize a tilted focusing sensor), determining an illumination profile for images from imaging sensor <NUM>, and/or determining an illumination profile for images from focusing sensor <NUM>. It should be understood that calibration step <NUM> may be performed only once for a particular system <NUM>, or periodically for the system <NUM> if recalibration is needed or desired.

The real-time focusing process may begin in step <NUM>, in which one or more, and preferably a plurality of three or more, focus points are acquired using a focus-buffer method. Each focus point may comprise an X, Y, and Z position, where the X and Y positions represent a position in a plane of sample <NUM>, and the Z position represents a focus height of objective lens <NUM>. In an embodiment, each focus point is obtained by positioning objective lens <NUM> over an X-Y position on sample <NUM>, sweeping objective lens <NUM> from one end of its height range to the other end of its height range to determine the focus height providing the best focus (e.g., peak of a contrast function) at the X-Y position.

In step <NUM>, a reference plane is created using the focus points obtained in step <NUM>. It should be understood that a reference plane can be created from as few as three focus points. When there are more than three focus points, focus points that are outliers with respect to a flat reference plane may be discarded. Otherwise, all focus points may be used to fit a reference plane. Alternatively, instead of a reference plane, a focal surface may be created from any plurality of focus points. Different embodiments for creating a reference plane or focal surface are described in <CIT> and issued as <CIT>, and <CIT> and issued as <CIT>.

In step <NUM>, objective lens <NUM> is moved to a Z position defined by the reference plane as a function of the X-Y position to be scanned.

In step <NUM>, a focusing image is acquired from focusing sensor <NUM>. Similarly, in step <NUM>, a main image is acquired from imaging sensor <NUM>.

In step <NUM>, the illumination in the focusing image acquired in step <NUM> is corrected using any well-known illumination-correction technique. Similarly, in step <NUM>, the illumination in the main image acquired in step <NUM> is corrected using any well-known illumination-correction techniques. The illumination correction for the focusing image may be based on the illumination profile for focusing sensor <NUM> that was determined in calibration step <NUM>, and the illumination correction for the main image may be based on the illumination profile for imaging sensor <NUM> that was determined in calibration step <NUM>.

In step <NUM>, an absolute gradient of the illumination-corrected focusing image is calculated. Similarly, in step <NUM>, an absolute gradient of the illumination-corrected main image is calculated.

In step <NUM>, the rows in the focusing image gradient calculated in step <NUM> are averaged. Similarly, in step <NUM>, the rows in the main image gradient calculated in step <NUM> are averaged.

In step <NUM>, a low-pass filter is applied to the focusing image gradient. Similarly, in step <NUM>, a low-pass filter is applied to the main image gradient.

In step <NUM>, it is determined whether or not the background area (i.e., the area of the image without tissue) in the main image is less than the tissue area (i.e., the area of the image with tissue) in the main image. If the background area is greater than the tissue area in the main image (i.e., "No" in step <NUM>), the process may return to step <NUM>. Otherwise, if the background area is less than the tissue area in the main image (i.e., "Yes" in step <NUM>), the process may proceed to step <NUM>.

In step <NUM>, ratio(s) are calculated between the focusing image gradient and the main image gradient. For example, the focusing image gradient may be divided by the main image gradient.

In step <NUM>, a peak is fit to the ratio(s) calculated in step <NUM> with minimal error. For example, a best-fit curve may be found for the ratio(s).

In step <NUM>, the peak of the fitting in step <NUM> is determined. For example, in an embodiment in which a best-fit curve is found for the ratio(s) in step <NUM>, the peak of the best-fit curve may be identified in step <NUM>.

In step <NUM>, if the peak identified in step <NUM> is not at the parfocal point P, objective lens <NUM> is moved until the peak is at the parfocal point P, for example, using a feedback loop as described elsewhere herein.

In step <NUM>, it is determined whether or not the scan is complete. If the scan is not complete (i.e., "No" in step <NUM>), the process returns to steps <NUM> and <NUM>. Otherwise, if the scan is complete (i.e., "Yes" in step <NUM>), the process ends.

<FIG> and <FIG> are block diagrams illustrating example microscope slide scanners, according to an embodiment, and <FIG> is a block diagram illustrating example linear sensor arrays, according to an embodiment. These three figures will be described in more detail below. However, they will first be described in combination to provide an overview. It should be noted that the following description is just an example of a slide scanner device and that alternative slide scanner devices can also be employed. <FIG> and <FIG> illustrate example microscope slide scanners that can be used in conjunction with the disclosed sensor arrangement. <FIG> illustrates example linear sensors, which can be used in any combination as the disclosed sensors (imaging sensor <NUM> or focusing sensor <NUM>).

For example, imaging sensor <NUM> and focusing sensor <NUM> may be arranged, as discussed above, using line scan camera <NUM> as primary imaging sensor <NUM>. In one embodiment, line scan camera <NUM> may include both focusing sensor <NUM> and imaging sensor <NUM>. Imaging sensor <NUM> and focusing sensor <NUM> can receive image information from a sample <NUM> through the microscope objective lens <NUM> and/or the focusing optics <NUM> and <NUM>. Focusing optics <NUM> for focusing sensor <NUM> may comprise the various beam splitters <NUM>, mirrors <NUM>, and glass blocks <NUM> illustrated in <FIG>. Imaging sensor <NUM> and focusing sensor <NUM> can provide information to, and/or receive information from, data processor <NUM>. Data processor <NUM> is communicatively connected to memory <NUM> and data storage <NUM>. Data processor <NUM> may further be communicatively connected to a communications port, which may be connected by at least one network <NUM> to one or more computers <NUM>, which may in turn be connected to display monitor(s) <NUM>.

Data processor <NUM> may also be communicatively connected to and provide instructions to a stage controller <NUM>, which controls a motorized stage <NUM> of slide scanner <NUM>. Motorized stage <NUM> supports sample <NUM> and moves in one or more directions in the X-Y plane. In one embodiment, motorized stage <NUM> may also move along the Z axis. Data processor <NUM> may also be communicatively connected to and provide instructions to a motorized controller <NUM>, which controls a motorized positioner <NUM> (e.g., a piezo positioner). Motorized positioner <NUM> is configured to move objective lens <NUM> in the Z axis. Slide scanner <NUM> also comprises a light source <NUM> and/or illumination optics <NUM> to illuminate sample <NUM>, either from above or below.

<FIG> is a block diagram of an embodiment of an optical microscopy system <NUM>, according to an embodiment. The heart of system <NUM> is a microscope slide scanner <NUM> that serves to scan and digitize a specimen or sample <NUM>. Sample <NUM> can be anything that may be interrogated by optical microscopy. For instance, sample <NUM> may comprise a microscope slide or other sample type that may be interrogated by optical microscopy. A microscope slide 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. 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 microarrays. Sample <NUM> may be a microtiter plate, for example a <NUM>-well plate. Other examples of sample <NUM> include integrated circuit boards, electrophoresis records, petri dishes, film, semiconductor materials, forensic materials, or machined parts.

Scanner <NUM> includes a motorized stage <NUM>, a microscope objective lens <NUM>, a line scan camera <NUM>, and a data processor <NUM>. Sample <NUM> is positioned on motorized stage <NUM> for scanning. Motorized stage <NUM> is connected to a stage controller <NUM> which is connected in turn to data processor <NUM>. Data processor <NUM> determines the position of sample <NUM> on motorized stage <NUM> via stage controller <NUM>. In an embodiment, motorized stage <NUM> moves sample <NUM> in at least the two axes (x/y) that are in the plane of sample <NUM>. Fine movements of sample <NUM> along the optical z-axis may also be necessary for certain applications of scanner <NUM>, for example, for focus control. Z-axis movement may be accomplished with a piezo positioner <NUM>, such as the PIFOC from Polytec PI or the MIPOS <NUM> from Piezosystem Jena. Piezo positioner <NUM> is attached directly to microscope objective <NUM> and is connected to and directed by data processor <NUM> via piezo controller <NUM>. A means of providing a coarse focus adjustment may also be needed and can be provided by Z-axis movement as part of motorized stage <NUM> or a manual rack-and-pinion coarse focus adjustment (not shown).

In one embodiment, motorized stage <NUM> includes a high-precision positioning table with ball bearing linear ways to provide smooth motion and excellent straight line and flatness accuracy. For example, motorized stage <NUM> could include two Daedal model <NUM> tables stacked one on top of the other. Other types of motorized stages <NUM> are also suitable for scanner <NUM>, including stacked single-axis stages based on ways other than ball bearings, single- or multiple-axis positioning stages that are open in the center and are particularly suitable for trans-illumination from below the sample, or larger stages that can support a plurality of samples. In one embodiment, motorized stage <NUM> includes two stacked single-axis positioning tables, each coupled to two millimeter lead-screws and Nema-<NUM> stepping motors. At the maximum lead screw speed of twenty-five revolutions per second, the maximum speed of sample <NUM> on the motorized stage <NUM> is fifty millimeters per second. Selection of a lead screw with larger diameter, for example five millimeters, can increase the maximum speed to more than <NUM> millimeters per second. Motorized stage <NUM> can be equipped with mechanical or optical position encoders which has the disadvantage of adding significant expense to the system. Consequently, such an embodiment does not include position encoders. However, if one were to use servo motors in place of stepping motors, then one would have to use position feedback for proper control.

Position commands from data processor <NUM> are converted to motor current or voltage commands in stage controller <NUM>. In one embodiment, stage controller <NUM> includes a <NUM>-axis servo/stepper motor controller (Compumotor 6K2) and two <NUM>-amp microstepping drives (Compumotor OEMZL4). Microstepping provides a means for commanding the stepper motor in much smaller increments than the relatively large single <NUM> degree motor step. For example, at a microstep of <NUM>, sample <NUM> can be commanded to move at steps as small as <NUM> micrometer. In an embodiment, a microstep of <NUM>,<NUM> is used. Smaller step sizes are also possible. It should be understood that the optimum selection of motorized stage <NUM> and stage controller <NUM> depends on many factors, including the nature of sample <NUM>, the desired time for sample digitization, and the desired resolution of the resulting digital image of sample <NUM>.

Microscope objective lens <NUM> can be any microscope objective lens commonly available. One of ordinary skill in the art will recognize that the choice of which objective lens to use will depend on the particular circumstances. In an embodiment, microscope objective lens <NUM> is of the infinity-corrected type.

Sample <NUM> is illuminated by an illumination system <NUM> that includes a light source <NUM> and illumination optics <NUM>. In an embodiment, light source <NUM> includes a variable intensity halogen light source with a concave reflective mirror to maximize light output and a KG-<NUM> filter to suppress heat. However, light source <NUM> could also be any other type of arc-lamp, laser, light emitting diode ("LED"), or other source of light. In an embodiment, illumination optics <NUM> include a standard Köhler illumination system with two conjugate planes that are orthogonal to the optical axis. Illumination optics <NUM> are representative of the bright-field illumination optics that can be found on most commercially-available compound microscopes sold by companies such as Leica, Carl Zeiss, Nikon, or Olympus. One set of conjugate planes includes (i) a field iris aperture illuminated by light source <NUM>, (ii) the object plane that is defined by the focal plane of sample <NUM>, and (iii) the plane containing the light-responsive elements of line scan camera <NUM>. A second conjugate plane includes (i) the filament of the bulb that is part of light source <NUM>, (ii) the aperture of a condenser iris that sits immediately before the condenser optics that are part of illumination optics <NUM>, and (iii) the back focal plane of the microscope objective lens <NUM>. In an embodiment, sample <NUM> is illuminated and imaged in transmission mode, with line scan camera <NUM> sensing optical energy that is transmitted by sample <NUM>, or conversely, optical energy that is absorbed by sample <NUM>.

Scanner <NUM> is equally suitable for detecting optical energy that is reflected from sample <NUM>, in which case light source <NUM>, illumination optics <NUM>, and microscope objective lens <NUM> must be selected based on compatibility with reflection imaging. A possible embodiment may therefore include illumination through a fiber optic bundle that is positioned above sample <NUM>. Other possibilities include excitation that is spectrally conditioned by a monochromator. If microscope objective lens <NUM> is selected to be compatible with phase-contrast microscopy, then the incorporation of at least one phase stop in the condenser optics that are part of illumination optics <NUM> will enable scanner <NUM> to be used for phase contrast microscopy. To one of ordinary skill in the art, the modifications required for other types of microscopy such as differential interference contrast and confocal microscopy should be readily apparent. Overall, scanner <NUM> is suitable, with appropriate but well-known modifications, for the interrogation of microscopic samples in any known mode of optical microscopy.

Between microscope objective lens <NUM> and line scan camera <NUM> are situated line scan camera focusing optics <NUM> that focus the optical signal captured by microscope objective lens <NUM> onto the light-responsive elements of line scan camera <NUM> (e.g., imaging sensor <NUM>). In a modern infinity-corrected microscope, the focusing optics between the microscope objective lens and the eyepiece optics, or between the microscope objective lens and an external imaging port, comprise an optical element known as a tube lens that is part of a microscope's observation tube. Many times the tube lens consists of multiple optical elements to prevent the introduction of coma or astigmatism. One of the motivations for the relatively recent change from traditional finite tube length optics to infinity corrected optics was to increase the physical space in which the optical energy from sample <NUM> is parallel, meaning that the focal point of this optical energy is at infinity. In this case, accessory elements like dichroic mirrors or filters can be inserted into the infinity space without changing the optical path magnification or introducing undesirable optical artifacts.

Infinity-corrected microscope objective lenses are typically inscribed with an infinity mark. The magnification of an infinity corrected microscope objective lens is given by the quotient of the focal length of the tube lens divided by the focal length of the objective lens. For example, a tube lens with a focal length of <NUM> millimeters will result in 20x magnification if an objective lens with <NUM> millimeter focal length is used. One of the reasons that the objective lenses manufactured by different microscope manufacturers are not compatible is because of a lack of standardization in the tube lens focal length. For example, a 20x objective lens from Olympus, a company that uses a <NUM> millimeter tube lens focal length, will not provide a 20x magnification on a Nikon microscope that is based on a different tube length focal length of <NUM> millimeters. Instead, the effective magnification of such an Olympus objective lens engraved with 20x and having a <NUM> millimeter focal length will be <NUM>. 2x, obtained by dividing the <NUM> millimeter tube lens focal length by the <NUM> millimeter focal length of the objective lens. Changing the tube lens on a conventional microscope is virtually impossible without disassembling the microscope. The tube lens is part of a critical fixed element of the microscope. Another contributing factor to the incompatibility between the objective lenses and microscopes manufactured by different manufacturers is the design of the eyepiece optics, the binoculars through which the specimen is observed. While most of the optical corrections have been designed into the microscope objective lens, most microscope users remain convinced that there is some benefit in matching one manufacturers' binocular optics with that same manufacturers' microscope objective lenses to achieve the best visual image.

Line scan camera focusing optics <NUM> include a tube lens optic mounted inside of a mechanical tube. Since scanner <NUM>, in an embodiment, lacks binoculars or eyepieces for traditional visual observation, the problem suffered by conventional microscopes of potential incompatibility between objective lenses and binoculars is immediately eliminated. One of ordinary skill will similarly realize that the problem of achieving parfocality between the eyepieces of the microscope and a digital image on a display monitor is also eliminated by virtue of not having any eyepieces. Since scanner <NUM> also overcomes the field of view limitation of a traditional microscope by providing a field of view that is practically limited only by the physical boundaries of sample <NUM>, the importance of magnification in an all-digital imaging microscope such as provided by scanner <NUM> is limited. Once a portion of sample <NUM> has been digitized, it is straightforward to apply electronic magnification, sometimes known as electric zoom, to an image of sample <NUM> in order to increase its magnification. Increasing the magnification of an image electronically has the effect of increasing the size of that image on the monitor that is used to display the image. If too much electronic zoom is applied, then the display monitor will be able to show only portions of the magnified image. However, it is not possible to use electronic magnification to display information that was not present in the original optical signal that was digitized in the first place. Since one of the objectives of scanner <NUM>, in an embodiment, is to provide high quality digital images, in lieu of visual observation through the eyepieces of a microscope, the content of the images acquired by scanner <NUM> should include as much image detail as possible. The term resolution is typically used to describe such image detail and the term diffraction-limited is used to describe the wavelength-limited maximum spatial detail available in an optical signal. Scanner <NUM> provides diffraction-limited digital imaging by selection of a tube lens focal length that is matched according to the well-known Nyquist sampling criteria to both the size of an individual pixel element in a light-sensing camera such as line scan camera <NUM> and to the numerical aperture of microscope objective lens <NUM>. It is well known that numerical aperture, not magnification, is the resolution-limiting attribute of a microscope objective lens.

An example will help to illustrate the optimum selection of a tube lens focal length that is part of line scan camera focusing optics <NUM>. Consider again the 20x microscope objective lens <NUM> with <NUM> millimeter focal length discussed previously, and assume that this objective lens has a numerical aperture of <NUM>. Assuming no appreciable degradation from the condenser, the diffraction-limited resolving power of this objective lens at a wavelength of <NUM> nanometers is approximately <NUM> micrometers, obtained using the well-known Abbe relationship. Assume further that line scan camera <NUM>, which in an embodiment has a plurality of <NUM> micrometer square pixels, is used to detect a portion of sample <NUM>. In accordance with sampling theory, it is necessary that at least two sensor pixels subtend the smallest resolvable spatial feature. In this case, the tube lens must be selected to achieve a magnification of <NUM>, obtained by dividing <NUM> micrometers, which corresponds to two <NUM> micrometer pixels, by <NUM> micrometers, the smallest resolvable feature dimension. The optimum tube lens optic focal length is therefore about <NUM> millimeters, obtained by multiplying <NUM> by <NUM>. Line scan focusing optics <NUM> with a tube lens optic having a focal length of <NUM> millimeters will therefore be capable of acquiring images with the best possible spatial resolution, similar to what would be observed by viewing a specimen under a microscope using the same 20x objective lens. To reiterate, scanner <NUM> utilizes a traditional 20x microscope objective lens <NUM> in a higher magnification optical configuration (about 47x in the example above) in order to acquire diffraction-limited digital images. If a traditional 20x magnification objective lens <NUM> with a higher numerical aperture were used, say <NUM>, the required tube lens optic magnification for diffraction-limited imaging would be about <NUM> millimeters, corresponding to an overall optical magnification of 68x. Similarly, if the numerical aperture of the 20x objective lens were only <NUM>, the optimum tube lens optic magnification would only be about 28x, which corresponds to a tube lens optic focal length of approximately <NUM> millimeters. Line scan camera focusing optics <NUM> may be modular elements of scanner <NUM> that can be interchanged as necessary for optimum digital imaging. The advantage of diffraction-limited digital imaging is particularly significant for applications, for example bright field microscopy, in which the reduction in signal brightness that accompanies increases in magnification is readily compensated by increasing the intensity of an appropriately designed illumination system <NUM>.

In principle, it is possible to attach external magnification-increasing optics to a conventional microscope-based digital imaging system to effectively increase the tube lens magnification so as to achieve diffraction-limited imaging as has just been described for scanner <NUM>. However, the resulting decrease in the field of view is often unacceptable, making this approach impractical. Furthermore, many users of microscopes typically do not understand enough about the details of diffraction-limited imaging to effectively employ these techniques on their own. In practice, digital cameras are attached to microscope ports with magnification-decreasing optical couplers to attempt to increase the size of the field of view to something more similar to what can be seen through the eyepiece. The standard practice of adding de-magnifying optics is a step in the wrong direction if the goal is to obtain diffraction-limited digital images.

In a conventional microscope, different power objectives lenses are typically used to view the specimen at different resolutions and magnifications. Standard microscopes have a nosepiece that holds five objectives lenses. In an all-digital imaging system, such as scanner <NUM>, there is a need for only one microscope objective lens <NUM> with a numerical aperture corresponding to the highest spatial resolution desirable. Thus, in an embodiment, scanner <NUM> consists of only one microscope objective lens <NUM>. Once a diffraction-limited digital image has been captured at this resolution, it is straightforward using standard digital image processing techniques, to present imagery information at any desirable reduced resolutions and magnifications.

One embodiment of scanner <NUM> is based on a Dalsa SPARK line scan camera <NUM> with <NUM> pixels (picture elements) arranged in a linear array, with each pixel having a dimension of <NUM> by <NUM> micrometers. Any other type of linear array, whether packaged as part of a camera or custom-integrated into an imaging electronic module, can also be used. The linear array in one embodiment effectively provides eight bits of quantization, but other arrays providing higher or lower level of quantization may also be used. Alternate arrays based on three-channel red-green-blue (RGB) color information or time delay integration (TDI), may also be used. TDI arrays 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 arrays can comprise multiple stages of linear arrays. TDI arrays are available with <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or even more stages. Scanner <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. Appropriate, but well known, modifications to illumination system <NUM> and line scan camera focusing optics <NUM> may be required to accommodate larger arrays. Linear arrays with a variety of pixel sizes can also be used in scanner <NUM>. The salient requirement for the selection of any type of line scan camera <NUM> is that sample <NUM> can be in motion with respect to line scan camera <NUM> during the digitization of sample <NUM>, in order to obtain high quality images, overcoming the static requirements of the conventional imaging tiling approaches known in the prior art.

The output signal of line scan camera <NUM> is connected to data processor <NUM>. In an embodiment, data processor <NUM> includes a central processing unit with ancillary electronics (e.g., a motherboard) to support at least one signal digitizing electronics board such as an imaging board or a frame grabber. In an embodiment, the imaging board is an EPIX PIXCID24 PCI bus imaging board. However, there are many other types of imaging boards or frame grabbers from a variety of manufacturers which could be used in place of the EPIX board. An alternative embodiment could be a line scan camera that uses an interface such as IEEE <NUM>, also known as Firewire, to bypass the imaging board altogether and store data directly on data storage <NUM> (e.g., a hard disk).

Data processor <NUM> is also connected to a memory <NUM>, such as random access memory (RAM), for the short-term storage of data, and to data storage <NUM>, such as a hard drive, for long-term data storage. Further, data processor <NUM> is connected to a communications port <NUM> that is connected to a network <NUM> such as a local area network (LAN), a wide area network (WAN), a metropolitan area network (MAN), an intranet, an extranet, or the Internet. Memory <NUM> and data storage <NUM> are also connected to each other. Data processor <NUM> is also capable of executing computer programs, in the form of software, to control critical elements of scanner <NUM> such as line scan camera <NUM> and stage controller <NUM>, or for a variety of image-processing functions, image-analysis functions, or networking. Data processor <NUM> can be based on any operating system, including operating systems such as Windows, Linux, OS/<NUM>, Mac OS, and Unix. In an embodiment, data processor <NUM> operates based on the Windows NT operating system.

Data processor <NUM>, memory <NUM>, data storage <NUM>, and communication port <NUM> are each elements that can be found in a conventional computer. One example would be a personal computer such as a Dell Dimension XPS T500 that features a Pentium III <NUM> processor and up to <NUM> megabytes (MB) of RAM. In an embodiment, the computer elements which include the data processor <NUM>, memory <NUM>, data storage <NUM>, and communications port <NUM> are all internal to scanner <NUM>, so that the only connection of scanner <NUM> to the other elements of system <NUM> is via communication port <NUM>. In an alternative embodiment of scanner <NUM>, the computer elements could be external to scanner <NUM> with a corresponding connection between the computer elements and scanner <NUM>.

In an embodiment, scanner <NUM> integrates optical microscopy, digital imaging, motorized sample positioning, computing, and network-based communications into a single-enclosure unit. The major advantage of packaging scanner <NUM> as a single-enclosure unit, with communications port <NUM> as the primary means of data input and output, are reduced complexity and increased reliability. The various elements of scanner <NUM> are optimized to work together, in sharp contrast to traditional microscope-based imaging systems in which the microscope, light source, motorized stage, camera, and computer are typically provided by different vendors and require substantial integration and maintenance.

Communication port <NUM> provides a means for rapid communications with the other elements of system <NUM>, including network <NUM>. One communications protocol for communications port <NUM> is a carrier-sense multiple-access collision detection protocol such as Ethernet, together with the TCP/IP protocol for transmission control and internetworking. Scanner <NUM> is intended to work with any type of transmission media, including broadband, baseband, coaxial cable, twisted pair, fiber optics, DSL, or wireless.

In one embodiment, control of scanner <NUM> and review of the imagery data captured by scanner <NUM> are performed on a computer <NUM> that is connected to network <NUM>. In an embodiment, computer <NUM> is connected to a display monitor <NUM> to provide imagery information to an operator. A plurality of computers <NUM> may be connected to network <NUM>. In an embodiment, computer <NUM> communicates with scanner <NUM> using a network browser such as Internet Explorer™ from Microsoft™, Chrome™ from Google™, Safari™ from Apple™, etc. Images are stored on scanner <NUM> in a common compressed format, such as JPEG, which is an image format that is compatible with standard image-decompression methods that are already built into most commercial browsers. Other standard or non-standard, lossy or lossless, image compression formats will also work. In one embodiment, scanner <NUM> is a web server providing an operator interface that is based on web pages that are sent from scanner <NUM> to computer <NUM>. For dynamic review of imagery data, an embodiment of scanner <NUM> is based on playing back, for review on display monitor <NUM> that is connected to computer <NUM>, multiple frames of imagery data using standard multiple-frame browser compatible software packages such as Media-Player™ from Microsoft™, Quicktime™ from Apple™, or RealPlayer™ from Real Networks™. In an embodiment, the browser on computer <NUM> uses the Hypertext Transfer Protocol (HTTP) together with TCP for transmission control.

There are, and will be in the future, many different means and protocols by which scanner <NUM> could communicate with computer <NUM>, or a plurality of computers. While one embodiment is based on standard means and protocols, the approach of developing one or multiple customized software modules known as applets is equally feasible and may be desirable for selected future applications of scanner <NUM>. Furthermore, there are no constraints that computer <NUM> be of any specific type such as a personal computer (PC) or be manufactured by any specific company such as Dell™. One of the advantages of a standardized communications port <NUM> is that any type of computer <NUM>, operating common network browser software, can communicate with scanner <NUM>.

If desired, it is possible, with some modifications to scanner <NUM>, to obtain spectrally-resolved images. Spectrally-resolved images are images in which spectral information is measured at every image pixel. Spectrally-resolved images could be obtained by replacing line scan camera <NUM> of scanner <NUM> with an optical slit and an imaging spectrograph. The imaging spectrograph uses a two-dimensional CCD detector to capture wavelength-specific intensity data for a column of image pixels by using a prism or grating to disperse the optical signal that is focused on the optical slit along each of the rows of the detector.

<FIG> is a block diagram of a second embodiment of an optical microscopy system <NUM>, according to an embodiment. In this system <NUM>, scanner <NUM> is more complex and expensive than the embodiment shown in <FIG>. The additional attributes of scanner <NUM> that are shown do not all have to be present for any alternative embodiment to function correctly. <FIG> is intended to provide a reasonable example of additional features and capabilities that could be incorporated into scanner <NUM>.

The alternative embodiment of <FIG> provides for a much greater level of automation than the embodiment of <FIG>. A more complete level of automation of illumination system <NUM> is achieved by connections between data processor <NUM> and both light source <NUM> and illumination optics <NUM> of illumination system <NUM>. The connection to light source <NUM> may control the voltage, or current, in an open or closed loop fashion, in order to control the intensity of light source <NUM>. Recall that light source <NUM> may be a halogen bulb. The connection between data processor <NUM> and illumination optics <NUM> could provide closed-loop control of the field iris aperture and the condenser iris to provide a means for ensuring that optimum Köhler illumination is maintained.

Use of scanner <NUM> for fluorescence imaging requires easily recognized modifications to light source <NUM>, illumination optics <NUM>, and microscope objective lens <NUM>. The embodiment of <FIG> also provides for a fluorescence filter cube <NUM> that includes an excitation filter, a dichroic filter, and a barrier filter. Fluorescence filter cube <NUM> is positioned in the infinity-corrected beam path that exists between microscope objective lens <NUM> and line scan camera focusing optics <NUM>. An embodiment for fluorescence imaging could include the addition of a filter wheel or tunable filter into illumination optics <NUM> to provide appropriate spectral excitation for the variety of fluorescent dyes or nano-crystals available on the market.

The addition of at least one beam splitter <NUM> into the imaging path allows the optical signal to be split into at least two paths. The primary path is via line scan camera focusing optics <NUM>, as discussed previously, to enable diffraction-limited imaging by line scan camera <NUM> (which may include imaging sensor <NUM>). A second path is provided via an area scan camera focusing optics <NUM> for imaging by an area scan camera <NUM>. It should be readily apparent that proper selection of these two focusing optics can ensure diffraction-limited imaging by the two camera sensors having different pixel sizes. Area scan camera <NUM> can be one of many types that are currently available, including a simple color video camera, a high performance, cooled, CCD camera, or a variable integration-time fast frame camera. Area scan camera <NUM> provides a traditional imaging system configuration for scanner <NUM>. Area scan camera <NUM> is connected to data processor <NUM>. If two cameras are used, for example line scan camera <NUM> and area scan camera <NUM>, both camera types could be connected to the data processor using either a single dual-purpose imaging board, two different imaging boards, or the IEEE1394 Firewire interface, in which case one or both imaging boards may not be needed. Other related methods of interfacing imaging sensors to data processor <NUM> are also available.

While the primary interface of scanner <NUM> to computer <NUM> is via network <NUM>, there may be instances, for example a failure of network <NUM>, where it is beneficial to be able to connect scanner <NUM> directly to a local output device such as display monitor <NUM> and to also provide local input devices such as a keyboard and mouse <NUM> that are connected directly into data processor <NUM> of scanner <NUM>. In this instance, the appropriate driver software and hardware would have to be provided as well.

The second embodiment shown in <FIG> also provides for a much greater level of automated imaging performance. Enhanced automation of the imaging of scanner <NUM> can be achieved by closing the focus-control loop comprising piezo positioner <NUM>, piezo controller <NUM>, and data processor <NUM> using well-known methods of autofocus. The second embodiment also provides for a motorized nose-piece <NUM> to accommodate several objectives lenses. The motorized nose-piece <NUM> is connected to and directed by data processor <NUM> through a nose-piece controller <NUM>.

There are other features and capabilities of scanner <NUM> which could be incorporated. For example, the process of scanning sample <NUM> with respect to microscope objective lens <NUM> that is substantially stationary in the X-Y plane of sample <NUM> could be modified to comprise scanning of microscope objective lens <NUM> with respect to a stationary sample <NUM> (i.e., moving microscope objective lens <NUM> in an X-Y plane). Scanning sample <NUM>, or scanning microscope objective lens <NUM>, or scanning both sample <NUM> and microscope objective lens <NUM> simultaneously, are possible embodiments of scanner <NUM> which can provide the same large contiguous digital image of sample <NUM> as discussed previously.

Scanner <NUM> also provides a general purpose platform for automating many types of microscope-based analyses. Illumination system <NUM> could be modified from a traditional halogen lamp or arc-lamp to a laser-based illumination system to permit scanning of sample <NUM> with laser excitation. Modifications, including the incorporation of a photomultiplier tube or other non-imaging detector, in addition to or in lieu of line scan camera <NUM> or area scan camera <NUM>, could be used to provide a means of detecting the optical signal resulting from the interaction of the laser energy with sample <NUM>.

Turning now to <FIG>, line scan camera field of view <NUM> comprises the region of sample <NUM> of <FIG> that is imaged by a multitude of individual pixel elements <NUM> that are arranged in a linear fashion into a linear array <NUM>. Linear array <NUM> of an embodiment comprises <NUM> of the individual pixel elements <NUM>, with each of pixel elements <NUM> being <NUM> micrometers square. In an embodiment, the physical dimensions of linear array <NUM> are <NUM> millimeters by <NUM> micrometers. Assuming, for purposes of discussion of the operation of the scanner <NUM>, that the magnification between sample <NUM> and line scan camera <NUM> is ten, then line scan camera field of view <NUM> corresponds to a region of sample <NUM> that has dimensions equal to <NUM> millimeters by <NUM> micrometers. Each pixel element <NUM> images an area about <NUM> micrometers by <NUM> micrometers.

In one embodiment of scanner <NUM>, the scanning and digitization is performed in a direction of travel that alternates between image strips. This type of bi-directional scanning provides for a more rapid digitization process than uni-directional scanning, a method of scanning and digitization which requires the same direction of travel for each image strip.

The capabilities of line scan camera <NUM> (e.g., comprising imaging sensor <NUM>) and focusing sensor <NUM> typically determine whether scanning and focusing can be done bidirectionally or uni-directionally. Uni-directional systems often comprise more than one linear array <NUM>, such as a three channel color array <NUM> or a multi-channel TDI array <NUM> shown in <FIG>. Color array <NUM> detects the RGB intensities required for obtaining a color image. An alternative embodiment for obtaining color information uses a prism to split the broadband optical signal into the three color channels. TDI array <NUM> could be used in an alternate embodiment of scanner <NUM> to provide a means of increasing the effective integration time of line scan camera <NUM>, while maintaining a fast data rate, and without significant loss in the signal-to-noise ratio of the digital imagery data.

<FIG> is a block diagram illustrating an example wired or wireless system <NUM> that may be used in connection with various embodiments described herein. For example system <NUM> may be used as or in conjunction with one or more of the mechanisms, processes, methods, or functions described above, and may represent components of slide scanner <NUM>, such as data processor <NUM>. System <NUM> can be any processor-enabled device that is capable of wired or wireless data communication. Other computer systems and/or architectures may be also used, as will be clear to those skilled in the art.

System <NUM> preferably includes one or more processors, such as processor <NUM>. Additional processors may be provided, such as an auxiliary processor to manage input/output, an auxiliary processor to perform floating point mathematical operations, a special-purpose microprocessor having an architecture suitable for fast execution of signal processing algorithms (e.g., digital signal processor), a slave processor subordinate to the main processing system (e.g., back-end processor), an additional microprocessor or controller for dual or multiple processor systems, or a coprocessor. Such auxiliary processors may be discrete processors or may be integrated with the processor <NUM>. Examples of processors which may be used with system <NUM> include, without limitation, the Pentium® processor, Core i7® processor, and Xeon® processor, all of which are available from Intel Corporation of Santa Clara, California.

Processor <NUM> is preferably connected to a communication bus <NUM>. Communication bus <NUM> may include a data channel for facilitating information transfer between storage and other peripheral components of system <NUM>. Communication bus <NUM> further may provide a set of signals used for communication with processor <NUM>, including a data bus, address bus, and control bus (not shown). Communication bus <NUM> may comprise any standard or non-standard bus architecture such as, for example, bus architectures compliant with industry standard architecture (ISA), extended industry standard architecture (EISA), Micro Channel Architecture (MCA), peripheral component interconnect (PCI) local bus, or standards promulgated by the Institute of Electrical and Electronics Engineers (IEEE) including IEEE <NUM> general-purpose interface bus (GPIB), IEEE <NUM>/S-<NUM>, and the like.

System <NUM> preferably includes a main memory <NUM> and may also include a secondary memory <NUM>. Main memory <NUM> provides storage of instructions and data for programs executing on processor <NUM>, such as one or more of the functions and/or modules discussed above. It should be understood that programs stored in the memory and executed by processor <NUM> may be written and/or compiled according to any suitable language, including without limitation C/C++, Java, JavaScript, Perl, Visual Basic,. NET, and the like. Main memory <NUM> is typically semiconductor-based memory such as dynamic random access memory (DRAM) and/or static random access memory (SRAM). Other semiconductor-based memory types include, for example, synchronous dynamic random access memory (SDRAM), Rambus dynamic random access memory (RDRAM), ferroelectric random access memory (FRAM), and the like, including read only memory (ROM).

Secondary memory <NUM> may optionally include an internal memory <NUM> and/or a removable medium <NUM>. Removable medium <NUM> is read from and/or written to in any well-known manner. Removable storage medium <NUM> may be, for example, a magnetic tape drive, a compact disc (CD) drive, a digital versatile disc (DVD) drive, other optical drive, a flash memory drive, etc..

Removable storage medium <NUM> is a non-transitory computer-readable medium having stored thereon computer-executable code (i.e., software) and/or data. The computer software or data stored on removable storage medium <NUM> is read into system <NUM> for execution by processor <NUM>.

In alternative embodiments, secondary memory <NUM> may include other similar means for allowing computer programs or other data or instructions to be loaded into system <NUM>. Such means may include, for example, an external storage medium <NUM> and a communication interface <NUM> (e.g., communication port <NUM>), which allows software and data to be transferred from external storage medium <NUM> to system <NUM>. Examples of external storage medium <NUM> may include an external hard disk drive, an external optical drive, an external magneto-optical drive, etc. Other examples of secondary memory <NUM> may include semiconductor-based memory such as programmable read-only memory (PROM), erasable programmable read-only memory (EPROM), electrically erasable read-only memory (EEPROM), or flash memory (block-oriented memory similar to EEPROM).

As mentioned above, system <NUM> may include a communication interface <NUM>. Communication interface <NUM> allows software and data to be transferred between system <NUM> and external devices (e.g. printers), networks, or other information sources. For example, computer software or executable code may be transferred to system <NUM> from a network server via communication interface <NUM>. Examples of communication interface <NUM> include a built-in network adapter, network interface card (NIC), Personal Computer Memory Card International Association (PCMCIA) network card, card bus network adapter, wireless network adapter, Universal Serial Bus (USB) network adapter, modem, a network interface card (NIC), a wireless data card, a communications port, an infrared interface, an IEEE <NUM> fire-wire, or any other device capable of interfacing system <NUM> with a network or another computing device. Communication interface <NUM> preferably implements industry-promulgated protocol standards, such as Ethernet IEEE <NUM> standards, Fiber Channel, digital subscriber line (DSL), asynchronous digital subscriber line (ADSL), frame relay, asynchronous transfer mode (ATM), integrated digital services network (ISDN), personal communications services (PCS), transmission control protocol/Internet protocol (TCP/IP), serial line Internet protocol/point to point protocol (SLIP/PPP), and so on, but may also implement customized or non-standard interface protocols as well.

Software and data transferred via communication interface <NUM> are generally in the form of electrical communication signals <NUM>. These signals <NUM> may be provided to communication interface <NUM> via a communication channel <NUM>. In an embodiment, communication channel <NUM> may be a wired or wireless network, or any variety of other communication links. Communication channel <NUM> carries signals <NUM> and can be implemented using a variety of wired or wireless communication means including wire or cable, fiber optics, conventional phone line, cellular phone link, wireless data communication link, radio frequency ("RF") link, or infrared link, just to name a few.

Computer-executable code (i.e., computer programs or software) is stored in main memory <NUM> and/or the secondary memory <NUM>. Computer programs can also be received via communication interface <NUM> and stored in main memory <NUM> and/or secondary memory <NUM>. Such computer programs, when executed, enable system <NUM> to perform the various functions of the disclosed embodiments as described elsewhere herein.

In this description, the term "computer-readable medium" is used to refer to any non-transitory computer-readable storage media used to provide computer-executable code (e.g., software and computer programs) to system <NUM>. Examples of such media include main memory <NUM>, secondary memory <NUM> (including internal memory <NUM>, removable medium <NUM>, and external storage medium <NUM>), and any peripheral device communicatively coupled with communication interface <NUM> (including a network information server or other network device). These non-transitory computer-readable mediums are means for providing executable code, programming instructions, and software to system <NUM>.

In an embodiment that is implemented using software, the software may be stored on a computer-readable medium and loaded into system <NUM> by way of removable medium <NUM>, I/O interface <NUM>, or communication interface <NUM>. In such an embodiment, the software is loaded into system <NUM> in the form of electrical communication signals <NUM>. The software, when executed by processor <NUM>, preferably causes processor <NUM> to perform the features and functions described elsewhere herein.

In an embodiment, I/O interface <NUM> provides an interface between one or more components of system <NUM> and one or more input and/or output devices. Example input devices include, without limitation, keyboards, touch screens or other touch-sensitive devices, biometric sensing devices, computer mice, trackballs, pen-based pointing devices, and the like. Examples of output devices include, without limitation, cathode ray tubes (CRTs), plasma displays, light-emitting diode (LED) displays, liquid crystal displays (LCDs), printers, vacuum florescent displays (VFDs), surface-conduction electron-emitter displays (SEDs), field emission displays (FEDs), and the like.

System <NUM> also includes optional wireless communication components that facilitate wireless communication over a voice network and/or a data network. The wireless communication components comprise an antenna system <NUM>, a radio system <NUM>, and a baseband system <NUM>. In system <NUM>, radio frequency (RF) signals are transmitted and received over the air by antenna system <NUM> under the management of radio system <NUM>.

In one embodiment, antenna system <NUM> may comprise one or more antennae and one or more multiplexors (not shown) that perform a switching function to provide antenna system <NUM> with transmit and receive signal paths. In the receive path, received RF signals can be coupled from a multiplexor to a low noise amplifier (not shown) that amplifies the received RF signal and sends the amplified signal to radio system <NUM>.

In alternative embodiments, radio system <NUM> may comprise one or more radios that are configured to communicate over various frequencies. In an embodiment, radio system <NUM> may combine a demodulator (not shown) and modulator (not shown) in one integrated circuit (IC). The demodulator and modulator can also be separate components. In the incoming path, the demodulator strips away the RF carrier signal leaving a baseband receive audio signal, which is sent from radio system <NUM> to baseband system <NUM>.

If the received signal contains audio information, then baseband system <NUM> decodes the signal and converts it to an analog signal. Then the signal is amplified and sent to a speaker. Baseband system <NUM> also receives analog audio signals from a microphone. These analog audio signals are converted to digital signals and encoded by baseband system <NUM>. Baseband system <NUM> also codes the digital signals for transmission and generates a baseband transmit audio signal that is routed to the modulator portion of radio system <NUM>. The modulator mixes the baseband transmit audio signal with an RF carrier signal generating an RF transmit signal that is routed to antenna system <NUM> and may pass through a power amplifier (not shown). The power amplifier amplifies the RF transmit signal and routes it to antenna system <NUM> where the signal is switched to the antenna port for transmission.

Baseband system <NUM> is also communicatively coupled with processor <NUM>, which may be a central processing unit (CPU). Processor <NUM> has access to data storage areas <NUM> and <NUM>. Processor <NUM> is preferably configured to execute instructions (i.e., computer programs or software) that can be stored in main memory <NUM> or secondary memory <NUM>. Computer programs can also be received from baseband processor <NUM> and stored in main memory <NUM> or in secondary memory <NUM>, or executed upon receipt. Such computer programs, when executed, enable system <NUM> to perform the various functions of the disclosed embodiments. For example, data storage areas <NUM> or <NUM> may include various software modules.

Those of skill in the art will appreciate that the various illustrative logical blocks, modules, circuits, and method steps described in connection with the above described figures and the embodiments disclosed herein can often be implemented as electronic hardware, computer software, or combinations of both. Skilled persons can implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the invention. In addition, the grouping of functions within a module, block, circuit, or step is for ease of description. Specific functions or steps can be moved from one module, block, or circuit to another without departing from the invention.

Any of the software components described herein may take a variety of forms. For example, a component may be a stand-alone software package, or it may be a software package incorporated as a "tool" in a larger software product. It may be downloadable from a network, for example, a website, as a stand-alone product or as an add-in package for installation in an existing software application. It may also be available as a client-server software application, as a web-enabled software application, and/or as a mobile application.

Claim 1:
A system (<NUM>) for scanning a sample (<NUM>) to acquire a digital image of the sample (<NUM>), the system (<NUM>) comprising:
a stage configured to support the sample (<NUM>);
an objective lens (<NUM>) having a single optical axis that is orthogonal to the stage;
an imaging sensor (<NUM>);
a focusing sensor (<NUM>);
at least one first beam splitter (<NUM>) optically coupled to the objective lens (<NUM>) and configured to receive a field of view corresponding to the optical axis of the objective lens (<NUM>), and simultaneously provide at least a first portion of the field of view to the imaging sensor (<NUM>) and at least a second portion of the field of view to the focusing sensor (<NUM>); and
focusing optics (<NUM>) comprising at least one second beam splitter (<NUM>) that is optically coupled to the at least one first beam splitter (<NUM>) and splits a beam (<NUM>), conveying the second portion of the field of view, into a plurality of separate optical paths (610A, 610B, 610C),
wherein the first portion of the field of view is a first region of the field of view, and wherein the second portion of the field of view is a second region of the field of view that is offset from the first region of the field of view; and
wherein one point (P) on the focusing sensor (<NUM>) is parfocal with the imaging sensor (<NUM>),
wherein the focusing sensor (<NUM>) comprises a plurality of regions, wherein each region of the focusing sensor (<NUM>) receives the second portion of the field of view along one of the separate optical paths (610A, 610B, 610C), wherein the focusing sensor (<NUM>) is tilted at an angle with respect to each of the separate optical paths, such that the second portion of the field of view is acquired, by each region of the focusing sensor (<NUM>), at a different focal distance than the other regions of the focusing sensor (<NUM>), and wherein at least one point on one of the plurality of regions is parfocal with the imaging sensor (<NUM>).